Cell Tissue Res DOI 10.1007/s00441-014-2049-8

REVIEW

Can the ‘neuron theory’ be complemented by a universal mechanism for generic neuronal differentiation Uwe Ernsberger

Received: 23 October 2014 / Accepted: 23 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract With the establishment of the ‘neuron theory’ at the turn of the twentieth century, this remarkably powerful term was introduced to name a breathtaking diversity of cells unified by a characteristic structural compartmentalization and unique information processing and propagating features. At the beginning of the twenty-first century, developmental, stem cell and reprogramming studies converged to suggest a common mechanism involved in the generation of possibly all vertebrate, and at least a significant number of invertebrate, neurons. Sox and, in particular, SoxB and SoxC proteins as well as basic helixloop-helix proteins play major roles, even though their precise contributions to progenitor programming, proliferation and differentiation are not fully resolved. In addition to neuronal development, these transcription factors also regulate sensory receptor and endocrine cell development, thus specifying a range of cells with regulatory and communicative functions. To what extent microRNAs contribute to the diversification of these cell types is an upcoming question. Understanding the transcriptional and post-transcriptional regulation of genes coding for cell type-specific cytoskeletal and motor proteins as well as synaptic and ion channel proteins, which mark differences but also similarities between the three communicator cell types, will provide a key to the comprehension of their diversification and the signature of ‘generic neuronal’ differentiation. Apart from the general scientific significance of a putative universal core instruction for neuronal development, the impact of this line of research for cell replacement therapy and brain tumor treatment will be of considerable interest.

Keywords Pan-neuronal . Sensory . Endocrine . Secretory . Sox . bHLH . MicroRNA U. Ernsberger (*) Max-Planck-Institute for Brain Research, Deutschordenstr. 46, D-60528 Frankfurt, Germany e-mail: [email protected]

Introduction The groundbreaking observation that transplantation of the dorsal blastpore lip in frog embryos induces the formation of an ectopic neural tube (Spemann and Mangold 1924; Spemann 1927, 1938) may well be considered the beginning of Developmental Neuroscience. In conjunction with the establishment of the ‘neuron theory’, that individual cellular entities called nerve cells or ‘neurons’ constitute the crucial morphological and functional units in nervous tissue (Waldeyer 1891; Ramon y Cajal 1894, 1906, 1909/ 1911, 1954; selected translations in Shepherd 1991), this prompted not only the question of the induction of the entire organ anlage but also of the generation of its most prominent constituents, the nerve cells. From the outset, the unifying term ‘neuron’ for a bewildering variety of morphologically, biochemically and functionally different cell populations emphasized a common principle of cell morphology and physiology that was later supported by the distinguishing property of electrical information processing and propagation. Here, I summarize the currently available literature on (1) key neuronal features such as ion channels, synaptic and cytoskeletal proteins, (2) Sox and basic helix-loop-helix (bHLH) transcription factors in vertebrate and invertebrate neural development, and (3) the transition from progenitor proliferation to neuronal differentiation, in the search for a regulatory mechanism conserved across vertebrate and possibly invertebrate neuronal development. Comparison with (4) sensory receptor cell and (5) endocrine cell differentiation in vertebrates and the role of Sox and bHLH proteins therein shows a large overlap in the developmental regulators employed by these cell lineages and neurons. Notable similarities are also observed with (6) secretory cell development. Significant differences in (7) microRNA equipment distinguish neurons from the endocrine and secretory cell types in vertebrates.

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Thus, a differentiation mechanism common to vertebrate neurons, which induces the developmental acquisition of key features shared across neuronal populations, the ‘generic’ neuron-specific properties, appears to entail not only certain Sox and bHLH proteins, but also microRNAs which may be involved in the diversion of neuronal, endocrine and secretory lineages. How far this extends to invertebrates is still open to analysis.

Sodium channel gene expression and the quest for the master regulator The characterization of the action potential as a universal information carrier in neurons and the critical role of voltage-dependent sodium conductances in its generation (Hodgkin and Huxley 1952a, b) provided a unique functional signature to neuronal information propagation. Distinct action potential waveforms and sodium conductance pharmacology between neuron populations, however, suggested mechanistic diversity, later confirmed by molecular characterization of multiple sodium channel protein isoforms and the corresponding gene family expressed in the nervous system (see Catterall et al. 2005, 2012 for reviews). However, action potential generation does not mark a unique feature of neurons. Muscle cells are also able to generate action potentials, albeit employing distinct sodium channel proteins from genes specifically expressed in different classes of muscle cells, and related to but different from those coding for neuronal sodium channels (see Catterall et al. 2005 for review). More importantly for our discussion, sensory receptor cells and endocrine cells display action potential activity. Pancreatic beta-cells fire action potentials in response to increasing glucose levels resulting in elevated calcium influx and enhanced insulin granule exocytosis (Drews et al. 2010; Rorsman and Braun 2013; Fridlyand et al. 2013 for reviews). Sodium channels also expressed in neurons are involved. Hair cells in the inner ear can spontaneously generate action potentials even before the cochlea responds reliably to sound (see Kennedy 2012 for review). These action potentials, however, are carried largely by calcium channels. Such a situation may also be found transiently in embryonic neurons (Desarmenien et al. 1993). The search for regulators of neuronal sodium channel gene expression uncovered the neuron-restrictive silencer factor (NRSF/REST), a zinc finger transcription factor initially considered a master regulator of neuronal gene expression governing not only transcription of sodium channel genes but also of a large number of other genes specifically detected in neurons (Schoenherr and Anderson 1995; Ballas and Mandel 2005 for review). However, largely normal development of neural and non-neural tissues in mice (Chen et al. 1998) and zebrafish (Kok et al. 2012) with mutational

inactivation of the gene coding for this transcriptional regulator disproved its key role in neural or neuronal specification. Yet the demonstration of NRSF/REST-binding motifs in a large variety of neuronally expressed genes drew attention to the questions as to how cell type-specific gene expression is coordinated during neuron differentiation, which aspects are shared between neuron populations, and what is the nature of the regulators. The former two issues attempt to define the characteristics of generic neuronal differentiation. Two features and target gene groups other than action potential generation and voltage-gated ion channels have attracted increasing attention in the search for the substrate and the regulators of generic nerve cell differentiation (Yang et al. 2011; Ernsberger 2012). On the one hand, there are neuronal morphology and cytoskeletal proteins, in particular expression of neuron-specific ßIII tubulin and microtubule-associated protein 2 (Map2) that are widely used as indicators of a neuronal phenotype. On the other hand, neurotransmission and synaptic proteins not only play a fundamental role in neuronal signal propagation but also promise to provide important information on divergence of neurons and related sensory and secretory cell types in development and evolution. In addition to the enhanced focus on the coordination of neuronal gene expression and the question as to which gene products constitute generic neuronal markers, characterization of the mechanism by which NRSF/REST mediates regulation of gene expression drew attention to the role of chromatin modification in neuronal differentiation. This emerging field promises to provide key insights into the problem of how the genome unfolds to allow divergent gene expression during differentiation of related cell types.

Synaptic proteins: evidence for a conserved neuron-specific gene expression program Synaptic protein expression during neurogenesis provides information on the developmental acquisition of a large set of molecular players crucial to neuronal function. A plethora of proteins involved in neurotransmitter release by vesicle fusion have been characterized and their critical roles in compartments of the neuronal synapse have been established in remarkable detail (Sudhof 2004, 2012). Proteome analysis allows the characterization of the protein inventory in these compartments (for review, see Laßek et al., this issue) and documents a similar protein equipment of glutamatergic and GABAergic synaptic vesicles (Boyken et al. 2013; Grønborg et al. 2010). By this means, a quantitative model of the neuronal synaptic vesicle was derived (Takamori et al. 2006). Yet synaptic proteins, including those involved in the formation of the SNARE complex required for vesicle fusion to the plasma membrane, such as synaptosomal-associated

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protein 25 (Snap-25) and vesicle-associated membrane protein 2 (Vamp2), do not qualify as exclusive neuronal markers. They are also expressed in cell types other than neurons such as endocrine (for reviews, see Pang and Südhof 2010; Lang and Jahn 2008) and sensory receptor cells (for reviews, see Zanazzi and Matthews 2009; Ramakrishnan et al. 2012). In addition to neuronal, sensory receptor, and endocrine cells, the in cell lineage-terms distant, mast cells also employ the SNARE complex for mediator release (Lorentz et al. 2012). The mast cell SNARE complex, however, operates with different protein isoforms such as Snap-23. Moreover, synaptotagmin I is not detectable. Thus, questions arise whether certain synaptic proteins define a group of related cell types involved in signal detecting and propagating functions, and how their expression is coordinated with other key features of the respective cell type. In diverse central and peripheral neuron populations of efferent, afferent and interneuron phenotypes, mRNA accumulation for selected synaptic proteins such as synaptotagmin I occurs in a characteristic pattern following expression of a set of genes coding for neuronal cytoskeletal proteins (see Ernsberger 2012 for review). This pattern is conserved between mammalian and avian neurons provoking the question whether it may be a constitutive part of a generic neuronal differentiation program. Importantly, enhancers linked to genes involved in axon guidance and synaptic transmission display a sequential recruitment of activity marks during mouse forebrain development (Nord et al. 2013). This is strong evidence that the mRNA accumulation patterns reflect a successive activation of different classes of genes expressed in neurons. Divergence in expression patterns and regulatory mechanisms between neurons and endocrine cells more precisely define essential features of the generic neuronal differentiation program. Sympathetic neurons and adrenal chromaffin endocrine cells share the expression of many synaptic proteins (Hou and Dahlström 1996; Stubbusch et al. 2013). Yet the relative amounts of transcripts and proteins as well as the developmental program driving embryonic and postnatal synaptic protein gene expression differ considerably between the two cell types. One key feature is the rapid embryonic accumulation of synaptotagmin I transcripts in mouse and chick sympathetic neurons as compared to the slow, predominantly postnatal expression onset in endocrine chromaffin cells (Patzke and Ernsberger 2000; Ernsberger et al. 2005; Stubbusch et al. 2013). While the involvement of transcription factors in the regulation of synaptic protein gene expression during neuronal and endocrine cell differentiation is largely unresolved, Dicer 1-dependent processes contribute to the differences in expression patterns between the two cell populations. Another crucial difference between the two cell types is the prominent expression of synaptotagmin VII in adrenal chromaffin cells which correlates with its superior role in

exocytosis as compared to synaptotagmin I (Schonn et al. 2008; Voets et al. 2001). Similar to adrenal chromaffin cells, analysis in null mutant mice shows that synaptotagmin VII is instrumental in insulin and glucagon secretion from pancreatic endocrine cells (Gustavsson et al. 2008, 2009). In beta-cells, it colocalizes with insulin-containing granules (Gauthier and Wollheim 2008). Synaptotagmin VII, II and IV can be detected in whole islets, while synaptotagmin I immunoreactivity is observed only in δ-cells despite expression in beta-cell lines (Brown et al. 2000; Gauthier and Wollheim 2008 for review). Whether the lack of detectable synaptotagmin I transcript signals (Jacobsson et al. 1994) reflects low expression levels, as suggested by the comparatively low message levels in pituitary and adrenal medulla, remains unclear. Similar to neurons and adrenal chromaffin cells, pancreatic islets express the SNARE complex subunits Snap-25, Vamp-2 and Syntaxin 1A (Jacobsson et al. 1994). Likewise, endocrine cells in the anterior and intermediate lobes of the pituitary demonstrate abundant transcripts for many critical regulators of vesicle cycle and fusion also found in neurons such as Vamp-2, Snap-25, Syntaxin 1A or Munc18 (Jacobsson and Meister 1996). Synaptotagmin I is detected by in situ hybridization (ISH) (Marquèze et al. 1995; Jacobsson and Meister 1996) and by immunohistochemistry (IHC) (Redecker et al. 1995; Jacobsson and Meister 1996), albeit at variable levels in different regions and cell populations. Quantification by reverse transcription/polymerase chain reaction (qRT-PCR) shows roughly 10-fold lower levels in pituitary than in the hypothalamus (Xi et al. 1999). Considerable similarity is observed in the protein equipment of neuronal synapses and ribbon synapses found in receptor cells such as retinal photoreceptors and inner ear hair cells. Among 19 synaptic proteins analyzed in mammalian retina by IHC, all but 2 are found in the outer plexiform layer of the rodent and bovine specimen (von Kriegstein et al. 1999). These include synaptotagmin I, Vamp-2, Snap-25 or Rab3a. These proteins are also found in hair cell ribbon synapses in the inner ear where synaptotagmin I is developmentally downregulated in mice (for reviews, see Zanazzi and Matthews 2009; Ramakrishnan et al. 2012). Different from neurons, complexin 1 and 2 are not detected in photoreceptors and hair cells while the protein ribeye appears unique to ribbon synapses. Thus, synaptic protein isoform expression, developmental mRNA accumulation profiles and coexpression with other cell type-specific proteins distinguish neurons among these functionally divergent classes of cells. Yet, Snap-25, Vamp-2 and Syntaxin expression patterns appear similar between neurons and endocrine cells of ectodermal as well as endodermal origin, in addition to sensory receptor cells. Taken together, several main issues emerge. On the one hand, the conserved sequence during neuronal differentiation

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in the expression of different genes belonging to distinct functional ontology categories draws attention to the possibility of a regulatory mechanism shared across neuron populations. On the other hand, expression of these genes in related cell types, albeit with different developmental profiles, prompts concern which aspects of the regulatory path to these cell types are shared and which are cell type-specific.

Vertebrate SoxB1 proteins: from pluripotency to neural lineage restriction The extraordinary interest in SoxB1 proteins and in particular the pluripotency factor Sox2 [SRY (sex determining region Y)-box 2] rests on a multitude of functional implications concerning basic and applied biomedical problems: it promotes neuroectodermal lineage selection in ES cells (Zhao et al. 2004; Thomson et al. 2011), plays a major role in reprogramming of non-neural cells to neural stem or precursor cells (Maucksch et al. 2013; Amamoto and Arlotta 2014), is critically involved in vertebrate neurogenesis and glial cell development (Reiprich and Wegner, this issue; Hoffmann et al. 2014), and orthologous proteins are found in the neurogenic regions of all animal phyla analyzed (see below). SOX2 haploinsufficiency in humans is associated with brain malformations as well as defects in multiple sensory systems including anophthalmia and hearing loss (Fantes et al. 2003; Hagstrom et al. 2005; Zenteno et al. 2005). A conserved role of SoxB1 proteins in neural development was already suggested more than a decade ago (Sasai 2001). Expression of SoxB1 genes in early vertebrae embryogenesis The SoxB1 genes, Sox1, Sox2 and Sox3, are expressed throughout the nervous system with strongest signals for Sox2 and Sox3 transcripts in the young embryos of all vertebrate classes analyzed. In the chick embryo, cSox3 is detected throughout the epiblast before neural induction, to then become restricted in the ectoderm to the nervous system (Uwanogho et al. 1995; Rex et al. 1997a, b; Uchikawa et al. 2003). cSox2 mRNA becomes apparent at the time of presumed neural induction and is restricted to the region that forms the neural plate. Thus, Sox3 expression has been referred to as ‘preneural’ and Sox2 as ‘pan-neural’ (Uchikawa et al. 2011). In mouse, however, Sox2 is expressed prior to the implantation stage while Sox3 follows after implantation (Avilion et al. 2003). Strong Sox2 and Sox3 signals cover the epiblast (Guo et al. 2010; Iwafuchi-Doi et al. 2011), to then become predominant in the prospective anterior neural plate (Wood and Episkopou 1999). Posterior neural plate formation can be seen by posterior extension of Sox2 expression. In zebrafish, six soxB1 genes have been identified and show similarities, but also differences, in embryonic

expression pattern (Okuda et al. 2006, 2010). In addition to sox1a/b, sox2 and sox3, sox19a and sox19b are present, and are also present in the Fugu rubripes genome (Koopman et al. 2004). sox19b is maternally supplied, sox3 and sox19a activated around the 1000-cell stage, and sox2 around the 30 % epiboly stage (Okuda et al. 2010). At the shield stage in zebrafish sox2, sox3 and sox19a are uniformly expressed in the future ectoderm (Okuda et al. 2006). At the 75–80 % epiboly stage, they are localized to a region of the future neuroectoderm. While chicken Sox2 is expressed throughout the neural plate at this stage (Hamburger-Hamilton stage 5, HH st5), zebrafish sox2 is strong in the presumptive forebrain but weak in the presumptive spinal cord. sox3 is expressed in presumptive forebrain, hindbrain and spinal cord while sox19a covers the entire presumptive neuroectoderm, thus resembling chicken Sox2. Neither zebrafish sox1a/b nor chicken Sox1 are detectably expressed at these stages. At early somite stages, zebrafish sox2 and sox3 show regionalized expression while chicken Sox2 and Sox3 are expressed throughout the entire CNS. Thus, SoxB1 gene products in vertebrates share expression associated with the neuroectoderm and the developing nervous system. Developmental time course and anterior–posterior distribution may, however, vary for individual group members among vertebrate classes. In addition, teleost fishes possess sox19 genes not known in birds and mammals. Sox2 ablation in mouse brain and retina While mice lacking either Sox1 or Sox3 show limited defects and are viable, Sox2 mutant embryos die around the preimplantation stage (Avilion et al. 2003; Masui et al. 2007). Strongly hypomorphic mutant mice carrying ß-galactosidase replacing one Sox2 allele, while having reduced expression from the second Sox2 allele due to an enhancer deletion, display cerebral malformation and neuronal degeneration (Ferri et al. 2004). Cultured neural stem cells from hypomorphic Sox2 mouse mutants carrying the enhancer deletion show compromised neuronal but normal astroglial differentiation in vitro (Cavallaro et al. 2008). ß-tubulin expression can be detected, albeit at reduced levels, but NeuN/Rbfox3, Map2 and neural cell adhesion molecule (NCAM) signals as well as neuritic arborzation are rarely observed. Sox2 protein staining as well as ß-galactosidase immunohistochemistry show expression in the early neural precursors of the ventricular zone, in dividing precursors of neurogenic regions, and a small proportion of differentiated neurons (Ferri et al. 2004). Robust expression of Sox2 is detected by IHC in neural stem and progenitor cells of the neocortical ventricular zone in mice and persists until withdrawal from the cell cycle (Bani-Yaghoub et al. 2006). At E13.5, more than 90 % of BrdU incorporating cells are Sox2 immunoreactive. After birth, Sox2 protein levels are massively downregulated to

