Journal of Neuroendocrinology, 2015, 27, 544–555 © 2015 British Society for Neuroendocrinology

REVIEW ARTICLE

Protocadherins and Hypothalamic Development: Do They Play An Unappreciated Role? G. M. Coughlin*† and D. M. Kurrasch*† *Department of Medical Genetics, University of Calgary, Calgary, AB, Canada. †Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB, Canada.

Journal of Neuroendocrinology

Correspondence to: D. M. Kurrasch, Cumming School of Medicine, Alberta Children’s Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Room HS2275, Calgary, AB T2N 4N1, Canada (e-mail: kurrasch@ucalgary. ca).

Normal brain development requires coordinated cell movements at precise times. It has long been established that cell–cell adhesion proteins of the cadherin superfamily are involved in the adhesion and sorting of cells during tissue morphogenesis. In the present review, we focus on protocadherins, which form the largest subfamily of the cadherin superfamily and mediate homophilic cell–cell adhesion in the developing brain. These molecules are highly expressed during neural development and the exact roles that they play are still emerging. Although, historically, protocadherins were considered to provide mechanical and chemical connections between adjacent cells, recent research suggests that they may also serve as molecular identity markers of neurones to help guide cell recognition and sorting, cell migration, outgrowth of neuronal processes, and synapse formation. This phenomenon of single cell diversity stems, in part, from the vast variation in protein structure, genomic organisation and molecular function of the protocadherins. Although expression profiles and genetic manipulations have provided evidence for the role of protocadherins in the developing brain, we have only begun to construct a complete understanding of protocadherin function. We examine our current understanding of how protocadherins influence brain development and discuss the possible roles for this large superfamily within the hypothalamus. We conclude that further research into these underappreciated but vitally important genes will shed insight into hypothalamic development and perhaps the underlying aetiology of neuroendocrine disorders. Key words: cadherins, protocadherins, cell–cell adhesion, neural development, hypothalamus

Introduction The development of the central nervous system (CNS) is an extraordinary process requiring dynamic cell movements, axonal growth, and synapse formation and maintenance. Each of these processes relies, in part, on specific interactions with neighbouring cells for accurate placement and stability. Within the hypothalamus, neurones are clustered into nuclei (instead of layers, such as in the cortex), whereby specialised cell types become subcompartmentalised into distinct domains within a particular nucleus (1). Hypothalamic development is broadly divided into four main events: (i) regionalisation of the hypothalamic prosencephalic territory [before embryonic day (e)10.5]; (ii) hypothalamic cell fate specification and differentiation (e10.5– 15.5); (iii) neuronal migration (e10.5–15.5); and (iv) coalescing of specialised hypothalamic neurones into subdomains within mature nuclei (e16.5–e18.5). Briefly, the hypothalamus derives from

doi: 10.1111/jne.12280

progenitors that line the ventricular zone along the third ventricle of the ventral prosencephalon, adjacent to the presumptive retina (2). By e9.5, just prior to the onset of hypothalamic neurogenesis, the presumptive hypothalamus and retina have acquired distinct regional identities as a result of the the combinatorial actions of extracellular signals (e.g. morphogens) and intrinsic factors (e.g. homeodomain transcription factors) and progenitor cells are awaiting instruction to exit the cell cycle and become differentiated neurones (3–10). Based on marker expression (11) and birthdating in both rats (12,13) and primates (14), hypothalamic neurogenesis follows general lateral-tomedial (i.e. outside-in) and dorsal-to-ventral (i.e. top-down) gradients. In the hypothalamus, an ‘outside-in’ differentiation pattern has been observed, with peak neuronal differentiation occurring at e13.5 (range: e10.5–e15.5) (15,16). In the mature nucleus, hypothalamic cell types localise to discrete regions of the proper nuclei in a strikingly precise fashion. Indeed, topographical errors (i.e. mis-positioned

Protocadherins and hypothalamic development

cells) are rare. This precision raises the question of whether cell-sorting mechanisms play an unrecognised role in placing these neurones into their correct subdomain. The paraventricular nucleus (PVN) provides a good example of this suborganisation, with magnocellular neurosecretory neurones residing in the posterior magnocellular (PVNpm) zone and parvocellular neurosecretory neurones clustered in the dorsomedial parvocellular, dorsal parvocellular, and ventromedial parvocellular zones, PVNmpd, PVNdp, and PVNmpv, respectively (Fig. 1). Proper sorting of these distinct neuronal phenotypes as they differentiate and migrate away from the shared ventricular zone might be, at least in part, the unappreciated work of protocadherins. We postulate