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negligible levels in the adult neocortex. NCAM-positive neuronal precursors are detected in a transitory location adjacent to the ventricular zone where Sox2 and NCAM are present in the same cell. Selection of NCAM-positive and A2B5-negative neuronal precursors by immunopanning shows distinct Sox2 expression albeit at reduced levels when compared to cells in the VZ (Bani-Yaghoub et al. 2006). The cells lose Sox2 and acquire ß-III tubulin within 3 days and Map2 within 7 days in vitro. Retrovirus-mediated overexpression of Sox2 in the cultured cells induces Notch1 and Hes5 expression and inhibits neurogenesis but not gliogenesis. In mice in which Sox2 is deleted under the control of the nestin promoter, brain abnormalities are limited (Miyagi et al. 2008; Favaro et al. 2009). Immunohistological analysis of nestin expression in the spinal cord of the mouse embryo at E10.5 demonstrates that virtually all cells are positive and mitotically active (Tanaka et al. 2004). The nestin-positive cells largely coexpress Sox2 and Pou3f2/Brn2 (POU domain, class 3, transcription factor 2), both localized to the nucleus. Sox1, Sox3 and the SoxC protein Sox11 show a similar distribution pattern. By E13.5, Sox2 and the other SoxB1 proteins are confined to the ventricular zone of the spinal cord. Sox11 is strong in the subventricular zone, extending into the intermediate zone at moderate levels. Thus, the restricted effects of the conditional Sox2 ablation may be due to compensation by other Sox proteins. In contrast to CNS ventricular zone precursors that also express Sox1 and Sox3, mouse retinal neural progenitors exclusively express Sox2 (Taranova et al. 2006; compare Collignon et al. 1996). Coincident with retinal neuronal differentiation, Sox2 is downregulated but is maintained in Müller glia. Consequently, its expression is mutually exclusive with ß-III tubulin and neurofilament. Conditional ablation of Sox2 in mouse retina by a Pax6 (paired box 6) regulatory element, that targets CRE expression to distal retinal progenitor cells, results in a marked reduction of BrdU incorporation and the number of cycling cells in this region. Notch1 and Hes5 [hairy and enhancer of split 5 (Drosophila)] are not expressed, Math5/Atoh7 [atonal homolog 7 (Drosophila)], NeuroD/NeuroD1 (neurogenic differentiation 1) and ß-III tubulin are absent. Thus, conditional ablation of Sox2 in a neural domain in the mouse embryo not expressing other SoxB1 genes abrogates neuronal development in a particular neural domain if it does not express other SoxB1 genes. Sox2 overexpression in chick neural tube and inner ear In the spinal cord of the chick embryo, the SoxB1 proteins Sox1–3 are expressed in an extensively overlapping fashion in all mitotically active cells that incorporate BrdU (Bylund et al. 20 03 ; G rah am e t al. 20 03 ) . W h e r e a s al l n e s t i n

immunoreactive and a high proportion of Ngn2/Neurog2 (neurogenin 2)-positive cells coexpress Sox1-3, expression is low in NeuroM/Math3-positive cells and absent in neurons expressing the differentiation markers NeuN/Rbfox3, ß-III tubulin, as shown by Tuj1 immunoreactivity, or p27kip1 (cyclin-dependent kinase inhibitor 1B). Electroporationmediated overexpression of Sox1, Sox2 or Sox3 correlates with the absence of NeuN/Rbfox3, Tuj1 or p27kip1 signals and strongly reduces NeuroM/Math3. Ngn2/Neurog2 expression and cell proliferation are slightly reduced, Proliferating cell nuclear antigen (PCNA) and Sox1 are retained. In contrast, overexpression of the proneural genes Ngn2/Neurog2 and Cash1/Ascl1 [chicken achaete-scute complex homolog 1 (Drosophila)] results in a rapid decrease of cell-cycle regulators cyclin D1, D2 and A, downregulation of Sox1-3, and commitment to terminal differentiation. Sox3 is able to block the NeuN/Rbfox3 or Tuj1 induction by Ngn2/Neurog2 (Bylund et al. 2003). In the inner ear of the chick embryo, Sox2 is markedly expressed and Ngn1/Neurog1 (neurogenin 1) and Neurod1/Neurod are detected in a subpopulation of cells in the neurosensory competence domain. Sox2 levels are much reduced in the cochleovestibular ganglion where Neurod1/Neurod but not Ngn1/Neurog1 is highly expressed. Overexpression of Sox2 in the chick inner ear induces the expression of Ngn1/Neurog1 but not N e u ro d 1 / N e u ro d a n d r e d u c e s t h e s i z e o f t h e cochleovestibular ganglion (Evsen et al. 2013). In contrast, Ngn1/Neurog1 overexpression induces Neurod1positive cells and downregulates Sox2. The repressive domain of the Nop-1 regulatory element in the Sox2 gene may mediate a direct repression by Ngn1/Neurog1 or Neurod1/Neurod. These studies in chick neural structures show that Sox2 overexpression blocks the acquisition of terminal markers of neuronal differentiation, variably affects the expression of the proneural genes Ngn1/Neurog1 and Ngn2/Neurog2, and modestly reduces progenitor proliferation. On the other hand, overexpression of proneural bHLH transcription factors downregulates SoxB1 proteins.

Sox2 in the mouse peripheral nervous system The neural crest is the source of neurons and glia in the enteric nervous system, sympathetic ganglia and dorsal root ganglia (DRG). Sox2 expression is detected at E9.5 throughout the mouse neural tube but is absent from the migrating neural crest (Wood and Episkopou 1999; Heanue and Pachnis 2011). Instead, Sox9 is transiently expressed in premigratory neural crest (Cheung and Briscoe 2003; Scott et al. 2010) and Sox10 expression marks migrating neural crest cells (Kuhlbrodt et al. 1998; Southard-Smith et al. 1998).

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Upon arrival at its destination, Sox2 expression may become upregulated again, as shown for the enteric nervous system (Heanue and Pachnis 2011) and the dorsal root ganglia (Cimadamore et al. 2011). All enteric Sox10-positive cells express Sox2 as shown at E11.5 through P1, but a small fraction of Sox2-positive cells lack Sox10 signal (Heanue and Pachnis 2011). TuJ1 signals are not observed in Sox2 and Sox10-positive cells but untypical punctate TuJ1 signal is found in Sox2-positive and Sox10-negative cells in acute enteric cell culture. In E11 DRG, however, Sox2 and Sox10 co-label with TuJ1 (Cimadamore et al. 2011). Mutational inactivation of Sox2 in neural crest derivatives results in a reduction of DRG neuron number. As Sox2 is shown to bind to Ngn1/ Neurog1 as well as Mash1/Ascl1 promoters in embryonic stem cell-derived neural crest-like cells, SoxB1>bHLH signaling may be involved.

Manipulation of SoxB1 levels in zebrafish Ectopic expression of exogenous sox3 in zebrafish induces neural tissue as well as, occasionally, a partial second axis and, in addition, may result in the loss of one or both eyes (Dee et al. 2008). sox2 and sox19b/31 domains are expanded. Blocking endogenous sox3 transcripts by morpholinos results in abnormal development of neuroectoderm and reduction in size of CNS structures. Approximately 40 % of cells are lost in brain and spinal cord. Expression of ngn1/neurog1, elavl3 (ELAVlike neuron-specific RNA binding protein 3), elavl4, and neurofilament M is substantially reduced. Loss of ngn1 is observed in all placodes, and otic vesicle development is severely disturbed. Morpholino-mediated knockdown in zebrafish of all four SoxB1 transcription factors, sox2/3/ 19a/19b, affects different genes expressed in the neuronal lineage differently (Okuda et al. 2010). While her3 (hairy-related 3) and neurog1/ngn1 expression is lost, ascl1a/zash1a (achaete-scute family bHLH transcription factor 1a) and alpha 1 tubulin (tuba1) are at least transiently upregulated. These studies in zebrafish point to the importance of sox3 for neuronal development and, together with the different expression onset during zebrafish and mouse embryogenesis, may indicate a changing relative importance of Sox2 and Sox3 in neural development of amniotes and fish. Taken together, these studies point to a crucial role of vertebrate Sox B1 proteins and in particular Sox2, not only for neural specification but also neuronal differentiation (see also Reiprich et al., this issue; Pevny and Nicolis 2010 for review). Yet expression is not restricted to neural tissue. For example, Sox2 is also detected in the anterior foregut endoderm of mice (Que et al. 2007),

chick (Ishii et al. 1998), and zebrafish (Muncan et al. 2007), as discussed later.

SoxB1 and SoxC proteins bind to the genomic loci of proneural as well as terminal neuronal differentiation genes Sox protein binding to genomic sites during development at different developmental stages has been reported from mouse embryonic stem (ES) cells, ES cell-derived neural progenitor cells (NPCs) and neurons, employing chromatin immunoprecipitation (ChIP) with antibodies against Sox2, Sox3 or Sox11 (Bergsland et al. 2011). In ES cells, Sox2 binds at neural lineage-specific genes that will be bound and activated by Sox3 in NPCs and later occupied and activated by Sox11. This progression correlates with alterations in the histone modification at these genes associated with changes in gene expression. Binding of all three Sox proteins is observed at ∼46-kb distance to the Ascl1/Mash1 gene, while binding near the Neurog1/Ngn1 gene is reported in this study for Sox3 and Sox11 but not Sox2, and near the Neurog2/Ngn2 gene for Sox2 and Sox3 but not Sox11. In human ES cell-derived neural crest cells, ChIP with antibodies against Sox2 indicates binding at ∼2-kb 3’ of the NGN1/NEUROG1 and ASCL1/ MASH1 promoters (Cimadamore et al. 2011). In addition, binding near terminal differentiation genes such as those coding for the synaptic proteins Snap-25 and neurexin I (Nrxn1) has been detected for all three Sox proteins (Bergsland et al. 2011), but the function of these binding sites is not characterized. At the ß-III tubulin gene, Sox3-bound regions in mouse NPCs are preferentially associated with the H3K27me3 mark characteristic for transcriptional repression, while Sox11-bound regions in neurons are preferentially associated with the H3K4me3 mark characteristic for transcriptional activation (Bergsland et al. 2011). Sox2 binds primarily to unique distal enhancer elements in mouse ES cells and ES-derived NPC (Lodato et al. 2013). Only a small set of chromatin and transcriptional regulators is bound by Sox2 in both ES cells and NPCs, while a larger set is occupied selectively in NPCs. Since the same consensus DNA motif is targeted in both cell types, different additional factors mediate cell-type specificity. Accordingly, many genomic sites are co-occupied by Sox2 and Oct4/POU5F1 (POU class 5 homeobox 1) in ES cells, but Sox2 binds many distal enhancers together with Brn2 in NPCs. This is the case for a known regulatory region 3’ of the Ascl1/Mash1 locus which also carries H3K4me1 and H3K27Ac marks in NPCs indicative of gene activity. Forced expression of Brn2 in ES cells leads to recruitment of Sox2 to a subset of NPC-specific targets.

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Upon in silico analysis of highly conserved non-coding regions (HCNR) from vertebrate genomes, enrichment of Sox, POU and homeodomain transcription factor binding sites, and linkage to genes expressed in the developing nervous system, are frequently found (Bailey et al. 2006). Within the nestin neural enhancer, a 30-bp sequence conserved from rodents to humans with adjacent and essential Sox and POU factor binding sites shows nervous system-specific and Sox and POU-dependent activity as analyzed after electroporation in chick embryos and transfection of a neural stem cell line (Tanaka et al. 2004; Jin et al. 2009). Upon analysis in liver cells, in combination with Brn2, all SoxB1 proteins (Sox1, Sox2, Sox3), but not SoxB2 proteins (Sox14, Sox21) nor the SoxE protein (Sox9), activate reporter expression. In combination with Sox2, class III POU proteins (Brn1, 2, 4 and Oct6) and class V (Oct3/4), but not class II (Oct2), are strongly activated. Brn2 is widely expressed in the spinal cord at this stage with high levels in the ventricular and subventricular zone, mostly in the nuclei, compared to moderate levels in the intermediate zone largely localized to the cytoplasm (Tanaka et al. 2004). Likewise, Brn2 and also Brn1 are detectable in the mouse neural tube (Jin et al. 2009) beginning at E9.5 when Oct4 expression is downregulated. EMSA with a probe to the Sox/POU binding site of the nestin neural enhancer in combination with Brn2- and Brn1-specific antibodies indicate binding at this developmental stage. Although still at an early stage, this kind of analysis provides several important insights. First, SoxB1 proteins bind to proneural but also to terminal neuronal differentiation genes. Second, the sites and co-bound factors may change with development. And third, chromatin modification at these binding sites changes with sequential SoxB1 and SoxC binding in correlation with gene expression.

SoxB proteins are associated with neurogenic regions throughout the animal kingdom The trend-setting observations originally made in Xenopus (Mizuseki et al. 1998a, b; Kishi et al. 2000) that Sox proteins from different groups, in particular the SoxB1 group, initiate neural induction and consolidation of neural cell identity complemented the finding that a closely related gene is essential for CNS development in Drosophila (Nambu and Nambu 1996). Drosophila SoxB proteins are critical for nervous system development and share target genes with mouse SoxB1 and SoxC proteins All the dipteran and the hymenoptera species Drosophila melanogaster, Drosophila pseudoobscura, Anopheles gambiae, and Apis mellifera possess each of the four genes

encoding SoxB proteins, SoxNeuro, Dichaete, Sox21a and Sox21b, the latter three of which are organized in a linked genomic cluster (McKimmie et al. 2005). In the honeybee Apis mellifera embryo, the Dichaete homolog AmSoxB1 is expressed along the ventral gastrulation folds and after gastrulation in neuroblasts arising from neuroectoderm (Wilson and Dearden 2008). Expression is also seen in the embryonic brain cephalic lobes. AmSox21b expression is detected in the late embryo in the CNS. In D. melanogaster, the SoxB genes Dichaete and SoxNeuro are expressed early in neuroectoderm in a largely non-overlapping manner (Zhao and Skeath 2002; Crémazy et al. 2000). Dichaete is initially expressed in seven transverse pair-rule stripes in early embryos and regulates segmentation (Nambu and Nambu 1996). During gastrulation, it is activated in the ventral half of the neuroectoderm. Within the neuroectoderm, expression begins during stage 7 and is detected uniformly in the ventral region (Zhao and Skeath 2002). It is restricted to medial and intermediate columns through late stage 12 and then expands to include the entire neuroectoderm. Newly formed medial and intermediate neuroblasts express weak levels of Dichaete which is not detectable in older neuroblasts. Lateral neuroblasts activate Dichaete at specific time points in their lineages. SoxNeuro expression in D. melanogaster embryonic development is likewise associated with development of the CNS (Crémazy et al. 2000; Buescher et al. 2002). A head-specific stripe is observed in early syncitial blastoderm, then expression becomes detectable laterally in the trunk and, in late blastoderm, two lateral stripes in the presumptive ventrolateral neuroectoderm are observed. During gastrulation, expression is found in cephalic and ventral neurogenic regions and extends more laterally than Dichaete signals. Forming neuroblasts initiate Dichaete and SoxNeuro coexpression or only one or the other. In late embryogenesis, most SoxNeuro staining is found in ventral and lateral epidermis, chordotonal organs and brain. It colocalizes with Dichaete protein in chordotonal organs, while expression in the brain is mostly in distinct areas. In Dichaete mutants, achaete is derepressed in intermediate column neuroblasts while expression in the medial column appears normal (Zhao and Skeath 2002; Buescher et al. 2002). The development of late-forming neuroblasts in the medial and intermediate columns is disturbed. In the lateral neuroectoderm of D. melanogaster, in which SoxNeuro is uniquely expressed, achaete expression is lost or reduced in SoxNeuro mutants (Overton et al. 2002; Buescher et al. 2002). Many correctly specified neuroblasts therein are lost. SoxNeuro and Dichaete double mutants are characterized by a severe neural hypoplasia throughout the CNS but not a complete loss of neuronal cells as shown by residual staining for axonal and neuroblast markers. achaete expression is

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dramatically reduced in proneural clusters and medially derived neuroblasts. Substantial Dichaete binding is found by genome-wide binding analysis with the DNA adenine methyltransferase identification (DamID) method across the achaete–scute complex and all four genes in the complex are downregulated in Dichaete mutants (Aleksic et al. 2013). Binding is also detected at the prospero (pros) gene which is upegulated in mutants. In addition, other targets including components of the Notch, Wnt and EGFR signaling pathways have been identified (Shen et al. 2013). Using two complementary approaches, the DamID method and chromatin immunoprecipitation (ChIP), combined with genome-wide tiling arrays, SoxN binding across the Drosophila genome was identified (Ferrero et al. 2014). The list of shared targets is enriched for transcription factors and effectors with roles in CNS development. More than 40 % of Sox2-bound genes in mouse are also bound by SoxN in Drosophila which, however, accounts for only approximately 13.5 % of SoxN-bound genes. A much larger overlap is observed with mouse Sox11. Over a third of the SoxN bound genes (34 %, 1,092 genes) in Drosophila have mouse orthologues bound by Sox11 in neural precursors or differentiating neural cells.

Caenorhabditis Sox proteins are involved in neuronal transdifferentation and specification In Caenorhabditis elegans, sox-2 and sox-3 are expressed in the developing nervous system and intestine, and sox-3 also in the pharynx (www.wormbase.org). sox-2 mutants are not reported to show neuronal cell loss or defects and sox-2/sox-3 double mutant data are not available. Yet, sox2 is involved in cell type transdifferentiation towards a neuronal fate in vivo. The C. elegans Y cell, located in the rectum and displaying typical epithelial ultrastructure as well as markers for apical-basolateral polarity, but devoid of neural characteristics, differentiates into the PDA neuron devoid of residual epithelial features but with typical axonal process as well as pan-neuronal markers (Jarriault et al. 2008). This process does not require cell division and progresses through a dedifferentiated state. An RNAi screen targeting early steps of Y-to-PDA transdifferentiation, as shown by a persistent Y-cell phenotype, identified egl-27, sem-4/SALL, ceh-6/ OCT and sox-2, but not sox-3a or egl-13/soxD as important (Kagias et al. 2012). Since sox-2 mutants are not reported to show neuronal cell loss or defects, this affects initiation of transdifferentiation rather than neural fate promotion. egl-13/soxD is required for establishment of the correct neuronal fate in the Q neuroblast lineage (Gramstrup Petersen et al. 2013; Feng et al. 2013).

Sea urchin SoxB protein expression is associated with ectodermal and endodermal neuron production The neuroectoderm in the sea urchin embryo comprises the anterior neuroectoderm (ANE) and the ciliary body neuroectoderm with the ANE consisting of a central disc called animal plate and a surrounding cell torus (see Angerer et al. 2011 for review). Neurons in the larval nervous system appear as neuroblasts in these domains (see Burke et al. 2006 for review). Genes expressed in but not confined to the animal plate ectoderm in Strongylocentrotus purpuratus embryogenesis include SpSoxB1 and SpSoxB2. Genes expressed exclusively in the neurogenic ectoderm at the animal pole include achaete–scute (Sp-Ac-Sc) and homeobrain (Sp-Hbn), neurogenin and NeuroD. Surprisingly, some pharyngeal neurons appear to develop from the endoderm in a region where SpSoxB1 is also expressed (Wei et al. 2011). Cnidaria SoxB and SoxC protein expression resembles neural patterning In the cnidarian Nematostella vectensis, 14 Sox genes have been identified (Magie et al. 2005). NvSoxB1 is expressed at the gastrula stage broadly in the aboral half of the embryo and in a domain surrounding the blastopore. Later, aboral expression becomes restricted to the apical tuft and oral expression to the pharyngeal ectoderm. NvSoxB2 and NvSox2 are expressed in a salt-and-pepper pattern in the early embryo. Further analysis is required to investigate whether the pattern resembling the neural net in cnidarians represents neuronal expression. In the coral Acropora millepora, AmSoxB1 and AmSoxBb, the likely orthologues of NvSoxB1 and NvSox3, are assigned to the SoxB clade (Shinzato et al. 2008). They are uniformly distributed in the unfertilized egg and early cleaving embryos. With gastrulation, transcripts become depleted in the presumptive endoderm. AmSoxC is initially expressed in scattered cells and then, during gastrulation, more uniformly in a subset of cells in the presumptive ectoderm. The morphology of some of the cells at later stages is consistent with that of type 1 sensory neurons. Nematostella NvSoxC has a similar distribution but appears later. Sponges and ctenophores: two special cases Expression of SoxB and SoxC proteins in various groups of invertebrates is related to neural and neuronal development from ectoderm and in sea urchins also from endoderm. Yet, in sponges that do not have neurons, SoxB and SoxC genes have also been described (Larroux et al. 2006, 2008). In addition, basic helix-loop-helix (bHLH) proteins have been detected in sponges (Simionato et al. 2007) with 16 bHLH protein genes in the genome of the demosponge Amphimedon

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queenslandica. AmqbHLH1 shows proneural activity in Xenopus and Drosophila (Richards et al. 2008). In contrast, ctenophores that develop nerve cells appear devoid of genes encoding key proneural bHLH proteins (Moroz et al. 2014) but possess Sox proteins (Jager et al. 2006). SoxB and SoxE protein-coding genes are found among others in the ctenophore Pleurobrachia pileus in which they are detected in adult neurosensory structures (Jager et al. 2008). Genes coding for SoxB (MleSox1), SoxC (MleSox2) and SoxE (MleSox3, MleSox4) proteins are characterized in the ctenophore Mnemiopsis leidyi (Schnitzler et al. 2014). Expression of MleSox1 is related to developing comb plate and comb rows as well as the apical sensory organ. MleSox2 expression becomes restricted to the pharynx, tentacle bulbs, and the apical sense organ. MleSox3 and MleSox4 transcripts are also found in these structures. Taken together, SoxB gene expression is related to neurosensory development throughout the animal kingdom including the highly debated ctenophores. Moreover, SoxB and SoxE gene expression points to their possible involvement in the generation of neurosensory tissues (see below). In addition to neurogenic ectoderm, SoxB protein expression in vertebrates and invertebrates is associated with non-neural endoderm derivatives.

The hierachical action of Sox proteins in neuronal differentiation Multiple classes of Sox proteins are involved in neurogenesis in subsequent stages on the path from neuroectodermal stem cell to the differentiated neuron (Reiprich and Wegner, this issue; Wegner and Stolt 2005; Wegner 2011). This cascade of Sox protein action is, so far, largely documented for vertebrates. Sox21: a SoxB protein with repressor function expressed already in embryonic progenitors and required in adult neurogenesis Sox21 appears involved in intermediate stages of neuronal differentiation before the onset of expression of markers for terminal neuronal differentiation. In neural stem cells, Sox21 is strongly upregulated by Sox2 (Kuzmichev et al. 2012). It attracts particular interest for its proliferation inhibiting activity in neural progenitors (see below) and induction of neuronal differentiation in brain tumor cells (Ferletta et al. 2011; Caglayan et al. 2013; for review, see Swartling et al., this issue). In both chick and mouse embryos, Sox21 expression initiates in the anterior neural plate and then extends to the posterior neural plate, but lags behind that of Sox2

(Uchikawa et al. 2011). Sox21 expression is positionally restricted within the CNS as documented in chick (Rex et al. 1997b; Uchikawa et al. 1999). Longitudinal stripes are found for transcript and protein in the spinal cord and a more complex pattern is seen in the brain. In Xenopus, sox21 is found throughout the developing central nervous system, including the olfactory placodes, with strongest expression at the boundary between the midbrain and hindbrain (Cunningham et al. 2008). Zebrafish sox21, sox11A and sox11B transcripts are accumulated in the egg, are present in all cells until gastrulation, and become restricted later to the developing CNS (Rimini et al. 1999). sox21b is predominantly expressed in the telencephalon, hypothalamus, and mesencephalon, sox21a is solely expressed in the midbrain-hindbrain boundary, olfactory placode and lateral line, and both genes are expressed in the hindbrain, spinal cord and ear (Lan et al. 2011). Ectopic expression of sox21a leads to dorsalization of the embryos, and a subset of the dorsalized embryos show an ectopic neural tube (Argenton et al. 2004). Sox21 IR is confined to Sox1-3-positive cells in the spinal cord of chick embryos and detected in all BrdU-incorporating cells (Sandberg et al. 2005). Most Ngn2/Neurog2-positive cells express Sox21, while NeuroM/NeuroD4 immunoreactivity is found only in the most lateral Sox21-positive cells. Additional Ngn2/Neurog2 and NeuroM/NeuroD4immunoreactive cells are observed lateral to the Sox21positive cells in the intermediate zone. NeuN/Rbfox3, NF1 and ß-III tubulin as shown by TuJ1 staining are generally not coexpressed with Sox21. Electroporation-mediated misexpression of Sox21 in the spinal cord of chick causes cell cycle exit as detected by lack of BrdU incorporation (Sandberg et al. 2005). Sox3 gets downregulated, and NeuN/Rbfox3 but not ß-III tubulin and NF1 is prematurely detectable. Ngn2/Neurog2 is not upregulated. Electroporation-mediated misexpression of Ngn2/ Neurog2 or Ascl1/Cash1, however, induces high levels of Sox21 before NF1 and ß-III tubulin upregulation. siRNAmediated Sox21 knockdown reduces the number of NeuroM/NeuroD4-positive but not Ngn2/Neurog2-positive cells. The number of Ngn2/Neurog2-positive cells incorporating BrdU is increased. Since a fusion protein of the HMG domain with the EnR repressor domain, in contrast to the VP16 activator domain, mimics the overexpression effects of Sox21 full-length protein, Sox21 is considered to act as a transcriptional repressor (Sandberg et al. 2005). In embryonic mouse brain, Sox21 is exclusively expressed in the ventricular zone of the E15.5 cerebral cortex, populated by BrdU-incorporating and Ki67-positive neural stem and progenitor cells but not β-III tubulin-positive neurons (Matsuda et al. 2012). Similar Sox21 expression patterns were also observed in the progenitor population in the E14.5 spinal cord and P7 cerebellum. In Sox21 mutant mice, adult but not

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embryonic neurogenesis is impaired. In adult mouse hippocampus, loss of Sox21 reduces production of new neurons by repression of Hes5 gene expression. Sox 4 and Sox11: SoxC proteins with activator function required for embryonic and adult neurogenesis Sox11 and Sox4 appear to be involved in terminal neuronal differentiation from embryonic as well as adult neural progenitors. Sox11 mutant mice develop small and disorganized brains, accompanied by proliferation deficits in NPCs (Wang et al. 2013a). In the adult hippocampus, cells deficient for Sox4 and Sox11 fail to initiate or maintain a full neuronal differentiation program (Mu et al. 2012). Sox11 is expressed in the neural epithelium of the chick embryo and becomes transiently upregulated in maturing neurons after they leave the neural epithelium (Uwanogho et al. 1995). In addition, strong mesodermal expression is found. In zebrafish, sox11a and sox11b are also expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage onward (de Martino et al. 2000). Regions that display both transcripts include the ventral portion of the presumptive telencephalon and diencephalon as well as the cerebellum. The midbrain– hindbrain border expresses neither. Thus, expression is positionally restricted and in part opposite to sox21 (Rimini et al. 1999). In the spinal cord of chick embryos, Sox11 is first detected coincident with the appearance of Tuj1 signal for ß-III tubulin (Bergsland et al. 2006). It is expressed medial to and within the domain of Tuj1-positive neurons. In the ventricular zone, Sox11 is restricted to Sox3-positive cells and most of these Sox11-positive cells coexpress Ngn2/Neurog2. In the intermediate zone, Sox11-positive cells express NeuroM/ NeuroD4. Sox11 is also found in more differentiated neurofilament NF1-positive cells. Sox4 largely overlaps with Sox11 but becomes progressively weaker in differentiated neurons in lateral aspects of the marginal zone. Electroporation-mediated misexpression of Sox11 or Sox4 in the spinal cord of chick embryos induces ectopic expression of ß-III tubulin and Map2 immunoreactivities after 24 h and NF1 or SCG10 after 48 h (Bergsland et al. 2006). No alterations are detected after 24 h in the expression of Ngn2/ Neurog2 or NeuroM/NeuroD4 but many transfected cells express Sox3. The frequency of cells in a self-renewing state is comparable to control as shown by BrdU incorporation and PCNA staining. However, a high incidence of Tuj1/Sox3 as well as Tuj1/BrdU double-positive cells indicates a precocious upregulation of a generic neuronal differentiation marker. Conversely, siRNA-mediated Sox11 knockdown reduces the number of Tuj1, NF1 or SCG10-positive cells but also of NeuroM/NeuroD4 and p27 Kip1 . Ngn2/Neurog2 and Sox3 expression as well as BrdU incorporation appear

unaffected. While 27 % of NeuroM/NeuroD4-positive cells coexpress Sox3 and 70 % coexpress NF1 in control, in Sox11 siRNA-treated spinal cord, most NeuroM/Math3-positive cells coexpress Sox3 and only a few NF1. Since a fusion protein of the HMG domain with the VP16 activator domain, in contrast to the EnR repressor domain, mimics the overexpression effects of Sox11 full-length protein, Sox11 is considered to act as transcriptional activator (Bergsland et al. 2006). As the ß-III tubulin gene contains a genomic fragment with potential Sox4 and 11 binding sites, the effects on the Tuj1 signal may occur via direct transcriptional regulation of the gene. Electroporation-mediated misexpression of Ngn2/Neurog2 or Ascl1/Mash1 induces high levels of Sox11 and Sox4 before upregulation of p27Kip1, NF1 and Tuj1 signal (Bergsland et al. 2006). The division cycle is exited by many cells. After electroporation-mediated misexpression of Ngn2 together with Sox4 and Sox11 siRNAs, the division cycle is still exited by many transfected cells and p27Kip1 becomes upregulated but few have induced NF1 and Tuj1 immunoreactivity. In mouse embryos at E10.5, Sox4 and Sox11 proteins are both expressed uniformly at high levels throughout the spinal cord (Thein et al. 2010). Then, VZ levels begin to decrease, and at E14.5 Sox4 also starts to decrease in the mantle zone followed with temporal delay by Sox11. Expression is also present in glial cells albeit at lower levels. At birth, these SoxC proteins have almost disappeared from the spinal cord. Thus, they are expressed during neuronal development from neuroepithelial precursors to not fully mature neurons. Sox4 and Sox11 double mutant mice show severe hypoplasia of developing spinal cord (Thein et al. 2010). Dorsoventral patterning at E11.5 appears normal as judged by Nkx2.2, Olig2, Irx3 and Nkx6.1 expression. Sox2-positive neuroepithelial precursors are found at all dorsoventral levels from E10.5 to 14.5, but 10–25 % fewer than normal. Oligodendrocyte progenitors and astrocyte progenitors are reduced but the strongest effect is seen on neurons. TUNEL staining shows dramatically increased cell death in VZ, SVZ and mantle zone at all ages until E16.5. Conditional deletion of both Sox4 and Sox11 in neural progenitors of the cerebral cortex disrupts cortical lamination and leads to a severely stunted or missing hippocampus (Miller et al. 2013). Similarly, combined deletion of Sox4 and Sox11 completely disrupts the overall structure of the retina with the three nuclear layers reduced to one thin layer and the inner and outer plexiform layers abolished (Jiang et al. 2013). RGCs appear completely lost and all other retinal cell types are dramatically reduced. Sox4 and Sox11 double-deficient sympathetic ganglia are hypoblastic due to early proliferation defects and later apoptotic cell loss (Potzner et al. 2010). Yet, neither Sox4 nor Sox11 predominantly function by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons.

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Sox9: a SoxE protein expressed in neural stem cells Sox9 is involved in the development of many organs and tissues, most notably testes development and sex determination, cartilogenesis and skeletal development, or duodenal, pancreatic and liver development. During neural development, Sox9 is essential for maintenance of pluripotency and required for astrocyte formation (Reiprich and Wegner, this issue; Wegner and Stolt 2005; Wegner 2011). Germline deletion of Sox9 in mice leads to death at E11.5, morphological abnormalities in the CNS, and severe NSC defects (Scott et al. 2010). Nestin/Cre-mediated deletion results in death at birth. At E14.5, S100-positive astrocytes, NG2-positive oligodendrocytes and TuJ1-positive neurons are reduced while neuroblasts positive for PSA-NCAM appear increased. At E18.5, no astrocytes remain. Lateral ventricles are enlarged similar to heterozygous mutations of SOX9 in humans. Sox9 induction in neural cells coincides with the appearance of neural stem cells in the CNS that are not present in substantial numbers before E10.5 in mouse and E5 in chick embryos (Scott et al. 2010). Expression in the spinal cord of mouse embryos begins in a few Sox2-expressing cells at E10.5 (Scott et al. 2010). From E11.5 onward, it is expressed with Sox2 in almost all cells of the developing CNS. Nearly all BrdU incorporating cells express Sox9 in adult animals. Sox9 is a target of miR-124 in the adult SVZ (Cheng et al. 2009). It is expressed in ependymal cells, all SVZ astrocytes, part of EGFR-positive transit amplifying cells, and transcript, but no protein is detected in doublecortin (DCX)-positive neuroblasts. Overexpression of Sox9 maintains SVZ cells as GFAP-positive astrocytes but eliminates neuron production. Taken together, the SoxC proteins Sox4 and Sox11 appear most relevant for embryonic neurogenesis among the nongroup B proteins. Sox11 expression overlaps with Sox3 but also with the terminal differentiation markers neuron-specific ß-III tubulin and neurofilament. Effects on progenitor proliferation, pan-neuronal differentiation and survival are documented. In Sox4 and Sox11 double-deficient mice, survival defects prevail and their strength seems to depend on the neuron population. Yet, a complete loss of neuronal differentiation is not observed. Drosophila Sox14 acts in cell survival as well as axonal and dendritic pruning (Kirilly et al. 2009; Osterloh and Freeman 2009). Since DSox14 was initially described as most similar to mouse Sox4 and to human Sox11 (Sparkes et al. 2001), this may point to a conservation of its role in advanced neuronal development. The interaction of SoxB and SoxC proteins and basic helix-loop-helix transcription factors is one key aspect of neuronal development in vertebrates and important invertebrate groups.

Proneural beta helix-loop-helix transcription factors: from neural progenitor to neuron The study of proneural factors involved in successive steps of neurogenesis from neuronal commitment of precursors to neuronal differentiation in invertebrates (see García-Bellido and de Celis 2009; Ghysen and Dambly-Chaudière 1988 for reviews) and vertebrates (see Bertrand et al. 2002 for review) demonstrates that, already by the very early stages of neuronal differentiation, population-specific characters become apparent. Mutational disruption of proneural gene function attracted attention due to the loss of distinct sensory organ types (RuizGómez and Modolell 1987) and neuron populations (Guillemot et al. 1993) in Drosophila and mouse, respectively. Accordingly, the gene products of different proneural genes, related members of the basic helix-loop-helix (bHLH) transcription factor family, are expressed in different domains of the developing sensory and nervous system. Thus, the bHLH gene expression demonstrates diversification within the neurogenic regions of the embryo preceding neuronal differentiation proper. Partial functional equivalence of proneural bHLH proteins An important question was whether the different bHLH proteins in the various neuron populations could in principle exert the same function albeit in different prespecified progenitor cell groups. Replacement gene targeting in mice shows that proneural bHLH proteins impose neuronal specification according to the region of origin of the ectopically expressed factor in some but not all neuron populations. While Ascl1/Mash1 expression from the Ngn2/Neurog2 locus, which directs high Ascl1/Mash1 expression levels to the dorsal telencephalon, induces in a subset of cells properties of ventral telencephalic neurons (Fode et al. 2000), Ngn2/ Neurog2 expression from the Ascl1/Mash1 locus fully compensates the proneural function of Ascl1/Mash1 in the ventral telencephalon but does not change the identity of the neurons (Parras et al. 2002). In the spinal cord, Ascl1/Mash1 expression from the Ngn2/ Neurog2 locus allows largely normal appearance of motoneuron and V2 interneuron markers, while Ngn2/Neurog2 expression from the Ascl1/Mash1 locus cannot rescue the defect in V2 interneuron development seen in Ascl1/Mash1 mutant mice (Parras et al. 2002). In DRG, however, Ascl1/Mash1 is unable to functionally replace Ngn2/Neurog2, suggesting incomplete equivalence of the proneural function as well (Parras et al. 2002). In sympathetic ganglia, Ngn2/Neurog2 expression from the Ascl1/ Mash1 locus allows noradrenergic neuron differentiation including pan-neuronal marker expression and induces no properties of DRG neurons. Neuroblast proliferation cannot be maintained, however.

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The data show only partially overlapping, but also population-specific, functions of proneural bHLH proteins. bHLH cascades in vertebrate neurogenesis Different bHLH proteins act sequentially during differentiation of a neuron. Initially, this was described in mouse olfactory neuron progenitors, where Ascl1/Mash1 expression is followed by Ngn1/Neurog1, and NeuroD/NeuroD1 becomes expressed at the onset of neuronal differentiation (Cau et al. 1997, 2002). Mutational inactivation of Ascl1/Mash1 results in the loss of Ngn2/Neurog2 and NeuroD/NeuroD1 as well as most neurons in the olfactory epithelium. In placode-derived sensory ganglia, a cascade of Ngn1/Neurog1 and Ngn2/ Neurog2>Math3/NeuroD4>NeuroD/NeuroD1>Nscl1/Nhlh1 expression is detected (Fode et al. 1998). In Ngn2/Neurog2 mutant mice, Math3/NeuroD4, NeuroD/NeuroD1 and Nscl1/ Nhlh1, as well as neurofilament, SCG10 and ß-III tubulin, are missing in regions of geniculate and petrosal precursors and ganglia but remain present in nodose placode and ganglion. A similar sequence is observed in placode-derived ganglia in the chick with notable differences in the expression periods and the neurogenin prevalence (Abu-Elmagd et al. 2001). While Ngn1/Neurog1 is strongly expressed in the trigeminal region and Ngn2/Neurog2 more prevalent in the epibranchial region of the mouse embryo, the situation in chick is the opposite. Also different from mouse, the general sequence of activation in chick epibranchial placodes is Ngn1/Neurog1>NeuroD/ NeuroD1>NeuroM/NeuroD4. In chick spinal cord and optic tectum, NeuroM/NeuroD4 expression precedes that of NeuroD/NeuroD1 and is restricted to cells that have ceased proliferation (Roztocil et al. 1997). In the progenitor domain of motoneurons in the chick spinal cord, the bHLH protein Olig2 is expressed in virtually all cells while Ngn2/Neurog2 appears later and displays more scattered distribution (Novitch et al. 2001). NeuroM/ NeuroD4 is also prominently expressed but in more lateral aspects of the pMN domain. After retrovirus-mediated overexpression of Olig2, the Ngn2/Neurog2 expression domain expands, transfected cells become located more laterally, and the number of BrdU incorporating cells decreases. Similar results are seen with Ngn2/Neurog2 misexpression, but cell cycle exit occurs more rapidly. The situation differs in the cerebellum where Math1/Atoh1 is expressed in the precursors of the external granular layer (EGL) (Akazawa et al. 1995; Ben-Arie et al. 1996). Mutational inactivation results in a failure to form cerebellar granule cells and the lack of the EGL (Ben-Arie et al. 1997). Math1/Atoh1-deleted cells in the EGL lack a range of neuronal differentiation regulators such as Pax6, NeuroD1/NeuroD, or Nhlh1/2, as well as TuJ1 as pan-neuronal marker (Klisch et al. 2011). Identification of direct Math1/Atoh1 target genes in P5 mouse cerebellum reveals the Math1/Atoh1 gene itself as

well as NeuroD1/NeuroD, NeuroD2, Nhlh1/Nscl1 and Nhlh2/ Nscl2 among 600 high confidence targets (Klisch et al. 2011). Yet, terminal differentiation genes related to neuronal excitability and synaptic transmission or the pan-neuronal markers of the neuronal cytoskeleton do not figure prominently among these targets. NeuroD/NeuroD1 expression follows primarily in the postmitotic inner zone of the EGL, and mutational inactivation results in a neuronal deficit in the cerebellar granule layer (Miyata et al. 1999). Nscl1/Nhlh1 but not Nscl2/Nhlh2 transcript levels are reduced in the mutant cerebellum (Kim 2012). These studies document different sets of bHLH genes expressed in distinct neuron populations which may also diverge between vertebrate classes. Proneural functions in cycling progenitors are associated, with Ascl1/Mash1, Ngn1/ Neurog1 and Ngn2/Neurog2 also active in cortical neurogenesis (Britz et al. 2006). Postmitotic differentiation processes are associated with NeuroM/NeuroD4 and NeuroD/NeuroD1 expression, with the notable extension of NeuroD/NeuroD1 expression in postnatal mammalian olfactory bulb, cerebellar and hippocampal development combined with the detection already in proliferating neuroblasts (Lee et al. 2000). Consolidation of neuronal fate and withdrawal from cell cycle in neuronal progenitors were recognized as important aspects of proneural activity (see below). bHLH protein function and progenitor proliferation The relationship between proneural function and progenitor proliferation, initially considered to entail withdrawal from the mitotic cycle in parallel to induction of differentiation by the bHLH transcription factors, turned out to be more complex (Castro and Guillemot 2011). Genes promoting and terminating the cell cycle can both be direct targets of proneural proteins. The gene encoding the Notch ligand Delta1, a common target of Ascl1/Mash1, Ngn1/Neurog1 and Ngn2/Neurog2, contains two proximal enhancers bound by Ascl1/Mash1 and Ngn2/Neurog2 and required for expression in Ascl1/ Mash1 or Ngn1/Neurog1 and Ngn2/Neurog2 expressing brain regions, respectively (Castro et al. 2006). Comparing gene expression profiles in the telencephalon of Ngn2/Neurog2 and Ascl1/Mash1 mutant mouse embryos with those of cortices overexpressing Ngn2/Neurog2 or Ascl1/Mash1 indicates Dll1 [delta-like 1 (Drosophila)], Hes5 [hairy and enhancer of split 5 (Drosophila)] and Elav4 as common targets (Gohlke et al. 2008). ChIP-chip from mouse ventral telencephalon at E12.5 with an Ascl1/Mash1 antibody combined with profiling by arrays covering ∼8 kb of proximal promoter region identified 339 likely direct targets of Ascl1/Mash1 (Castro et al. 2011). In addition to members of the Notch signaling pathway, not only negative but also positive regulators of the cell cycle, such as E2F1 (E2F transcription factor 1), the main

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transcriptional activator of genes promoting G1/S transition, are found. Similar to Atoh1/Math1 in the brain, terminal differentiation genes related to neuronal excitability and synaptic transmission or the pan-neuronal markers of the neuronal cytoskeleton do not figure prominently among these Ascl1/ Mash1 targets. Reduction of cells in S and G2/M phase of the cell cycle in SVZ but not VZ of E14.5 Ascl1/Mash1 mutant mice indicates a loss of cycling intermediate progenitors. In the neural stem cell line NS5, all cell cycle target genes of Ascl1/Mash1 are known to promote cell proliferation. Cell cycle arrest genes are induced only after Ascl1/Mash1 overexpression. Cell cycle-regulated phosphorylation of bHLH proteins that differently affects transcription of distinct target genes (Ali et al. 2011, 2014; Hindley et al. 2012; for review, see Hardwick et al., this issue) allows for different regulatory outcomes depending on cell cycle length and phosphorylation status. The remarkable observation that bHLH protein as well as mRNA levels rapidly cycle in relation to the cell cycle in proliferating progenitors, but persistently increase in differentiating neurons (Imayoshi et al. 2013), provides a framework to understand the various aspects of proneural function (for review, see Kageyama et al., this issue).

Cell cycle withdrawal and the transition from proliferation to differentiation The exit from cell cycle by neuronal progenitors is of particular interest for a number of reasons, including its intimate association with neuronal differentiation in many neuron populations as much as the clinical consequences of failed coordination between proliferation and differentiation. The relevance for the understanding of and future treatment strategies against brain tumor formation is one key aspect (see Swartling et al., this issue, for review). The question of the mechanistic link between progenitor cycling and specification of neuronal phenotype is another (see Laguesse et al., this issue, and Hardwick et al., this issue, for reviews). Cyclins and cyclin-dependent kinases (cdks) in the core cell cycle machinery and their role in the regulation of differentiation via the duration of cell cycle phases are one focus of interest (see Hardwick et al., this issue; Lange and Calegari 2010 for reviews). The demonstration of bHLH and Hes protein interaction during the transition from cycling progenitors to differentiation of neurons provides a key element in this puzzle (see Kageyama et al., this issue, for review). The role of regulators of the core cell cycle machinery such as Cdc25 phosphatases (see Agius et al., this issue, for review) and the CKI proteins are of interest for their involvement in the regulation of proliferation and neuronal subtype specification.

The interaction of Cyclins and Cyclin-dependent kinases with proneural factors Pharmacological manipulation of cell cycle length in telencephalic neuroepithelial cells by inhibition of Cyclin-dependent kinases induces premature neurogenesis as shown by TIS21 and Map2 expression (Calegari and Huttner 2003). In support of the ‘cell cycle-length hypothesis’ of neuronal differentiation derived from this observation, the length of the cell cycle and in particular G1 and S phase is altered upon commitment of stem and progenitor cells to neuron production (Calegari et al. 2005; Dehay and Kennedy 2007; Arai et al. 2011). Reduction of G1 duration by forced expression of Cyclin D1 and Cyclin E promotes cell cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6-positive precursors (Pilaz et al. 2009). Shortening of the G1 phase in cortical progenitors by Cyclin D1 and Cyclin-dependent kinase 4 (cdk4) overexpression delays neurogenesis, whereas lengthening of G1 by RNAi-mediated knockdown has the opposite effect (Lange et al. 2009). Cyclin Ds regulate the G1 phase of the cell cycle and link extracellular signals to the cell cycle machinery (Lobjois et al. 2004). In the chick spinal cord, Cyclin D1 and D2 are initially detected by IHC in an overlapping manner in the ventricular zone (Lukaszewicz and Anderson 2011). Signals then become non-overlapping and, while Cyclin D2 remains restricted to the ventricular progenitor domain, Cyclin D1 also becomes located in the adjacent zone, detectable in TuJ1 and NeuN/Rbfox3-positive, PCNA-negative newborn neurons. Knockdown of Cyclin D1 but not of Cyclin D2 by siRNA reduces the ratio of newly differentiated HB9 and NeuN/Rbfox3positive neurons in the analyzed motoneuron domain. Conversely, overexpression of Cyclin D1 but not of Cyclin D2 enhances generic neurogenesis. While Cyclin D1 knockdown reduces Hes6-2 mRNA, Cyclin D2 knockdown reduces Hes5 mRNAs (Lukaszewicz and Anderson 2011). Electroporation-mediated overexpression of Cyclin D1 and Cyclin D2 for extended time periods results in proliferating cells outside the ventricular zone that do not express progenitor markers but instead go through neuronal differentiation and axonal extension (Lobjois et al. 2008). Thus, coupling of cell cycle withdrawal and differentiation in the motoneuron lineage is neither mandatory nor required for proper cell fate choice. Mass spectrometric analysis of Cyclin D1-binding proteins identified not only cell cycle partners like Cyclin D1dependent kinases cdk4 and cdk6 or Cip/Kip family members p27Kip1 and p57Kip2, but also transcriptional regulators (Bienvenu et al. 2010). In developing mouse retina, a regulatory region of the Notch gene is bound by Cyclin D1 where it recruits CBP resulting in increased histone acetylation and transcription. Thus, in addition to its function as core cell

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cycle regulator, Cyclin D1 may affect neurogenesis via transcriptional regulation. Ngn2/Neurog2, Ascl1/Mash1 and NeuroM/NeuroD4 misexpression in the spinal cord of chick embryos results in rapid cell cycle exit (Novitch et al. 2001; Lee and Pfaff 2003; Castro et al. 2006). Selection of Ngn2/Neurog2 transfected cells early (6 h) after electroporation and expression profiling using microarrays shows downregulation of the transcripts for core regulators of G1 and S phase such as Cyclin D1, E1, E2 and A2 (Lacomme et al. 2012). Additional targets belong to the Notch signaling pathway such as Hes1, Hes5, Dll1, Jag1 and Jag2. Cotransfection of Ngn2/Neurog2 with Cyclin D1 increases the number of EdU-incorporating TuJ1-positive cells. Conversely, combined Cyclin A and cdk2 overexpression inhibits neurogenesis induced by ectopic xNgn2 mRNA in Xenopus embryos. Ngn2/Neurog2 is a highly sensitive substrate of Cyclin A- and Cyclin B-dependent kinases, cdk1 and cdk2 (Ali et al. 2011). Importantly, phosporylation continues over a considerable period, indicating that some Ngn2 sites are phosphorylated before others. Consequently, Ngn2/ Neurog2 phosphorylation can be regulated during the cell cycle depending on cell cycle kinetics. Strikingly, mutation of phosphorylation sites enhances its ability to induce neuronal differentiation in Xenopus embryos or mouse P19 cells, showing the importance of the phosphorylation state for Ngn2/Neurog2 activity. Phosphorylation status affects Ngn2/ Neurog2 stabilization by E protein binding and DNA affinity as shown for NeuroD/NeuroD1 and Dll1 promoters. In Xenopus embryos or mouse P19 cells, the NeuroD/Neurod1 promoter is markedly more sensitive to the Ngn2/Neurog2 phosphorylation status than the Delta promoter (Hindley et al. 2012). In addition, inhibition of Ngn2/Neurog2 by the Notch intracellular domain is lost with mutation of Ngn2/Neurog2 phosphorylation sites. Cyclin-dependent kinase inhibitor, bHLH and Sox2 regulatory circuits Activity of bHLH proteins is under the control of cell cycle effectors. Conversely, bHLH proteins regulate Cyclin expression and thus may affect cell cycle kinetics. Also, Cyclindependent kinase inhibitor (CKI) p27Kip1 and p57Kip2 expression is under the control of bHLH transcription factors. Overexpression of Ngn2/Neurog2, Ascl1/Mash1 and NeuroM/Math3 in chick embryo spinal cord for longer time periods (1 day or more) results in upregulation of p27Kip1, lack of BrdU incorporation, and precocious activation of ß-tubulin and neurofilament expression (Novitch et al. 2001; Lee and Pfaff 2003). In addition, overexpression of Ngn2/Neurog2 in chick results in activation of p57Kip2 mRNA expression (Gui et al. 2007). Forced overexpression in chick embryo spinal cord of all three Cip/Kip family members arrests cell cycle

progression as shown by the total absence of BrdU incorporation in transfected cells. p27Kip1 inhibits in particular cyclin-dependent kinase 2 (Besson et al. 2008). In p27Kip1 null mice, main phenotypes include organ hyperplasia, pituitary tumors and retinal dysplasia (Nakayama et al. 1996; Fero et al. 1996; Kiyokawa et al. 1996). In brain and retina of these animals, Sox2 mRNA levels are elevated as compared to wild-type and p27Kip1 associates together with a repressive p130-E2F4-SIN3A complex to a Sox2 gene enhancer during development (Li et al. 2012). In mouse cortex at E14, p27Kip1 transcripts are expressed at significant levels in VZ, SVZ, IZ, and CP (Nguyen et al. 2006), and p21Cip1 and p57Kip2 transcripts in CP. p27Kip1 protein is detected throughout the cerebral cortex with moderate levels in a subset of VZ progenitors and elevated levels in neurons migrating through IZ into CP. While VZ progenitors show predominantly cytoplasmic expression, CP neurons have largely nuclear localization. p21Cip1 and p57Kip2 proteins are found at low levels in a subset of CP cells. In p27Kip1 null mutants, neurons are aberrantly distributed in the cortex and the generation of HuC/D-positive neurons is reduced (Nguyen et al. 2006). Conversely, overexpression of p27Kip1 produces increased numbers of HuC/D and ß-III tubulin-positive cells. In VZ/SVZ, p27Kip1 and Ngn2/ Neurog2 proteins are extensively coexpressed with a sharp downregulation of Ngn2/Neurog2 in cells leaving the germinal zone. In p27Kip1 null mutants, reduction of Ngn2/ Neurog2 protein signals but not of transcript distribution is observed. Stabilization of Ngn2/Neurog2 protein by p27Kip1 is the likely cause (Nguyen et al. 2006). Similarly, in Xenopus embryos, p27Xic1, the only family member, promotes primary neuron formation by neurogenin protein stabilization (Vernon et al. 2003). In embryonic mouse spinal cord at E9.5–13.5, all three CKI factors are expressed in overlapping but distinct sets of cells at the lateral border of the VZ as detected by IHC (Gui et al. 2007). p27Kip1 has the broadest expression along the dorsoventral axes, being present in nearly every cell in the mantle zone but not in progenitors. p21Cip1 on the other hand is detected medial to the V2 interneuron domain. p57Kip2 is detectable more broadly in 2 stripes of cells at the lateral margin of the VZ and proximal part of the MZ but specifically excluded from the V2 and MN domains. The majority of medial p57Kip2 -positive cells coexpress Cyclin D1, while laterally positioned cells coexpress p27Kip1, ß-III tubulin, NeuN and neurofilament. In p57Kip2 mutant mice, increased BrdU incorporation and phosphorylated histone H3 (pH3) indicate enhanced proliferation in the VZ (Gui et al. 2007). Notably, pH3, normally only seen in cells adjacent to the central canal, is also found in lateral cells adjacent to and within the proximal MZ, indicating that cells targeted for differentiation abnormally re-enter cell cycle. In p27Kip1 and

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p57Kip2 double mutant mice, most cells lack expression of all Cip/Kip family CKI proteins in the spinal cord (Gui et al. 2007). Proliferating cell number is increased and excess postmitotic differentiating neurons are generated. In adult mice, forebrain neural stem cell cycling is regulated by p21Cip1 (Kippin et al. 2005), which is also important for maintaining hematopoietic stem cell quiescence and self-renewal. p21Cip1 deficiency results in increased cell cycle reentry and subsequent exhaustion of the NSC pool. This correlates with a decrease in Sox2 mRNA mediated by direct enhancer binding and negative regulation by p21 Cip1 (Marqués-Torrejón et al. 2013). Sox2 in turn interacts with other players crucially involved in the progression from proliferating progenitors to differentiating neurons. MicroRNAs of the miR-200 family, whose expression is regulated by Sox2, in turn target Sox2 and E2F message (see Trümbach and Prakash, this issue, for review). In addition, Sox2 directly promotes Lin28 transcription thus affecting let-7 maturation that affects Cyclin D, Ascl1/Mash1, Ngn1/Neurog1 and E2F2 expression (Wulczyn, this issue, for review). Terminal mitosis and the expression of neuronal cytoskeletal proteins Analysis of the relationship of progenitor cell cycle exit and neuronal differentiation has strongly focused on proteins associated with the neuronal cytoskeleton. ß-III tubulin, due to the highly valued staining properties of the monoclonal antibody TuJ1, and doublecortin, became most frequently used in this context. In the developing mouse telencephalon, the expression of ß-III tubulin as recognized by TuJ1 was studied in relation to incorporation of bromodeoxyuridin (BrdU) as S phase marker in proliferating cells under different pulse chase schedules (Menezes and Luskin 1994). Single injections of BrdU followed by a 1-h survival period at E14–E17 show no incorporation by TuJ1-positive cells indicating that ß-III tubulin becomes expressed postmitotically. BrdU-positive nuclei are largely restricted to the ventricular zone with few positive examples in the subventricular zone. Yet, TuJ1 signal becomes detectable even in the ventricular zone at E12/13 and is abundant at the VZ/SVZ interface at E14. Map2, however, stained with antibodies recognizing all isoforms, is not observed in cells of the VZ or SVZ (Menezes and Luskin 1994). TuJ1 signals largely overlap with doublecortin (DCX) expression in the brain of developing mouse embryos and the rostral migratory stream destined for the olfactory bulb in postnatal animals (Gleeson et al. 1999). DCX transcripts and protein are, however, excluded from the VZ and only occasionally detected in the SVZ at E14. Yet, the related doublecortin-like kinase (DCLK) is detected in VZ/SVZ and also in cortical plate and the marginal zone of E13 to E17 mice

(Shu et al. 2006). DCLK is associated with the mitotic spindle in dividing cells. Coexpression with nestin is observed for DCLK but not DCX. In contrast to mouse, in chick both DCX and DCLK immunostaining is detected in the VZ from E8 to E14 (Capes-Davis et al. 2005). The developing cerebellum in mammals is characterized by a zone of proliferating granule neuron precursors in the outer part of the external granule layer (EGL). This zone is devoid of TuJ1 signal in humans (Katsetos et al. 2003a) and mice (Helms et al. 2001), which appears in deeper zones populated by postmitotic neurons. In the mouse cerebellum, DCX and DCLK proteins are highly expressed at P8 but nearly absent in adults (Shu et al. 2006). While DCX is expressed in the inner zone of the external granule layer at P8 together with NeuN/Rbfox3, DCLK is largely restricted to the outer zone and colocalizes with the mitotic marker pH3. Like DCX, NeuroD/NeuroD1 expression is detected in the inner EGL while Math1/Atoh1 immunoreactivity is found in the outer EGL (Miyata et al. 1999; Helms et al. 2001). Yet, one-third of the EGL cells labelled by a 2-h BrdU pulse are positive for NeuroD/NeuroD1 (Beta2) transcripts (Lee et al. 2000). Transgenic Math1/Atoh1 overexpression induces the appearance of cells positive for DCX and NeuroD/NeuroD1 transcripts in the outer EGL of neonatal mice (Helms et al. 2001). While the analysis of these large CNS domains indicates a frequent restriction of ß-III tubulin and doublecortin expression to postmitotic neurons during embryonic development, the situation is different during postnatal and, in particular, adult neurogenesis. ß-III tubulin as well as doublecortin has already been detected in mitotically active neuronal precursors (see von Bohlen und Halbach 2007 for review). These cells correspond to neuroblasts or type 3 cells, which also express polysialic NCAM, NeuN/Rbfox3 and Prox1 (see Encinas et al. 2013 for review) and miR-124 (Cheng et al. 2009). In the dentate gyrus of adult mice, EGFP expressed transgenically driven by a Sox2 promoter colocalizes with nestin and a few weakly EGFP-positive cells weakly express DCX (Kuwabara et al. 2009). No overlap of EGFP is observed with NeuroD1/NeuroD-positive cells the majority of which are DCX-immunoreactive but rarely nestin-positive, yet show the proliferation marker Ki67. In the adult rat brain, 60 % of the cells in the neurogenic regions labeled by a 2-h BrdU pulse are DCX-positive and DCX frequently colabels with Ki67 (Brown et al. 2003). These cells form clusters in the subgranular zone of the hippocampus and some of the cells appear without processes while others resemble processbearing neuroblasts. Similar colabeling of DCX and Ki67 is found in the subventricular zone where the cells are mostly bipolar with short processes (Kuwabara et al. 2009). Correspondingly, TuJ1-positive immature neurons destined for the olfactory bulb and migrating from the anterior SVZ in postnatal rats are mitotically active, as shown by BrdU incorporation with

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3-h survival time (Menezes et al. 1995). A significant proportion of cells labeled by a 2-h BrdU pulse also show NeuroD1/ NeuroD immunoreactivity (Roybon et al. 2009). In embryonic sympathetic ganglia of the PNS, proliferation of cells with neuron-specific properties was already demonstrated in the 1970s (Rothman et al. 1978). 3H-thymidine labeling in E7 chick embryos for a 2-h period labels cells in sympathetic ganglia expressing the neuron-specific tetanus toxin receptor and a ganglioside recognized by the monoclonal antibody Q211 (Rohrer and Thoenen 1987). In contrast, in DRG neurons, 18 hours pass between final 3H-thymidine incorporation and the appearance of these neuronal differentiation markers. In mouse sympathetic neurons, TuJ1 immunostaining is detectable from E10.5 onward during a phase of cell cycle prolongation but not termination (Gonsalvez et al. 2013). At E11.5, the differentiated cells resume proliferation, are positive for the proliferation indicators Ki67 and pH3, and incorporate BrdU in addition to neuronal marker expression. Thus, in the sympathoadrenal neural crest progeny of birds and mammals, proliferation of already differentiated neurons is observed in embryonic sympathetic ganglia (see Rohrer 2011; Young et al. 2011 for reviews) and the adrenal medulla acquires its mature complement of neuroendocrine cells by division of differentiated cells that extends into the postnatal period. Importantly, it is from this lineage that neuroblastoma, the most frequent extra-cranial solid tumor during childhood, is derived (for reviews, see Ilias and Pacak 2005; Fung et al. 2008). Of considerable interest is the observation that the cerebellum and the sympathoadrenal system are regions associated with neuroblastic tumor formation during childhood. Medulloblastoma are considered a fundamentally neuronal tumor phenotype (Katsetos et al. 2003b). ß-III tubulin expression in these tumors is associated with neuronal differentiation and reduced proliferation (Katsetos et al. 2003b). In contrast, in gliomas, typicallly found in adults, expression is associated with increased malignancy. The possible origin and transformation mechanisms of the respective tumor stem cells are under intense investigation (Swartling et al., this issue).

Vertebrate sensory receptor cells are closely related to neurons In two major vertebrate sensory organs, eye and inner ear, sensory receptor cells and neurons develop in immediate neighborhood. Morphology of the receptor cells with the signal receiving and transducing apparatus as well as the absence of axonal or dendritic processes mark major differences to neurons. Correspondingly, differences in cytoskeletal and motor proteins stand out with loss of myosin VIIa in hair cells and photoreceptors associated with sensory impairment

of the audiovestibular and visual systems (see Yan and Liu 2010; Williams and Lopes 2011 for reviews) and loss of myosin VI associated with hearing loss (Hilgert et al. 2008; see Friedman et al. 1999 for review). Synaptic protein and developmental regulator expression and function show great similarities to neurons. SOX2 haploinsufficiency in humans is associated with defects in multiple sensory systems including anophthalmia, hearing loss and brain malformations (Fantes et al. 2003; Hagstrom et al. 2005; Zenteno et al. 2005). Analysis in mice, chick and zebrafish shows transcription factors of the Sox and bHLH families that are active in neurogenesis to be also involved in the differentiation of sensory receptor cells. Sox and proneural bHLH proteins in neuron and hair cell production in the vertebrate inner ear Hair cell development in amniote vertebrates depends on Sox2 and Atonal homolog 1 (Atoh1/Math1) and involves direct binding of Sox2 protein to an Atoh1/Math1 enhancer and consequential activation of transcription (see Raft and Groves, this issue; Groves et al. 2013; Neves et al. 2013 for reviews). Neuron development from the inner ear neurosensory domain critically depends on Sox2 and Neurogenin1 (Neurog1/Ngn1). In Lcc/Lcc mice that are completely deaf, no prosensory domain is established in the inner ear, and neither hair cells nor supporting cells differentiate (Kiernan et al. 2005). The Sox2 locus is rearranged and transcripts from the intact Sox2 coding region are detected throughout the spinal cord but are absent from the otocyst, as analyzed by ISH at E9.5 and E14.5. No complementation occurs with a targeted Sox2 null allele. Math1/Atoh1 transcripts are not detected at E15.5 in Lcc/Lcc mice, while Sox2 protein is detected in Math1/Atoh1 mutants at E14.5. In addition to the absence of hair cells in Sox2 mutant mice, spiral ganglion neurons are absent, as analyzed at E15.5 and P0 (Puligilla et al. 2010). In Sox1 mutant animals, TuJ1 expression and ganglion cell morphology are unaltered, however. Sox2 is expressed in both neuronal and sensory progenitors and downregulated in differentiated neurons and hair cells in chick and mouse inner ear (Neves et al. 2007; Hume et al. 2007). Sox2 protein in the chick otocyst is first detected by IHC in the neurogenic region that gives rise to sensory neurons, to then become restricted to prosensory patches (Neves et al. 2007). It is not detected in delaminating and TuJ1positive neuroblasts at E2–3, and also, at later stages at E5, TuJ1-positive neurons appear negative for Sox2. Sox2 transcripts are detected in neuroblasts contributing to the cochleovestibular ganglion but at levels much reduced compared to the neurosensory domain (Evsen et al. 2013). Sox3 protein is expressed during otic neurogenesis and downregulated in sensory progenitors (Neves et al. 2007). In the mouse

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otocyst, Sox2 immunoreactivity is detected along with Neurog1/Ngn1 and NeuroD1/NeuroD protein in neuroblasts as early as E10.5 (Puligilla et al. 2010). At P0 in mice, Sox2, and also Sox1 but not Sox3, is observed in neurons as confirmed by double-labeling for TuJ1 (Puligilla et al. 2010). Sox2 protein is transiently detectable in hair cells of the chick inner ear, and its expression is retained in supporting cells (Neves et al. 2007). Sox2 electroporation induces ectopic hair cell differentiation within neurosensory as well as nonsensory domains of the otic epithelium as demonstrated by myosin VIIa immunostaining in chick (Neves et al. 2011) and myosin VI in mammals (Pan et al. 2013). The function of Sox2 in chick relies on the direct regulation of Atoh1/Math1 (Neves et al. 2012). A reporter carrying the Atoh1/Math1 enhancer elements that reside at 3’ of the Atoh1/Math1 coding sequence is active in the otic vesicle but spatially restricted to the Sox2-positive expression domain. Activity blockade by a dominant negative Sox2HMG–Engrailed fusion protein or mutation of the Sox transcription factor binding site indicates that Atoh1/Math1 transcriptional activity in the otic vesicle depends directly on Sox2. In addition, Sox2 overexpression is shown to promote neurogenesis in chick (Neves et al. 2011) and in mammalian inner ear (Puligilla et al. 2010). In mouse cochlear explants, electroporation of Sox2, but not Sox1, drives neuronal differentiation (Puligilla et al. 2010). Sox2 did not, however, induce hair cell properties in these experiments. With increasing age, a progressive decrease in neuron induction is observed. Surprisingly, Sox2-transfected cells express neither NeuroD1/NeuroD nor Neurog1/Ngn1. In chick embryos, Sox2 overexpression results in ectopic delamination of TuJ1positive cells combined with increased transcript levels of Neurog1/Ngn1 (Neves et al. 2011). The resultant enlarged cochleovestibular ganglion formation suggests that Sox2 is sufficient to specify neurogenic fate. Overexpression of Sox2 induces Neurog1/Ngn1 in and outside the neurosensory domain but not NeuroD1/NeuroD (Evsen et al. 2013). However, the size of the cochleovestibular ganglion in this study appeared reduced by approximately 50 %. In zebrafish otic vesicle, sox2 expression becomes detectable after atoh1 and is lost as hair cells mature but retained in support cells (Millimaki et al. 2010). Morpholino-mediated knockdown of sox2 compromises hair cell maintenance. In addition to Sox2, all SoxB genes except Sox14 are expressed in the primordium of the chick inner ear (Uchikawa et al. 1999). Sox2, Sox3 and Sox21 transcripts become detectable at Hamburger-Hamilton stage 9–10, Sox1 expression starts around stage 18. In all sensory epithelia, Sox2 and Sox21 transcripts are uniformly expressed while Sox3 is limited to the cristae. Sox1 expression in sensory epithelia except for the cristae is weak. In addition, other Sox transcripts including Sox4, Sox5 and Sox11 are found in the chick otocyst (Sinkkonen et al. 2011). Sox11 transcripts

appear specifically associated with prosensory domains of the basilar papilla and vestibular maculae. Sox21 transcripts are detected at E10.5 in the mouse otic vesicle (Hosoya et al. 2011). EGFP expression from the Sox21 locus shows overlapping expression with Sox2 protein in the otocyst, the forming spiral ganglion, and in the organ of Corti both in the hair cell and the supporting cell layer. Mutational inactivation of Sox21 shows only mild effects in hair cells with an increase in their number in some homozygous animals combined with disorganized polarity. In the chick inner ear, which displays a similar Sox21 expression pattern, overexpression at different stages leads to different results (Freeman and Daudet 2012). Retrovirus-mediated misexpression reduces Sox2 and Prox1 (prospero homeobox 1) expression and prevents neuron and hair cell differentiation as analyzed by TuJ1 IHC, which in the chick embryo labels both cell types. In organotypic cultures from vestibular epithelia at later stages, however, Sox21 biased progenitors to a hair cell fate. In addition, the SoxE protein Sox9 is expressed in the inner ear. A comparative analysis of Sox2 and Sox9 expression during mouse inner ear development shows both proteins in the prosensory region, Sox2 in hair cells of cristae, maculae and organ of Corti, and Sox9 in periotic mesenchyme, bony labyrinth, and supporting cells, and a more complex pattern of Sox2 and Sox9 expression in the cochleovestibular ganglion (Mak et al. 2009). In the E9.5 otocyst, Sox2 protein is detected in the proneural region while Sox9 IR is detected in the entire otic epithelium and faintly in the periotic mesenchyme. At E12.5, Sox2 marks the sensory primordia of cristae, maculae and cochlear duct while Sox9 is still detected in the entire otic epithelium and the periotic mesenchyme. Beginning at E14.5, when hair and supporting cell differentiation commences, Sox2 is expressed in both cell types while Sox9 becomes restricted to supporting cells. In the spiral ganglion, Sox9 is detected but not coexpressed with ß-III tubulin as shown by TuJ1 IHC. Among the bHLH proteins, Atoh1/Math1, Ngn1/Neurog1 and NeuroD/NeuroD1 have distinguished roles in the neurosensory development of the inner ear. Neurog1/Ngn1 and NeuroD1/NeuroD expression become detectable first, followed by Atoh1/Math1, corresponding to the sequence of neuron and hair cell generation. In the chick otocyst at E3, a subpopulation of cells in the neurosensory domain expresses Ngn1/Neurog1 and NeuroD/ NeuroD1 (Evsen et al. 2013). In the delaminated neuroblasts that will form the cochleovestibular ganglion, NeuroD/ NeuroD1 but not Ngn1/Neurog1 transcripts are detected at high levels. Expression of Ngn1/Neurog1 and NeuroD/ NeuroD1 in the mouse inner ear is detectable at E9.0 (Ma et al. 1998). Sox2 is expressed along with Ngn1/Neurog1 and NeuroD/NeuroD1 (Puligilla et al. 2010). The progeny of Neurog1/Ngn1-expressing cells is identified in a BAC transgenic mouse line where not only VIIIth cranial ganglion

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neurons but also sensory and non-sensory epithelia of the inner ear are marked (Raft et al. 2007). Derivatives are found as myosin VIIa-expressing hair cells in utricular and saccular maculae but not the organ of Corti. In Ngn1/Neurog1 mutant mice, displaying a neonatal lethal phenotype, the cochleovestibular ganglion is absent as analyzed by TuJ1 whole mount IHC at E10.5 (Ma et al. 1998). At E9.0, NeuroD/NeuroD1 transcripts are expressed in columnar epithelial cells of the wild-type otic placode but are undetectable in the mutant. Also, Nscl1/Nhlh1 transcripts are eliminated. In addition, Ngn1/Neurog1 mutant mice develop smaller inner ear sensory epithelia with morphologically normal hair cells (Ma et al. 2000). In particular, the saccule is affected. In the cochlea of newborn animals, approximately 22 % of inner and 44 % of outer hair cells are lost. The utricular macula, however, is expanded medially in Ngn1/Neurog1 mutants into a normally neurogenic region while the saccule is hypoblastic as shown by the number of Atoh1/Math1-positive cells at E13.5 (Raft et al. 2007). In heterozygous animals, Atoh1/ Math1-positive cell number is increased in both utricle and saccule, but not the lateral cristae. However, myosin VIIaexpressing cells are reduced in number in homozygous as well as heterozygous mutants. No evidence for apoptosis is detected. NeuroD/NeuroD1 mutant mice, rescued by reintroducing NeuroD/NeuroD1 in the pancreatic endocrine cells, are viable but deaf, as demonstrated by the absence of evoked auditory potentials (Kim et al. 2001). This correlates with a massive although not complete loss of sensory neurons different from the full loss of these neurons in Ngn1/Neurog1 mutant animals (Ma et al. 1998). While sensory neuron formation appears unaltered in NeuroD/NeuroD1 mutants at E9.5, by E10.5 a reduction of cells expressing lacZ from the NeuroD/NeuroD1 locus becomes apparent, and at E14.5 the cochlear ganglion is almost absent (Kim et al. 2001). This is accompanied by a loss of trkB and trkC expression and cell death as shown by TUNEL staining. NeuroD1 and NeuroD2 double mutants do not show aggravated effects. Importantly, conditional deletion of NeuroD1/NeuroD in the inner ear results in formation of hair cells within the inner ear sensory ganglia (Jahan et al. 2010). This correlates with spatiotemporally altered Atoh1/ Math1 expression in the NeuroD1/NeuroD mutant cochlea. A similar phenotype with premature expression of Atoh1/Math1 in the apex is observed in Neurog1/ Ngn1 mutant mice (Matei et al. 2005). In mouse cochlear explants, Ngn1/Neurog1 or NeuroD/ NeuroD1 electroporation drives neuronal differentiation from non-sensory epithelial cells as shown by neuronal morphology as well as TuJ1 and Map2 IR, while NFs and GAP-43 are not detected (Puligilla et al. 2010). These cells also generate action potentials. Hair cell markers are not induced by electroporation of Ngn1/Neurog1 or NeuroD/NeuroD1 but by Math1/Atoh1. Overexpression of Ngn1/Neurog1 in the chick

inner ear results in downregulation of Sox2 IR, broad ectopic delamination of NeuroD1/NeuroD-positive cells from the entire otocyst, and also NeuroD1/NeuroD expression in the otic epithelium (Evsen et al. 2013). The data show a preferential but not exclusive action of Ngn1/Neurog1 and NeuroD/NeuroD1 in the neuronal branch of inner ear neurosensory development. The key regulator of inner ear hair cell formation is atonal homolog 1, Atoh1/Math1, the homolog of Drosophila atonal, ato. Math1/Atoh1 mutant mice fail to generate cochlear and vestibular hair cells while supporting cells develop (Bermingham et al. 1999). The hair cell marker myosin VI is absent from mutant inner ear at E13.5 and E18.5. Expression of Math1/Atoh1 in Drosophila by the UAS-Gal4 system results in ectopic external sense organs on the notum and the wing blade (Ben-Arie et al. 2000). Like ato, it produces additional chordotonal organs. In ato mutants chordotonal organ formation can be partially rescued by Math1/Atoh1. Expression of lacZ replacing the Math1/Atoh1 coding region in mice is first detected in the otic vesicle at E12.5 and continues until E18.5 throughout much of the sensory epithelia (Ben-Arie et al. 2000). Expression of ß-galactosidase or EGFP-tagged Atoh1 from a knock-in at the mouse Atoh1/ Math1 locus shows expression in the cochlea at E13.5, several days before the appearance of hair cell differentiation markers (Woods et al. 2004; Cai et al. 2013). As differentiation proceeds, Atoh1/Math1 becomes restricted to myosin VI-labeled hair cells. Conditional deletion shows its role in precursor and hair cell survival as well as hair bundle formation. Ectopic expression of Math1/Atoh1 in nonsensory regions of cochlear explants from mouse embryos is sufficient to induce the formation of sensory clusters that contain both hair cells, as detected by myosin VI or myosin VIIa as hair cell markers, and supporting cells (Woods et al. 2004). Similary, atoh1 overexpression in zebrafish otic vesicle induces hair cell differentiation, albeit only in sites of sox2 expression (Millimaki et al. 2007). When sox2 is additionally overexpressed, atoh1-induced formation of ectopic hair cells can be observed throughout the otic vesicle. In Math1/Atoh1 mutant mice, excess neural precursors from utricle and saccule contribute to an enlarged VIIIth cranial ganglion (Raft et al. 2007). In mice in which the Neurog1/Ngn1 coding sequence replaces Atoh1/Math1, patches of organ of Corti precursor cells displaying microvilli but not stereocilia-bearing cells indicate a partial rescue in hair cell precursor survival but no transdifferentiation to neurons (Jahan et al. 2012). In summary, Neurog1/Ngn1 and Atoh1/Math1 expression mark the paths to neuronal and sensory receptor cells, respectively, in the inner ear. NeuroD/NeuroD1 supports neuron development and suppresses hair cell differentiation. Pou4f3/ Brn3c/Brn3.1 mutation is associated with hearing loss in

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humans (Vahava et al. 1998) and hair cell loss in mice (Keithley et al. 1999). The binding of Atoh1/Math1 to the Pou4f3/Brn3c/Brn3.1 gene enhancer correlates with the induction of hair cells and is reduced by NeuroD/NeuroD1 (Ikeda et al. 2014). Among these regulators, NeuroD/ NeuroD1 is also involved in retina development but promotes rather than suppresses sensory receptor development. Retinal photoreceptor development SOX2 haploinsufficiency in humans is associated with defects in multiple sensory systems including anophthalmia (Fantes et al. 2003; Hagstrom et al. 2005; Zenteno et al. 2005). Gene dosage allelic series of Sox2 mutations in mice show eye phenotypes related in severity to the Sox2 expression levels in progenitors of the neural retina (Taranova et al. 2006). Reduction of expression levels to less than half of control levels results in variable microphthalmia. Conditional deletion of Sox2 under control of a retina-specific Pax6 regulatory element reduces BrdU incorporation in the targeted distal parts of the E13.5 retina. In addition, Notch1 and Hes5 transcripts are not detected, Math5 and NeuroD/NeuroD1 mRNAs as well as ß-III tubulin IR are absent. In mice in which the Sox2 coding region is replaced by EGFP, expression in retinal progenitor cells coincides with the proliferation marker PCNA and is downregulated upon neuronal and sensory differentiation such that expression is mutually exclusive with ß-III tubulin, neurofilament and rhodopsin (Taranova et al. 2006). In contrast, Müller glia retain Sox2 expression. Different from EGFP as well as Sox2 immunostaining, Sox1 and Sox3 signals are not detected in mouse retinal progenitors. In the retina of the chick embryo, however, the four SoxB genes Sox1, Sox2, Sox3 and Sox21, are expressed (Uchikawa et al. 1999). In addition, SoxC genes are expressed in developing retina. Sox11 transcripts are already widely expressed in mouse optic cup and retina at E10.5 (Jiang et al. 2013). At E11, some Sox11-expressing cells exhibit ß-III tubulin signals (Usui et al. 2013). Sox4 becomes detectable at E11.5 and both are modestly expressed in the neuroblast layer as compared to high levels in the ganglion cell layer. Retrovirus-mediated Sox11 overexpression in retinal explant cultures appears to induce increased generation of RXRγ-positive cone cells that do not, however, pass through final maturation as shown by the lack of M-opsin and cone arrestin 4 (Usui et al. 2013). The generation of rhodopsin-positive rod cells is suppressed. Sox11 null mutant mice display microphthalmia and RXRγ-positive cone cells differentiate in reduced numbers at E13 but achieve normal counts at E18 due to a shift in birth date. The number of apoptotic cells is increased at E12–E18. Sox11 and Sox4 double mutant mice show reduction of the retina to one thin layer, but markers for all retinal cell types including rods and cones are found (Jiang et al. 2013).

The SoxE protein Sox9 is detected by IHC in mouse retina and at E11.5 yields low signals as compared to the strong signals in surrounding tissue (Zhu et al. 2013). In zebrafish sox9b but not sox9a mutants, the number of photoreceptors and Müller glia cells is reduced (Yokoi et al. 2009). Expression of neurod is severely reduced and sox4a is not detectable. Taken together, the Sox protein inventory of retina and inner ear shows many similarities so that differences between sensory receptor cells and neurons are not immediately recognizable. Sox2 appears required for progenitors of all these cell types in mice. Expression patterns in chick indicate a similar situation. The protein playing this role in zebrafish remains to be identified. Neurog2/Ngn2 expression is first detected in the dorsocentral retina at E11 in mouse embryos (Hufnagel et al. 2010). Similarly, first Atoh7/Math5-expressing cells are detected in the dorso-central retina at E11 (Brown et al. 1998, 2001), while Ascl1/Mash1 expression begins by E12.5 (Hufnagel et al. 2010). Neurog2/Ngn2-expressing cells are capable of adopting all retinal fates (Ma and Wang 2006), including that of cone photoreceptors (Hufnagel et al. 2010). GFP expressed from the Neurog2/Ngn2 locus colocalizes with Pax6 and Sox2 (Hufnagel et al. 2010). Differentiating neurons, appearing from E11 onward, highly coexpress ß-III tubulin and p27kip1 and all positive cells co-label with GFP. In Neurog2GFP/+; Atoh7lacZ/+ mice, Neurog2/Ngn2 expression is detected first by IHC or GFP at E11 in the dorso-central retina (Hufnagel et al. 2010). At that time, Neurog2GFP and Atoh7lacZ are extensively coexpressed. Upon peripheral expansion of the expression domain, the Neurog2GFP domain extends more peripherally and includes more cells than the Atoh7lacZ domain. Neurog2/ Ngn2 is mainly detected in S-phase progenitors, Atoh7/ Math5 not during S but in G2/M-phase (Hufnagel et al. 2010). They are differently regulated by Notch signaling (Maurer et al. 2014). Neurog2/Ngn2 mutants show a delay in the spread of neurogenesis that gets restored at later stages (Hufnagel et al. 2010). This delay can be rescued by Ascl1/Mash1 expressed from the Neurog2/Ngn2 locus. Cone photoreceptors occur in normal numbers in Neurog2/Ngn2 mutants but are increased in Atoh7/Math5 mutants. This increase is not prevented by Ascl1/Mash1 expressed from the Atoh7/Math5 locus (Hufnagel et al. 2013). Combinatorial bHLH gene mutations in mice affect distinct retinal neuron populations differently (Akagi et al. 2004). Interestingly, a triple combination of Ngn2/Neurog2, Mash1/ Ascl1 and Math3/Neurod4 null mutations in mice significantly alters the prevalence of every neuron class in the retina without eradicating any of them. In addition, it leaves the number of photoreceptors unaltered. Two other bHLH proteins,

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encoded by Atoh7/Ath5 and NeuroD/NeuroD1, appear involved in vertebrate photoreceptor development. NeuroD/NeuroD1 null mutant mice show a delay in amacrine cell differentiation, an increase in bipolar neurons, and a loss of a subset of rod photoreceptors (Morrow et al. 1999). In addition, Müller glial cell numbers are increased. NeuroD1/NeuroD also regulates cone photoreceptor development such that all cones express S-opsin and none expresses M-opsin in mutant mice (Liu et al. 2008). The expression of other bHLH genes appears unaffected in NeuroD/NeuroD1 mutants, indicating that the observed effect is not due to altered regulation of related proteins (Akagi et al. 2004). In Math3/Neurod4 and NeuroD/NeuroD1, double-mutant retina amacrine cells are completely missing (Inoue et al. 2002). In the embryonic rat retina, NeuroD/NeuroD1 ISH signal is weak at E13 and becomes strong at E17 during the peak of amacrine cell generation (Morrow et al. 1999). No signal is detected at these stages in the retinal ganglion cell layer. At E20, the majority of cells that incorporate BrdU do not express NeuroD/NeuroD1 (Ahmad et al. 1998). At P0, transcripts are detectable in the region of prospective photoreceptors (Morrow et al. 1999). In mice with lacZ targeted into the NeuroD/NeuroD1 locus, the expression pattern is highly similar. Anti-NeuroD1 antibodies give weak labeling of retinal progenitor cells in the E12.5 mouse retina, and at E14.5 expression tends to cluster close to the presumptive photoreceptor sites (Mao et al. 2013). Infection of rat retinal explants with the NeuroD/NeuroD1-recombinant virus increases the proportion of cells expressing opsin (Ahmad et al. 1998). NeuroD/NeuroD1 can interact with an opsin promoter E box element in vitro. NeuroD/NeuroD1 is involved in photoreceptor formation not only in mice and rats but also in chick (Yan and Wang 2004) and zebrafish (Ochocinska and Hitchcock 2009; Ochocinska et al. 2012), suggesting functional conservation in vertebrates. Two pathways, one via Ngn2/Neurog2 and the other via Ath5/Atoh7, appear to converge on NeuroD/ NeuroD1 during photoreceptor specification (see Yan et al. 2005a for review). In the embryonic chick retina, coexpression of Cath5/ Atoh7 and NeuroD/NeuroD1 occurs in a region adjacent to young photoreceptors (Ma et al. 2004). Retrovirus-mediated Ath5/Atoh7 overexpression increases the population of photoreceptor and retinal ganglion cells (RGC). In mice, Math5/ Atoh7-expressing cells mostly develop into retinal ganglion cells, horizontal cells, photoreceptors, and amacrine cells as shown by a GFP reporter activated by a Math5-Cre knock-in allele (Feng et al. 2010) or BAC transgene (Brzezinski et al. 2012). HA-tagged Atoh7/Math5 expressed from the Atoh7/ Math5 locus is detected in the central part of the E12.5 mouse retina where neurogenesis begins, and at E14.5 expression is found in twice as many cells as NeuroD1/NeuroD1 (Mao et al. 2013).

In the absence of Math5/Atoh7 in the mouse retina, cells are diverted from RGC fate to become other retinal cell types. At E11.5, mutant cells are unable to assume the earliest fates, in particular that of RGCs (Wang et al. 2001; Le et al. 2006). The abundance of cone photoreceptors, however, is significantly increased in Math5/Atoh7 mutant retinae (Brown et al. 2001). Insertion of Atoh7/Math5 into the NeuroD1/NeuroD locus reprograms cells destined to produce photoreceptors and amacrine cells into RGCs (Mao et al. 2013). In Math5 and Brn3b double mutant mice, the number of RGCs is reduced by more than 99 % (Moshiri et al. 2008). Also, in zebrafish lakritz mutants, mutation of the ath5 locus leads to a complete loss of RGCs (Kay et al. 2001). Thus, in Drosophila and mouse, the specification of the first neural cell type in the visual sense organ, the R8 photoreceptors and RGCs, respectively, depends on atonal and its vertebrate homologue Ath5/Atoh7 (Hsiung and Moses 2002). In Drosophila atonal mutants, Xenopus Xath5/Atoh7 rescues photoreceptor development (Sun et al. 2003). Mouse Math5/ Atoh7 is much less effective. Yet, for photoreceptor specification in mice, it appears to play an inhibitory role in favor of retinal ganglion cell differentiation. As for Sox proteins, a simple diversion of bHLH proteins into those required for sensory receptor cells and neurons is not apparent. In addition to Sox and bHLH proteins, homeobox transcription factors play a crucial role in photoreceptor development and disease (see Zagozewski et al. 2014; Swaroop et al. 2010 for reviews). In vertebrates, paired box 6 (Pax6), retina and anterior neural fold homeobox (Rax), orthodenticle homolog 2 (Otx2) and cone-rod homeobox (Crx) are of particular relevance. Importantly, Pax6 directly regulates Neurog2/Ngn2 and Atoh7/Math5 gene expression while Crx targets photoreceptor-specific genes such as rhodopsin (see Zagozewski et al. 2014; Hennig et al. 2008 for reviews). Rax and Pax6 are expressed in retinal progenitor cells and are associated with an Eyeless and Small Eye phenotype in mutant mice, respectively, or anophthalmia and aniridia in human disease, respectively. In addition, forebrain defects are associated with mutations of Pax6 (see Georgala et al. 2011; Hébert and Fishell 2008 for reviews) and Rax (see Zagozewski et al. 2014; Muranishi et al. 2012 for reviews). Otx2 and Crx are expressed in cones, rods and bipolar cells (see Zagozewski et al. 2014; Swaroop et al. 2010 for reviews). Crx regulates photoreceptor differentiation and maintenance and targets photoreceptor-specific genes (see Zagozewski et al. 2014; Hennig et al. 2008 for reviews). While Otx2 mutation is associated with microphthalmia in mice and humans, Crx mutation comes with photoreceptor degeneration and dystrophy, respectively. In addition, Otx2 mutant mice have forebrain, midbrain and hindbrain defects pointing to its importance in neuronal in addition to sensory receptor tissues (see Beby and Lamonerie 2013 for review). In Crx

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mutant mice bipolar cell development is compromised (see Swaroop et al. 2010 for review). Taken together, Sox, bHLH and homeobox transcription factors in sensory receptor cell and neuron development overlap to a large extent. How this contributes to the similarities in synaptic protein expression and differences in cytoskeletal and motor protein equipment remains to be determined. Importantly, the combination of Rax, Otx2 and Crx with NeuroD/NeuroD1 has the potential to reprogram fibroblasts to photoreceptors (Seko et al. 2014).

Endocrine cells employing vesicle-mediated hormone release share many transcriptional regulators with neurons Endocrine cells employing hormone release from secretory storage vesicles share many functional and molecular features with neurons. Yet, morphological differences are profound and the release of the mediators into blood capillaries or the synaptic cleft represents the key distinction of the two cell types. Accordingly, there is a considerable overlap in the constituents of the vesicle fusion machinery and differences in the cytoskeletal protein complement. This correlates with distinct similarities in Sox and bHLH developmental regulators and differences in microRNAs. Sox and bHLH proteins in pituitary development Sox2 and Sox3 are required during early pituitary organogenesis (see Alatzoglou et al. 2009 for review). As Sox3, associated with X-linked hypopituitarism in mice and humans, is not expressed in Rathke’s pouch but in the ventral hypothalamus and infundibulum (Rizzoti et al. 2004), effects on pituitary development appear to be indirect. Heterozygous Sox2 mutations in humans are associated with severe eye defects and hypopituitarism, and in heterozygous mice abnormal anterior pituitary development is manifest (Kelberman et al. 2006, 2008). Expression in the pituitary anlage during development points to its direct role. At E11.5 in mice, Sox2 is uniformly expressed in Rathke’s pouch and by E18.5 in proliferating cells of the dorsal zone as well as in scattered cells of the anterior pituitary (Fauquier et al. 2008; Rizzoti et al. 2013). There is no colocalization with endocrine cell markers. In the adult pituitary, Sox2 is detected in a small population of cells: 3–5 % of the cells in the anterior lobe (Fauquier et al. 2008). The population remains mitotically active but the proportion of cells labeled by a 90-min BrdU pulse is much smaller than at E12.5. Sox9 is expressed in Rathke’s pouch in only a few Sox2-positive cells and at low levels at E12.5. More cells are positive at E18.5, and in adults extensive but not complete overlap of Sox9 and Sox2 is

found. While the Sox2-positive cells coexpressing Sox9 are considered transit-amplifying cells, Sox2-positive but Sox9negative cells are slowly dividing multipotent progenitor/stem cells. In the adult pituitary, the Sox2 and Sox9-positive progenitors can self-renew and generate endocrine cells in vivo (Rizzoti et al. 2013; Andoniadou et al. 2013). Heterozygous Sox2 mutant mice have smaller pituitaries at E18.5 and reduced numbers of somatotrophs and gonadotrophs (Kelberman et al. 2006). Conditional deletion of Sox2 in Rathke’s pouch by Hesx1-Cre results in severe anterior lobe hypoplasia and drastically reduced Pit1/POU1F1 (POU domain, class 1, transcription factor 1) expression (Jayakody et al. 2012). Somatotroph and thyrotroph differentiation from late-born precursors is strongly compromised, while corticotrophs, lactotrophs and gonadotrophs from precursors exciting the cell cycle at early stages are less affected. In addition to SoxB1 and SoxE proteins, the SoxC protein Sox4 is detected in the developing pituitary. It is one of the main transcription factors expressed in human fetal pituitary as analyzed by RT-PCR and ISH (Ma et al. 2009). In zebrafish, Sox4b is expressed in pituitary anlagen and pituitary (Quiroz et al. 2012). Morpholino knockdown or dominantnegative Sox4 mutant expression reduces growth hormone (gh) levels among others, but not prolactin (prl) or proopiomelanocortin (pomc). In mice, Ascl1/Mash1 is expressed in the developing anterior pituitary and is still intensely sustained in the intermediate lobe as seen by ISH at E17.5 (Liu et al. 2001). It exerts roles in terminal differentiation of thyrotrophs, gonadotrophes and corticotrophes (unpublished, cited in Zhu et al. 2006). Ngn2/Neurog2 expression is observed in a small number of cells at E12.5 and E13.5, while no Ngn1/Neurog1 or Ngn3/Neurog3 IR is detected from E10.5 to E15.5 (Lamolet et al. 2004). Mice mutant for Ngn1/ Neurog1 and Ngn2/Neurog2 display a grossly normal pituitary anlage at E13.5 as well as normal NeuroD1/ NeuroD and Pomc expression. In normal adult human pituitary, NGN2/NEUROG2 IR is observed in scattered cells of the adenohypophysis and colocalizes predominantly with corticotroph and somatotroph but not gonadotroph markers (Fratticci et al. 2007). NeuroD1/NeuroD expression in the mouse pituitary starts at E10.5, before Pomc, within the anterior lobe, as analyzed by IHC and correlating strongly with ISH (Poulin et al. 2000; Liu et al. 2001). From E16 onward, it appears no longer detectable. Transcript but no protein is found in the adult. At E14, all NeuroD1/NeuroD-positive cells are also ACTH/ Pomc-positive. In mice deficient for NeuroD1/BETA2, corticotroph terminal differentiation as assessed by Pomc expression is delayed (Lamolet et al. 2004). ACTH/Pomc IHC shows a decrease in positive cell numbers at E13.5 while αGSU/Cga (glycoprotein hormones, alpha subunit) and ßTSH/Tshb (thyroid-stimulating hormone beta subunit) are unaffected. At E16.5, however, Pomc-positive cell number has fully recovered to control levels.

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In the zebrafish pituitary, ascl1a/zash1a is required for endocrine differentiation and cell survival (Pogoda et al. 2006). At 26 hpf, ascl1a/zash1a transcripts are uniformly detectable throughout the entire adenohypophyseal anlage, while ascl1b/zash1b is excluded from the adenohypophysis. In pia/ascl1a mutants, no transcripts for prl, gh, tsh, pomc or gsu can be detected at 26 and 48 hpf. neurod/neurod1, which is expressed throughout the adenohypophysis in wild-type embryos, is absent in the pituitary of pia/ascl1a mutants at all stages studied. Remaining cells retain the ultrastructure of precursors, not of differentiated endocrine cells.

Differences in Sox and proneural bHLH protein usage between fish and mammals in pancreatic endocrine development Sox1, Sox2 and Sox3 are undetectable by RT-PCR from mouse pancreas at E12.5–E18.5 (Lioubinski et al. 2003). While Sox2 expression is observed in the developing mouse duodenum it appears specifically excluded from the pancreatic buds, as analyzed by ß-galactosidase expression from the Sox2 locus (Wilson et al. 2005). Correspondingly, global gene expression analysis on purified endodermal progenitors from morphologically distinct endodermal domains in mouse embryos shows Sox2 present in anterior domains while expression ceases at the level of the dorsal and ventral pancreatic buds (Sherwood et al. 2009). In contrast, Sox4, Sox9 and Sox11 among others are detected by RT-PCR in embryonic mouse pancreas (Lioubinski et al. 2003). Sox4 and Sox9 transcripts are found in the pancreatic epithelium and later in islets, and Sox11 in the mesenchyme surrounding pancreatic buds. Sox4 is detected by ISH and IHC broadly in the early pancreatic buds and restricted to the nuclei of all islet cells in adult mice (Wilson et al. 2005). At E10.5 and E12.5, Sox9 is expressed in mouse primary pancreatic progenitors but not in differentiated glucagon-expressing cells (Lynn et al. 2007a). At E15.5, Ngn3/Neurog3 is detected in a subset of Sox9-positive cells, and Sox9 can bind to and stimulate reporter expression from the Ngn3/Neurog3 promoter. In the adult, Sox9 expression persists in the cells along the major ducts and patchy IR is detected in the cytoplasm of betacells. Sox9 is expressed in undifferentiated cells but is absent from endocrine or differentiated endocrine and acinar cells (Seymour et al. 2007). Lineage tracing in mice demonstrates that Sox9 marks a pool of multipotent progenitors that give rise to both endocrine and exocrine cells (Seymour et al. 2008). In E18.5 the alpha- and beta-cell mass of haploinsufficient animals is reduced by 50–60 %. Mature Sox9 haploinsufficient animals display reduced beta-cell mass and glucose intolerance (Dubois et al. 2011). In zebrafish, sox9b is expressed in pancreatic ducts (Manfroid et al. 2012; Delous et al. 2012).

However, endocrine and acinar compartments appear unaffected in mutant embryos. Sox4 mutant mice display normal endocrine cell differentiation up to E12.5 but fail to form normal islets showing scattered endocrine cells (Wilson et al. 2005). An insulin secretory defect is observed in adult mice (Goldsworthy et al. 2008). In zebrafish, sox4b is strongly expressed in the pancreatic anlage, mostly restricted to precursors of the endocrine compartment but not maintained in differentiated cells (Mavropoulos et al. 2005). sox4b knockdown leads to drastic reduction of glucagon but not insulin expression. Ngn1/Neurog1 and Ngn2/Neurog2 expression is not detected in embryonic mouse pancreas (Gradwohl et al. 2000; Schwitzgebel et al. 2000), and in Mash1/Ascl1 null mutant mice, differentiation or distribution of cells expressing insulin, glucagon, somatostatin and pancreatic polypeptide appears normal (Schwitzgebel et al. 2000). Mice lacking functional Ngn3/Neurog3, however, fail to generate any pancreatic endocrine cells, and islets of Langerhans are missing (Gradwohl et al. 2000). Expression of NeuroD/NeuroD1 and Islet1 is lost. The absence of cell death as analyzed by TUNEL staining suggests that Ngn3/ Neurog3 specifies an endocrine fate. Conversely, overexpression of Ngn3/Neurog3 under control of the pancreatic and duodenal homeobox 1 (Pdx1) promoter in mouse pancreatic progenitors results in premature endocrine differentiation (Apelqvist et al. 1999; Schwitzgebel et al. 2000). It results in a marked increase in the number of glucagon-producing cells and depletion of precursors due to premature differentiation coupled with inhibition of proliferation (Schwitzgebel et al. 2000; Miyatsuka et al. 2011). Early and late transient expression in Xenopus laevis endoderm favors different endocrine cell fates (Oropez and Horb 2012). Cells positive for Ngn3/Neurog3 transcripts are detected by ISH as early as E9.5 in the pancreas, and subsequently are located within or adjacent to pancreatic ducts (Gradwohl et al. 2000). Coexpression with the islet differentiation factors NK6 homeobox 1 (Nkx6.1) and Nkx2.2 demonstrates the expression in immature cells of the islet lineage (Schwitzgebel et al. 2000). Positive cells do not coexpress glucagon or insulin and Ngn3/Neurog3 transcripts are not found in exocrine tissue. NeuroD1/NeuroD is expressed in endocrine cells of the pancreas and mutational inactivation in mice leads to a failure in the development of normal islets accompanied by a diabetic phenotype (Naya et al. 1997). Its expression is preceded by and requires Ngn3/Neurog3 (Gradwohl et al. 2000). NeuroD1/NeuroD is required for insulin gene transcription (see Chu et al. 2001; Fu et al. 2013 for reviews). In addition to NeuroD1/ NeuroD, NeuroD2 is expressed in the pancreas of mouse embryos at E15.5 (Gasa et al. 2008; but see Schwitzgebel et al. 2000 for NeuroD2). In NeuroD2 null mutant mice, differentiation and distribution of cells

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expressing insulin, glucagons, somatostatin and pancreatic polypeptide appears normal (Gasa et al. 2008). In zebrafish, neurog3/ngn3 is not expressed in the pancreas and null mutants display no apparent endocrine defects (Flasse et al. 2013). Instead, ascl1b is transiently detected in midtrunk endoderm and required for the first pancreatic endocrine precursors. In ascl1b/zash1b morphants, sox4b expression appears delayed and reduced. In contrast, neurod1/ neurod expression is correctly initiated. In ascl1b/zash1b morphants, the number of all types of pancreatic endocrine cells is reduced. neurod1/neurod is expressed after ascl1b/ zash1b and neurod1/neurod morphants display normal sox4b initiation but later loss. Differentiation of late-appearing endocrine cells is blocked in neurod1 morphants as shown by the almost complete depletion of glucagon (α) and grehlin (ɛ) cells. Simultaneous inactivation of both genes blocks initiation of endocrine differentiation and results in the loss of all hormone-producing cells. The sympathoadrenal lineage between neuronal and endocrine differentiation The neurons of the autonomic sympathetic ganglia and the endocrine cells of the adrenal medulla are derived from the same progenitors in the neural crest and thus originate from SoxB1-positive neuroectoderm. Upon neural crest cell generation in the dorsal neural tube, SoxE proteins become critically important. Sox9 is transiently expressed in premigratory trunk neural crest cells as shown in mice, chick, Xenopus and zebrafish (Cheung et al. 2005; McKeown et al. 2005; Spokony et al. 2002; Yan et al. 2005b). Migrating neural crest cells express Sox10 (Kuhlbrodt et al. 1998; Britsch et al. 2001; Cheung and Briscoe 2003). In Sox10lacZ/lacZ mice, adrenal chromaffin cells are absent, and no Sox10, Sox8, Phox2b or TH IR is detectable at E12.5 in adrenal anlagen (Reiprich et al. 2008). Similarly, in Sox10 mutant mice, prevertebral and caudal paravertebral sympathetic ganglia are lacking entirely (Kapur 1999; Britsch et al. 2001). The sympathetic neuronal deficiency is mild at the superior side where SCG are present but hypoplastic while lower thoracic and lumbar ganglia are completely absent. Also, at later stages, both cell types share a virtually identical set of transcription factors exerting overlapping but not identical functions during differentiation (for review, see Huber et al., this issue). The most prominent difference is observed in the time course of Mash1/Ascl1 expression which becomes rapidly downregulated in sympathetic neurons but is maintained in embryonic chromaffin cells (Huber et al. 2002). Different time courses are also observed for potential target genes. During initial differentiation, pan-neuronal markers such as neurofilament M transcripts are rapidly induced to high levels in neurons while expression in the adrenal endocrine lineage is moderate and transient in mice (Stubbusch

et al. 2013) or largely absent in chick (Ernsberger et al. 2005). Mice with mutational inactivation of the Ascl1/Mash1 gene show delayed but apparently normal differentiation of sympathetic neurons (Pattyn et al. 2006), while cells in the adrenal medulla retain neurofilament expression and lack the secretory granules typical for chromaffin endocrine cells (Huber et al. 2002). Taken together, there is an astonishing overlap in the Sox and bHLH transcription factor equipment of vertebrate neurons and endocrine cells operating with hormone release by vesicle fusion. Differences are, however, observed in the absence of Sox2 from the adrenal and pancreas anlagen in contrast to its strong expression in Rathke’s pouch. A remarkable swap in proneural bHLH protein usage is found in pancreatic endocrine development between mouse and zebrafish. Yet, the most striking difference among developmental regulators between neurons and endocrine cells currently concerns microRNAs. miR-375 is highly abundant in endocrine pancreas, pituitary and adrenal but not CNS and PNS tissue (Zhang et al. 2013; own unpublished observations). The regulatory embedment of this microRNA, already recognized for its importance in pancreatic endocrine development and function (for reviews, see Baroukh and Van Obberghen 2009; Li 2014), deserves further attention.

Endoderm-derived secretory cells in vertebrates Apart from pancreatic endocrine cells, a spectrum of other endocrine and in addition secretory cells originates from endoderm and during development share Sox and bHLH transcription factors with neuronal lineages. Yet, no neurons are known to be derived from endoderm in vertebrates. Distinct cell types characterize the epithelia of the mammalian respiratory and digestive tract with endocrine cells present in both systems. Three domains relevant to this discussion and analyzed in some detail are the trachea, the glandular stomach and the intestine. The tracheal lumen is lined by a pseudostratified epithelium composed of three major cell populations, basal, ciliated, and secretory Clara cells, as well as two minor ones, neuroendocrine cells and mucus-producing goblet cells. The gastric epithelium as single cell layer forming pit gland units encompasses three main secretory cell types, the mucus-secreting pit cells, pepsinogen-secreting zymogenic cells, and acid-secreting parietal cells, in addition to a variety of endocrine cells concentrated in the glandular stomach. The intestinal epithelium is a folded monolayer composed of several cell types including the resorptive enterocytes and the secretory goblet and Paneth cells as well as enteroendocrine cells as main populations. Transcript profiles in isolated intestinal stem cells as well as secretory and resorptive progenitors indicate that each cell

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population is distinct and the progenitors specified (Kim et al. 2014). Distinct Sox proteins are involved in rostral and caudal gut development Sox2 expression is detected in the anterior gut epithelium from the esophagus to the stomach of mouse (Que et al. 2007; Sherwood et al. 2009), chick (Ishii et al. 1998), Xenopus (Chalmers et al. 2000) and zebrafish (Muncan et al. 2007) embryos. The posterior gut epithelium develops devoid of Sox2 but displays expression of the homeobox transcription factor caudal type homeobox 2 (Cdx2) that has the potential for Atoh1/Math1 induction (Mutoh et al. 2006). Ectopic expression of Sox2 in the posterior region of the primitive gut causes anteriorization of the gut toward a gastric-like phenotype (Raghoebir et al. 2012). In humans, SOX2 haploinsuffciency is associated with anophthalmia-esophageal-genital (AEG) syndrome where esophagus and trachea may fail to separate and the trachea is connected to the stomach (Williamson et al. 2006). Heterzygous mice appear normal, but further reduction of Sox2 by combination of hypomorphic alleles can result in tracheo-esophageal malformations, ectopic mucus-producing cells in esophagus and forestomach, and esophageal expression of gene products normally detected in glandular stomach and intestine (Que et al. 2007). In the mouse embryo at E9.5, before foregut separation, both Sox2 protein and Sox2EGFP, where the SOX2 coding region is replaced to code for EGFP, are expressed at higher levels dorsally, in the prospective esophagus, than ventrally, in the prospective trachea (Que et al. 2007). EGFP expression from the Sox2 locus is found in all endoderm cells of the undivided foregut, but becomes downregulated in the posterior, glandular stomach and is absent from the glandular corpus and antrum at E18.5 (Que et al. 2007). At E11.5 and E13.5, after foregut separation, levels appear higher in the esophagus than the trachea (Que et al. 2009). Between E15.5 and P0, Sox2 is expressed in virtually all epithelial cells of the trachea. In the adult, high Sox2 expression levels are retained in basal cells. In the developing lung Sox2 is expressed in the epithelium of the proximal airways but is absent from the distal tips and primitive alveoli. Conditional Nkx2.5 promoter-driven deletion of Sox2 in the ventral epithelial domain of the early anterior mouse foregut results in significantly more mucus-producing cells compared to wild-type, and fewer basal stem cells, as well as greatly reduced ciliated and Clara cell numbers in the trachea (Que et al. 2009). Conditional deletion in Clara cells utilizing Scgb1a1-Cre results in loss of Sox2 in the bronchioles during perinatal and postnatal development (Tompkins et al. 2009). The rate of bronchiolar cell proliferation is decreased and

associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of ciliated, Clara, and goblet cells. In the mouse intestine, Sox9 protein is robustly expressed from the duodenum to the distal colon (Blache et al. 2004) and already detectable in the gut epithelium at E8.5 (Bagheri-Fam et al. 2006). Sox9 expression is observed in immature cells and colocalizes with Ki-67 (Blache et al. 2004). In the fetal intestine, crypts are not developed and the proliferative cells are found in the intervillus region, whereas in the adult intestine, proliferation is restricted to the lower half of the crypts of Lieberkühn. In addition to the proliferative compartment, Sox9 is detected in the Paneth cells at the bottom of the crypts of the adult small intestine. Sox9 expression differentially marks stem/progenitor populations and, different from pancreatic endocrine cells, enteroendocrine cells of the small intestine (Formeister et al. 2009; Furuyama et al. 2011). Crypt-based columnar cells with stem cell properties display low EGFP expression from the Sox9 locus whereas cells with high EGFP levels express chromogranin A and substance P but do not express Ki67 indicative of postmitotic enteroendocrine cells (Formeister et al. 2009). Inactivation of Sox9 in the mouse intestinal epithelium under control of villin-Cre results in a strong decrease in the goblet cell population and an almost complete loss of Paneth cells, but no obvious alteration in chromogranin A-positive enteroendocrine and Cdx2-positive enterocyte cell abundance (Bastide et al. 2007). Distinct proneural bHLH proteins are involved in rostral and caudal gut development In addition to different Sox proteins, distinct bHLH proteins are involved in the development of the rostral and caudal gut derivatives. Ascl1/Mash1 is required for pulmonary neuroendocrine cell formation (see Ball 2004 for review), as well as for endocrine cell differentiation in the mouse stomach (Kokubu et al. 2008). In the mouse intestine, however, Math1/Atoh1 regulates the specification and segregation of the intestinal secretory lineage comprising goblet and Paneth cells as well as enteroendocrine cells. Here, a Math1/Atoh1>Neurog3/Ngn3>NeuroD1/NeuroD cascade governs the generation of enteroendocrine cells (see Li et al. 2011 for review). Different from the mouse, in the zebrafish intestine, ascl1a/zash1a is crucial for differentiation of all secretory cell types including enteroendocrine cells (Flasse et al. 2013; Roach et al. 2013) that in mutant animals appear to differentiate into enterocytes. Ascl1/Mash1 expression in lung development is reported from human fetus and mouse embryo. In the normal fetal human lung, hASH1/ASCL1 and synaptophysin IHC labels solitary neuroendocrine cells as well as neuroepithelial bodies (NEBs) (Miki et al. 2012). Ascl1/Mash1 IHC in the mouse

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lung at E12.5 shows numerous, solitary positive cells in proximal airway structures and protein gene product 9.5 (PGP9.5) at low concentrations in all airway lining cells (Li and Linnoila 2012). By E14.5, Ascl1/Mash1 and PGP9.5 are detected in solitary pulmonary neuroendocrine cells as well as in NEBs, whereas the neuroendocrine marker calcitonin generelated peptide (CGRP) is still negative. By E17.5, many NEBs are positive for all three neuroendocrine markers, Ascl1/Mash1, PGP9.5, and CGRP. NeuroD/NeuroD1 expression, as analyzed by RT-PCR, is not detected in embryonic but in neonatal and adult mouse lung (Ito et al. 2000). Lineage tracing with Ascl1/Mash1-Cre, permanently labeling all lineage-derived cells, identifies a variety of cells in both airway and non-airway compartments including pulmonary neuroendocrine cells, Clara and ciliated cells, as well as smooth muscle cells (Li and Linnoila 2012). Neither neuroendocrine cells nor NEBs form in the lung of Ascl1/Mash1 mutant mice (Borges et al. 1997; Ito et al. 2000; Guha et al. 2012). CGRP-, chromogranin-, synaptophysin- or neuron-specific enolase signals are missing in lungs of newborn mutant animals (Borges et al. 1997). Yet, Clara cells and type II pneumocytes appear normal, and endocrine cells in gut and pancreas are positive for CGRP, chromogranin, and synaptophysin. In the stomach of the mouse embryo, Mash1/Ascl1 is highly expressed in the glandular epithelium (Kokubu et al. 2008). At E12.5, enteric neurons display high transcript levels while the gastric epithelium appears devoid of signal. Epithelial expression is observed at E14.5 and into adulthood albeit in fewer cells than during embryonal development. At E18.5, the chromogranin A-positive cells are negative for Mash1/Ascl1 transcripts that partially overlap with Ki67 signal. Ngn3/Neurog3 transcripts are found in some cells at E12.5 and increase in number to E14.5 and E16.5. Again, the number is strongly reduced in the adult stomach. NeuroD/NeuroD1 transcripts are not detected at E12.5 but at E14.5 in the glandular stomach. Mash1/Ascl1expressing cells appear to outnumber cells positive for Ngn3/Neurog3 and NeuroD/NeuroD1 transcripts and, in subsets of cells, Mash1/Ascl1 is coexpressed with Ngn3/ Neurog3 and NeuroD/NeuroD1. No Ngn1/Neurog1, Ngn2/ Neurog2 or Math1/Atoh1 expression is observed in the stomach (Kokubu et al. 2008). In Mash1/Ascl1 mutant mice, the stomach is smaller, yet the wall of the glandular stomach is thicker than in control animals (Kokubu et al. 2008). The number of chromogranin A-positive gastric endocrine cells is dramatically decreased and TuJ1-positive enteric neurons are reduced, while chief cell, parietal cell and pit cell formation appears normal. Smooth muscle also appears normal and no ectopic goblet cells form. All gastrin-, glucagon-, serotonin-, and somatostatin-immunoreactive cells are missing and also ghrelin transcript-positive cells are reduced. Importantly,

neither Ngn3/Neurog3 nor NeuroD/NeuroD1 expression appears reduced in Mash1/Ascl1 mutants (Kokubu et al. 2008). LacZ expressed from the Math1/Atoh1 locus is restricted within the gut to the intestinal epithelium, while no expression is detected in the stomach, pancreas, or lung (Yang et al. 2001). Expression commences at E16.5 and is sustained until adulthood. Colocalization with Ki-67 indicates that Math1/ Atoh1 is expressed in a common progenitor for the intestinal secretory cell types. Transgenic expression of Math1/Atoh1 under control of a villin promoter targets expression to intestinal epithelial cells and results in an almost complete transformation of the epithelium to goblet cells and a nearly complete loss of enterocytes in some animals (VanDussen and Samuelson 2010). Moreover, endocrine cell number as analyzed by chromogranin A IHC and RT-PCR signal for glucagon and CCK transcripts, as well as endocrine progenitors as shown by Neurog3/Ngn3 IHC and RT-PCR, are increased. Also, Paneth-like cells expand. Math1/Atoh1 deletion in mice results in the depletion of intestinal enteroendocrine, goblet and Paneth cells but not enterocytes (Yang et al. 2001). The pan-endocrine markers chromogranin A and synaptophysin, as well as the specific endocrine markers glucagon, gastrin, somatostatin, neurotensin, and serotonin are absent, as analyzed at E18.5. Electron microscopy shows no granular, goblet or enteroendocrine cells in any region of the mutant intestine. Transcripts for the Paneth cell marker cryptidin-1 are not detected by RT-PCR. Yet, enterocytes develop normally and no evidence for premature cell death is found. NeuroD/ NeuroD1 expression is lost completely. Depletion of Atoh1/ Math1 even from specified secretory progenitor cells converts them into enterocytes (Kim et al. 2014). Flag ChIP-seq in secretory progenitor cells isolated from Atoh1/Math1Flag mice identifies thousands of genomic binding sites, most of them in highly conserved, distant enhancers carrying Atoh1/Math1 consensus motifs, and different from Atoh1/Math1 binding sites found in cerebellar neurons (Kim et al. 2014). Less than 1000 of the close to 9000 binding sites detected in secretory progenitors are also detected in brain. Strong binding is observed at Math1/Atoh1, Dll4, Dll1 and Neurog3/Ngn3 genes. Expression of Neurog3/Ngn3 commences at E12.5 in the mouse gut endoderm and precedes NeuroD/NeuroD1 (Jenny et al. 2002). Neurog3/Ngn3 transcripts are found in dividing, BrdU-incorporating cells and do not colabel with chromogranin A. In the adult intestine, transcripts and protein are found exclusively in the proliferative domain, the crypts, but not in the villi. NeuroD/NeuroD1 expressing cells, however, are found mainly in the villi and coexpress chromogranin A. In addition to the intestine, Neurog3/Ngn3 expression is found in the glandular part of the stomach at embryonic as well as postnatal stages and in the adult (Jenny et al. 2002; Kokubu et al. 2008).

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In mice mutant for Neurog3/Ngn3, no intestinal endocrine cells are generated (Jenny et al. 2002). The principal intestinal hormones, cholecystokinin (CCK), secretin, gastrin, serotonin, peptide YY (PYY), glucagon-like protein (GLP), gastric inhibitory protein (GIP) and somatostatin are not produced in homozygous mutant intestine. While Math1/Atoh1 transcripts are present, NeuroD/NeuroD1 and also Pax6 expression is lacking. However, enterocytes, goblet and Paneth cells are present. In the gastric epithelilum, gastrin- and somatostatinexpressing G and D cell differentiation is impaired while serotonin- and ghrelin-producing endocrine cell types are still present in the mutants. This strongly agrees with a different origin of serotonin-expressing and enterochromaffin-like cells in the glandular stomach (Li et al. 2014). In the mouse intestine, NeuroD/NeuroD1 (Beta2) is coexpressed with chromogranin A (Rindi et al. 1999; Jenny et al. 2002). It is observed in secretin-positive cells (Mutoh et al. 1997), and NeuroD/NeuroD1 (Beta2) protein interacts with p300 to activate secretin transcription (Mutoh et al. 1997). In addition, peptide YY and glucagon-positive cells are labeled by lacZ expressed from the NeuroD/NeuroD1 (Beta2) locus (Rindi et al. 1999). NeuroD/NeuroD1 (Beta2) mutant mice fail to develop cells positive for secretin as well as CCK but not serotonin, PYY, GIP or glucagon expressing enteroendocrine cells (Naya et al. 1997). Taken together, this complex situation is important for a number of reasons: (1) endoderm-derived secretory and endocrine cells show an overlapping Sox and bHLH repertoire with neurons during development; (2) the Sox repertoire differs, however, from rostral to caudal sections in a manner conserved from mammals to fish; and (3) the proneural bHLH proteins in caudal gut development differ between fish and mammals. Of particular relevance is the situation in the glandular stomach where Sox2, Mash1/Ascl1, and NeuroD/NeuroD1, and also Neurog3/Ngn3 are expressed. Different from Mash1/ Ascl1-dependent neurons, expression of NeuroD/NeuroD1 does not require Mash1/Ascl1. Together with the large difference between Math1/Atoh1-bound sites in intestinal secretory progenitors and brain, this points at different chromatin accessability and/or cofactor availability in endoderm as compared to neurectoderm preceding proneural bHLH protein action. Interestingly, synaptic proteins used in neurons are also found in endoderm-derived secretory cells. No complete inventory of the expressed genes is available for the diverse intestinal cell types. Yet, syntaxin 1 and Snap-25 are detected in the surfactant-producing alveolar type II cells, albeit at much lower levels than syntaxin 2 and Snap-23 and than the levels found in brain (Abonyo et al. 2004). Moreover, Vamp-2 is expressed in these alveolar epithelial cells, again at much lower levels than Vamp-8 and the levels found in brain (Wang

et al. 2012). A similar Vamp-8 and Vamp-2 ratio is observed in airway goblet cells (Jones et al. 2012) that in addition utilize synaptotagmin II (Tuvim et al. 2009). The overlap in transcription factor and synaptic protein expression between endodermal secretory, endodermal and ectoderm-derived endocrine cells, as well as ectodermderived neurons, prompts the question for the regulators of developmental lineage diversion. The microRNAs miR-375 enriched in secretory and endocrine lineages as compared to miR-124 in neuronal lineages may be instrumental.

MicroRNAs: emerging posttranscriptional regulators with instructive potential to segregate neural and endocrine lineages? With the advent of microRNAs, an unexpected layer of developmental regulation surfaced from an RNA fraction once considered degradation waste from sub-optimal nucleic acid preparations. Their impact reformats basic ideas on neural and endocrine cell differentiation (for reviews, see Sun et al. 2013; Tang et al. 2013), tumor formation (for reviews, see Adams et al. 2014; Swartling et al. 2013), and reprogramming (see Moradi et al. 2014; Krishnakumar and Blelloch 2013; Leonardo et al. 2012 for reviews). Their strength is illustrated by the reprogramming of fibroblasts to neurons by miR-124 and miR-9 (Yoo et al. 2011) and on the other hand to pancreatic endocrine cells by Sox2 and miR-375 (Lahmy et al. 2014). miR-124 - access to core regulators of vertebrate neuronal differentiation miR-124 received particular attention for its abundant expression in all analyzed vertebrate and invertebrate nervous systems, its full sequence conservation from Caenorhabditis elegans to humans, and its interaction with important regulators of neuronal development such as the neuron-restrictive silencer factor NRSF/REST, the chromatin remodeler subunit BAF53a, the Notch ligand Jag1, and the polypyrimidine tract binding proteins PTBP1/PTBP2 (for reviews, see Abernathy and Yoo, this issue; Sun et al. 2013; Akerblom and Jakobsson 2013). The remarkable observation that miR-124 together with miR-9 has the potential to reprogram fibroblasts into neurons (Yoo et al. 2011; Tang et al. 2013) has put these two microRNAs into the focus of interest. In addition to the core regulators of initial steps in neuronal differentiation, miR-124 is involved in more advanced aspects of neuronal differentiation. miR-124 affects Rac1 (RASrelated C3 botulinum substrate 1) and Cdc42 (cell division cycle 42) expression and distribution (Yu et al. 2008; Franke et al. 2012), thereby regulating axonal and dendritic growth

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via the small GTPase RhoG (ras homolog gene family, member G) (see Schumacher and Franke 2013 for review). In mouse brain, miR-124 appears to account for 25–50 % of all brain miRNAs (Lagos-Quintana et al. 2002). miR-124/ miR-124a is encoded by three genes in mouse and human genome, each associated with an NRSE/RE1 site (Conaco et al. 2006; Wu and Xie 2006) and expressed in CNS and PNS of mouse embryos (Makeyev et al. 2007; Visvanathan et al. 2007). In the neuroepithelial layer of mouse and chick spinal cord, miR-124 is expressed at low levels but substantially upregulated in differentiating and mature neurons. Expression levels in the forebrain are low in the precursors of the ventricular zone (VZ) and substantially higher in the differentiated forebrain regions from subventricular zone (SVZ) to cortical plate (Makeyev et al. 2007; Maiorano and Mallamaci 2009). In the SVZ of the adult mammalian brain, miR-124 is expressed at low levels and becomes greatly upregulated in differentiated neurons (Cheng et al. 2009). Expression is not detected by ISH in early stages of the neural lineage, or the dividing doublecortin (DCX)-negative transitamplifying cells but in DCX-positive neuroblasts. A similar pattern is observed in the germinal zone of the developing forebrain. Retrovirally mediated overexpression of miR-124 in the SVZ of adult mice causes a substantial decrease in the proportion of dividing cells and an increase in the number of ß-III tubulin-positive neurons (Cheng et al. 2009). Conversely, 2’OMe-antisense miR-124 increases the number of dividing cells as well as the number of neurospheres generated from transit-amplifying cells and decreases the number of postmitotic neurons in vitro. Somewhat surprisingly, however, mutational inactivation in C. elegans (Miska et al. 2007) and D. melanogaster (Weng and Cohen 2012; Sun et al. 2012), which in contrast to mammals possess only one gene coding for miR-124, leaves homozygous mutants viable and without gross abnormalities. In Drosophila, a range of mutant phenotypes can be found, notably compromised of neuronal progenitor proliferation with a decreased number of Elav-positive cells per type I neuroblast-derived clone (Weng and Cohen 2012), and incomplete transition from neuroblast to neuronal gene expression signature (Sun et al. 2012), but no complete failure of neuron generation. miR-9: a critical link to vertebrate proneural gene function Similar to miR-124, miR-9 is initially identified as a brainenriched microRNA (see Coolen et al. 2013 for review). Expression, however, is predominantly associated with neural progenitors in vertebrates. Like miR-124, miR-9 targets NRSF/REST and BAF53a and, in addition, members of the Hes gene family. Consequently, miR-9 can suppress proliferation and promote neuronal differentiation, although the pan-

neuronal factor elavl3 may be directly inhibited (Coolen et al. 2012). A plausible interpretation is the favoring by miR-9 of an ‘ambivalent’ progenitor state allowing progenitor maintenance as well as neuronal commitment, possibly corresponding to ‘oscillating neural progenitors’ (Coolen et al. 2013; Kageyama et al. 2008). miR-9 is expressed in neural progenitors throughout most of the CNS in zebrafish, chick and mouse (Wienholds et al. 2005; Darnell et al. 2006; Kloosterman et al. 2006; Kapsimali et al. 2007). In the mouse and human genomes, three loci code for the mature miR-9s: miR-9-1, miR-9-2 and miR-9-3. Whole mount ISH in mice at E11.5 shows high miR-9 expression largely restricted to the CNS (Tan et al. 2012). At E12.5 and E14.5 in mice, the most abundantly expressed miR-9-2 is detected throughout the telencephalon, while miR-9-1 and miR-9-3 are mostly localized to the proliferative zone (Shibata et al. 2011). In the VZ, pri-miR-9-2 is increased in Hes1 mRNA-negative cells (Bonev et al. 2012). miR-9-1 and miR-9-2 expression, as observed from primiR-9-1 and pri-miR-9-2 distribution, occurs in regions with oscillatory Hes1 expression but is lacking in areas of high and sustained Hes1 levels (Tan et al. 2012). Conversely, in neurons where miR-9 levels are high, Hes1 is downregulated (Shibata et al. 2011; Bonev et al. 2011). Mutational inactivation of miR-9-2 and miR-9-3 decreases mature miR-9 expression by >75 % and leads to marked reduction in cortical layers and ventricular zone (Shibata et al. 2011). Cajal-Retzius cells and early ß-III tubulinpositive neurons are reduced. BrdU incorporation and phosphorylated histone H3 (pH3) labeling is increased in VZ and SVZ. Overexpression in the dorsal telencephalon at E13.5 induces enhanced migration out of the ventricular zone, cell cycle exit as indicated by a reduction of Ki67 and pH3positive cells, and reduced Hes1 expression levels (Tan et al. 2012). Knockdown decreases the generation of TuJ1-positive neurons and stabilizes Hes1 expression levels. In c17.2 neural progenitor and NIH3T3 cells, overexpression of miR-9 decreases Hes1-mRNA lifetime and downregulation has the opposite effect (Bonev et al. 2012; Tan et al. 2012). A miR-9 binding site is found in the 3’-UTR of mammalian Hes1 and zebrafish her1 mRNAs (Bonev et al. 2011). In a luciferase reporter, this site confers miR-9 sensitivity (Tan et al. 2012). The binding of Hes1 to the putative miR-9-1 and miR-9-2 promoters and their repression establishes a double-negative feedback loop (Bonev et al. 2012). The stability of mature miR-9 compared to its primary transcripts allows accumulation of the mature microRNA over time. In zebrafish, miR-9 expression starts at 20–24 hpf and consistently spares the midbrain–hindbrain boundary (MHB) (Leucht et al. 2008). Along the mediodorsal axis, miR-9 is prominent in the ventricular zone adjacent to the HuC-positive

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domain with few cells expressing both. miR-9 overexpression promotes neurogenesis. Fewer mitotically active cells are observed, neurogenin 1 expression is enhanced, and the number of elav/huc homolog (HuC)-positive cells is increased. Conversely, miR-9 blockade reduces the HuC-positive area. Direct targeting of the transcripts of hairy-related genes her5 or her9 that regulate neurogenin expression appears to contribute to the miR-9-induced enhanced neurogenesis. In contrast to vertebrates, miR-9a in Drosophila is expressed in epithelia but not the ventral ectoderm and the central nervous system (see Yuva-Aydemir et al. 2011 for review). Four miR-9 family members are characterized, among which miR-9a is broadly expressed in neuroectodermal territory in the early proliferative phase and subsequently lost in the differentiating neuronal lineage (Cohen et al. 2006; Li et al. 2006). It reduces the expression of proneural genes such as senseless in cells with non-neural fate, and thus indirectly affects generation of neurons. The situation is complicated by the other related miR-9 family members such as miR-4 and miR-79. Thus, the cause of neurodevelopmental defects in C. elegans deficient for miR79 by dysregulation of epidermal cell metabolism (Pedersen et al. 2013) may support the conclusion that this microRNA family has changed its regulatory context between animal phyla. In the basal chordate amphioxus, miR-9 and miR-124 are expressed in the nervous system (Candiani et al. 2011). While miR-124 is already detected in neurulae, miR-9, different from vertebrates, is not found in embryos but appears later in larvae in scattered cells throughout the neural tube. In addition, miR7 expression is observed in the neurula stage and appears restricted to ventral cerebral regions and a part of the pharyngeal endoderm from which Hatschek’s pit develops, a putative homolog to the vertebrate adenohypophysis. microRNAs in sensory receptor cell development miR-124 is not only expressed in neuronal but also sensory lineages. In mice, miR-124 is detected in the cochlea (ElkanMiller et al. 2011) and retina (Karali et al. 2010; Sanuki et al. 2011). In the retina, the precursor pre-miR-124a-l is strongly observed in the presumptive photoreceptor layer at E17.5 when cone photoreceptors are generated (Sanuki et al. 2011). The pre-miR-124a-2 host gene, retinal non-coding RNA 3 (Rncr3), is, however, barely detectable and pre-miR124a-3 is absent in the photoreceptor precursor layer at E17.5, suggesting pri-miR124a-1 as the predominant source of miR124 at this stage. Yet, Rncr3 and miR-124 signals are strongly detected in ganglion cells and differentiating neurons at E13.5, and those signals increase until mice are 1 month old. The abundant miR-124 and Rncr3 signals in photoreceptor cells accumulate in the inner segment from postnatal day 1 to adulthood.

In Rncr3 mutant mice at 2 months of age, the cone cell number is reduced while rod photoreceptor and other retinal cell types are unaffected (Sanuki et al. 2011). At E17.5, however, cone cell numbers are unaltered. Neurod1 and Ngn2/Neurog2 expression is unaffected, indicating a postnatal role of miR-124a-2 and prompting the question for the prenatal role of miR-124a-1. In the adult mouse retina, the miR-183/96/182 cluster is specifically expressed in all photoreceptors and the inner nuclear layer (Xu et al. 2007; Karali et al. 2007; Lumayag et al. 2013). Expression is not detected at embryonic stages but commences at P2. It is not restricted to retina but detected in all sensory epithelia such as inner ear, olfactory and tongue epithelia as well as DRG. A null mutation results in the absence of alternatively spliced transcripts and all three mature microRNAs from retina, inner ear, olfactory and tongue epithelia as well as DRG (Lumayag et al. 2013). In the postnatal retina, progressive degeneration and ribbon synapse defects result in ERG disturbances. Postnatal photoreceptor development is compromised and at the age of 1 year M opsin is largely lost. Depletion of microRNAs from postnatal cone photoreceptors by conditional Drosha/Dgcr8 inactivation results in loss of cone opsins and outer segments that can be rescued by miR183 and miR-182 but not miR-124 (Busskamp et al. 2014). Moreover, overexpression of the miR-183/96/182 cluster in ES cell-derived cultures induces inner segment outgrowth and outer segment differentiation, as well as rhodopsin expression and light responses. In mouse inner ear, miR-183/96/182 expression is already detected at embryonic stages and found in ganglia as well as sensory epithelia (Friedman et al. 2009; Weston et al. 2011). Point mutations in the seed region of human miR-96 are associated with progressive non-syndromic hearing loss (Mencía et al. 2009; Lewis et al. 2009). In the mouse mutant diminuendo that has a single base change in the seed region of miR-96, hair cell development is arrested around the day of birth (Kuhn et al. 2011). They lack calcium-activated and negatively acting potassium currents, and overall potassium as well as calcium current development appears to stall around birth. Also, synaptic morphology remains immature. Also, in zebrafish, miR-183/96/182 family microRNAs promote otocyst, sensory patch and hair cell formation (Li et al. 2010). microRNAs in endocrine and secretory cell development Microarray profiling of microRNAs in adult mouse CNS displays strong miR-124 signals in all brain regions except the pituitary gland (Bak et al. 2008). Instead, miR-7 is highly detected in pituitary but not the different brain regions, with the exception of the hypothalamus with its neurosecretory cell populations. In addition, miR-375 is found at high levels in the

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mouse pituitary but not in brain (Zhang et al. 2013). miR-7 is, together with miR-375, also found in pancreas (for review, see Dumortier and Van Obberghen 2012). miR-7 and miR-375 are detected during mouse as well as human pancreatic development (Joglekar et al. 2009). Quantitative RT-PCR analysis in rat pancreas shows miR-375 as the most abundant microRNA in mature islets and miR-7 as the most enriched in endocrine islets as compared to exocrine acinar cells (Bravo-Egana et al. 2008). Data on miR-124 in mouse pancreas differ, however. Cloning and sequencing of microRNAs purified from E14.5 mouse pancreas revealed abundant miR-7 and miR-375 but not miR-124 (Lynn et al. 2007b). Microarray analysis and RTPCR at the same stage provided miR-124 signal (Baroukh et al. 2007). miR-7 knockdown in mice affects beta-cell differentiation but results differ (Nieto et al. 2012; Kredo-Russo et al. 2012). Mutational inactivation of miR-375 generates hyperglycemic mice with reduced beta-cell and increased alpha-cell number (Poy et al. 2009). In addition to endocrine cell development, insulin secretion from mature beta-cells is directly affected by miR-375 mutation (Poy et al. 2004). In zebrafish, morpholinomediated knockdown of miR-375 causes defects in the morphology of the pancreatic islets (Kloosterman et al. 2007). Importantly, in human ES cells and induced pluripotent stem cells, lentivirus-mediated overexpression of miR-375 induces endocrine markers found in pancreatic islets, in particular insulin (Lahmy et al. 2013, 2014). Apart from the pancreas, mature miR-7, as quantified by qRT-PCR, is enriched in human and mouse brain relative to other organs, while the primary transcript shows much less tissue specificity (Choudhury et al. 2013). Tissue-specific restriction of maturation is mediated by nonneural HuR/ Elavl1 (ELAV (embryonic lethal, abnormal vision)-like 1 (Hu antigen R)) and Musashi homolog 2. In the forebrain of zebrafish and medaka, miR-7 displays a highly restricted medial expression (Tessmar-Raible et al. 2007) in neurosecretory cells also found in the forebrain of the annelid Platynereis dumerilii. Despite its role in endocrine tissue, miR-375 is also detected in nervous tissue during development. While high miR-375 levels are detected by RT-PCR in E13 mouse telecephalon, the lowest levels among a sample of tissues are observed in adult brain as compared to skeletal muscle or heart (Abdelmohsen et al. 2010). Lentivirus-mediated overexpression in the mouse hippocampus reduces dendrite abundance accompanied by a reduction of HuD and RhoA levels. In addition to endocrine and developing nervous tissue, miR-375 is detected in secretory airway and intestinal epithelia. Mice deficient for miR-375 have normal intestinal goblet cell numbers, yet altered gene expression indicates a differentiation deficit (Biton et al. 2011). miR-375 is also detected in lung and affects alveolar epithelial cell trans-differentiation

and surfactant secretion (Wang et al. 2007, 2013b; Zhang et al. 2010). Similar to transcription factors, a large overlap exists in microRNAs used by neurons, endocrine and sensory receptor cells, and their progenitors. To what extent the regulatory networks involving miR-124, miR-183/96/182 and miR-375 will specify the segregation of neuronal, sensory, endocrine and secretory lineages will be an important upcoming question.

Conclusions and perspective During vertebrate neurogenesis, sequential expression of transcriptional regulators, most notably Sox and bHLH proteins, but also diverse groups of homeodomain and zinc finger proteins, goes hand-in-hand with successive neuronal specification and activation of specific gene expression programs. In particular, genes coding for neuron-specific cytoskeletal, synaptic and ion channel proteins are consecutively activated, and parts of both the target gene and the transcription factor programs are conserved among neuron populations, qualifying for a ‘generic’ neuronal differentiation program. In particular, the presence of SoxB1 proteins throughout the vertebrate neuroectoderm and their binding to a wide range of genes specifically expressed in neurons makes them prime candidates for key drivers of neural stem cell and progenitor specification during the first step in this program. The role of SoxC proteins during consecutive differentiation steps, which also bind to proneural as well as terminal neuronal differentiation genes, is less clearly established. The precocious upregulation of generic neuronal differentiation markers after overexpression points to a role in the progression from progenitor to neuroblast and neuron expressing ‘pan-neuronal’ cytoskeletal proteins. Apoptosis in mutant mice, however, impedes a straightforward interpretation. The expression of pan-neuronal cytoskeletal proteins, such as ß-III tubulin or Map2, follows withdrawal from proliferation in many but not all embryonic neuronal lineages. In postnatal and adult neuroblasts, ß-III tubulin expression may typically commence during the mitotically active phase. Even though SoxB1 proteins are a hallmark of embryonic and adult progenitors, their role in the regulation of cell cycling in neural progenitors is not fully disclosed. Inactivation as well as overexpression of Sox2 may compromise mitotic activity. Also, for bHLH proteins, the role in proliferation control appears more elaborate than initially anticipated. Both genes coding for proteins promoting cell cycle exit as well as cell cycle progession can be activated by proneural bHLH protein binding. The dynamic changes in bHLH and Hes proteins during progenitor cell cycle, Cyclin- and cdk-dependent phosphorylation of proneural bHLH proteins, and distinct phosphorylation state-

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dependent bHLH protein binding to promoters of different genes provide major progress to solve this issue. Less well defined is the regulation of pan-neuronal markers by the consecutively expressed bHLH proteins. Even though overexpression of proneural bHLH proteins effectively induces cell cycle withdrawal in progenitors combined with ßIII tubulin expression followed by Map2, qualitatively similar to unperturbed in vivo differentiation, the occupation by bHLH proteins of the regulatory regions driving gene expression for pan-neuronal cytoskeletal proteins is largely unexplored. Yet, for SoxB1 and SoxC proteins, binding at the ß-III tubulin gene is described and found to be associated with histone modification marks of transcriptional repression or activation, respectively. These observations suggest an early progenitor phase (1) of the ‘generic’ neuronal differentiation program in vertebrates characterized by SoxB1 expression, cell proliferation, the presence of nestin and absence of ß-III tubulin signal. In a subsequent phase (2), SoxB1 and nestin transcripts and protein are downregulated while SoxC expression commences, cells exit the mitotic cycle, and become positive for ß-III tubulin signal. Proneural bHLH gene expression is induced during this transition in a neuron population-specific manner, and the bHLH protein signals can overlap with SoxB1 as well as SoxC protein signals. The coupling of terminal cell cycle exit with neuronal cytoskeletal protein expression is not obligatory yet observed frequently in embryonic neuronal differentiation. With ongoing development, the appearance of gene transcripts for synaptic proteins subsequent to the expression of multiple pan-neuronal cytoskeletal proteins marks the acquisition of another set of gene products crucial for the neuronal phenotype. Conservation of this pattern across diverse mammalian and avian neuron populations indicates that this maturation step (3) also constitutes part of the ‘generic’ neuronal differentiation program. While SoxC protein binding is associated with histone modification marks of transcriptional activation at some of these genes, and Dicer 1 mutation indicates a role for microRNAs in this process, the molecular mechanisms of this transition are ill-defined. The situation for neuronally expressed ion channels appears even less clear, as knowledge on conserved induction patterns in vivo is lacking. Thus, the precise coupling of transcription factors and target gene expression is incompletely understood and requires more refined analysis of target gene enhancers and promoters, including their binding factor occupancy and modification during development. The recognition of different layers of regulation, from chromatin modification to transcription initiation, from transcriptional elongation to splicing, and from posttranscriptional to posttranslational processes, crucially refines our understanding of cell type differentiation. The emergent role of regulatory RNAs, in particular, currently, microRNAs, offers further conserved mechanisms involved in the transition during neuronal differentiation from

proliferating progenitors to postmitotic neurons expressing neuronal cytoskeletal proteins, synaptic proteins and neuronspecific ion channels. Importantly, a range of proteins central to neuronal function, in particular synaptic proteins and ion channels, are also detected and functionally relevant in related cell types, namely sensory receptor and endocrine cells. Even pan-neuronal cytoskeletal proteins can be expressed, as observed transiently during endocrine cell development. In line with these findings, developmental regulators also appear largely shared, as shown for Sox and bHLH proteins but also observed with other protein families. In addition, microRNA patterns overlap but differences such as the miR-124/miR-375 dichotomy between neurons and endocrine cells as well as miR-183/96/182 for sensory specification deserve further attention. Currently, the regulator networks described for vertebrate neurons, sensory receptor and endocrine cells provide enormous insight into cell type differentiation but appear insufficient to explain the diversification of the three communicator cell types. Moreover, Sox and bHLH expression display similarities with secretory cell differentiation from endoderm. The analysis of epigenetic mechanisms as well as transcriptional regulation at the genomic loci critically involved in neurite formation can be expected to provide crucial information on the core aspect of the ‘generic’ neuronal differentiation program segregating the neuronal branch from other secretory cell types. To what extent this applies to invertebrates is still far from resolved. The involvement of SoxB proteins in invertebrate neurogenesis is incompletely analyzed. Yet, the central role of Dichaete and SoxNeuro in the generation of the vast majority of Drosophila neurons and the role of Sox2 in transdifferentiation in C. elegans emphasize the similar function of SoxB proteins in vertebrates and invertebrates. Also, proneural bHLH protein function is shared between insects and vertebrates yet questioned for ctenophores. The conservation of the role of microRNAs in invertebrate neurogenesis is only partially established. Thus, conserved aspects of generic neuronal differentiation are becoming establishd for vertebrate neurons. They include the succession of Sox and bHLH function reported from mammals to fish, and the sequential activation of genes coding for cytoskeletal and synaptic proteins observed in mammals and birds. The conservation of the critical regulatory events among invertebrate neurons remains to be established. Acknowledgments Special thanks go to Klaus Unsicker for his encouragement and support with this Special Issue. Magdalena Götz, Simone Reiprich and Michael Wegner kindly commented the manuscript. Particular thanks go to Hermann Rohrer for an inspiring environment and steady support. The work of UE was supported by the Deutsche Forschungsgemeinschaft (grant RO 469/12-1) and the Gemeinnützige Hertie-Stiftung (grant 1.01.1/07/009). Ute Tönchen-Wagner provided invaluable aid in face of my fragile health status and made the work possible.

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