(A)

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that mechanisms that generate highly specific cell–cell interactions are vastly important for hypothalamic development, and that dysfunction in such processes will likely to lead to abnormal hypothalamic organisation and possibly disease. In Drosophila, specificity in neural development is partially achieved through the Down syndrome cell adhesion molecule 1 (Dscam1) system (17). Interestingly, alternative splicing of Dscam1 can generate more than 38 000 isoforms (the entire Drosophila genome only has 15 016 genes) and these isoforms go on to become cell adhesion molecules that preferentially interact with identical isoforms on apposing cells (17). This alternative splicing of Dscam1 leads to a unique expression of Dscam proteins on the sur-

During development (~E12.5): Waves of newly born neurones migrate away from the VZ VZ MZ

3V

(B)

In the mature nucleus (~P0): Phenotypically similar cells cluster into nuclear subdomains Magnocellular oxytocin Dorsal parvocellular 3V (PVNpm) (PVNdp)

Medial parvocellular (PVNmpd)

Magnocellular vasopressin (PVNpm)

Ventral parvocellular (PVNmpv)

Fig. 1. Cartoon representation of a potential role for protocadherins in cell sorting between subcompartments within a hypothalamic nucleus. (A) During development, newly-born neurones migrate away from progenitors that line the third ventricle. Although little is understood about migration of these differentiated neurones, it is speculated that the neuronal phenotypes are relatively mixed during this migratory process. However, the timing of neuronal birth might keep ‘like’ neurones migrating in close proximity and it is possible that protocadherins play a cell adhesion role during migration. (B) Cartoon representation of the subcompartments of the paraventricular nucleus. The first zoomed view illustrates the borders that separate cells into a distinct compartments. In the mature nucleus, neurones with similar phenotypes have become clustered into subdomains within the broader nucleus, although how this happens remains unknown. The second zoomed view demonstrates our hypothesis that neurones with similar identities might be sorted based on the expression of particular subsets of protocadherins. Distinct protocadherins are represented by different colors. 3V, third ventricle; VZ, ventricular zone; MZ, mantle zone; PVN, paraventricular nucleus; PVHmpd, dorsomedial parvocellular; PVNdp, dorsal parvocellular; PVNmpv, ventromedial parvocellular; PVNpm, posterior magnocellular. The cartoon representation of the paraventricular nucleus is adapted from The Autonomic Nervous System and The Hypothalamus, Susan Iversen, Leslie Iversen, and Clifford B Saper. Journal of Neuroendocrinology, 2015, 27, 544–555

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face of every neurone in the fly and serves to facilitate contactdependent, self-avoidance mechanisms required for normal circuitry establishment. Such self-avoidance mechanisms prevent crossing of outgrowing processes, thereby increasing the area that a process can access and preventing redundant connections from forming (17–19). The impressive size and complexity of this system in this relatively simple organism illustrates the demand for finely tuned cell–cell interactions for proper development. Surprisingly, vertebrates developed their own evolutionarily distinct process and, instead, rely on protocadherins as key molecules in the generation of such specific cell–cell interactions (20). Although these molecules are structurally distinct from Dscam1, they are considered to be functional analogues of the Dscam1 system (19,21). Protocadherins are members of the cadherin superfamily of cell adhesion molecules that display diverse spatiotemporal expression patterns in the developing brain (20,22,23), including within the hypothalamus (24–34). Moreover, this large subfamily demonstrates a diverse range of functions during neural development, which may be mediated though cell–cell adhesion mechanisms and/or via activation of intracellular signalling cascades (20,35). Complexity of the protocadherin family, however, has restricted the ability to conduct the functional studies required to shed insight into the precise roles for protocadherins in CNS development and, as such, a relatively narrow body of research into protocadherin function exists. Nonetheless, these underappreciated proteins likely serve vital roles in the development of the nervous system and, in the present review, we examine our understanding of the protocadherin superfamily, and attempt to determine how these complex molecules might help establish the exquisite suborganisation and connectivity of hypothalamic nuclei where appropriate.

Protocadherin diversity and function The cadherin superfamily Cadherins are transmembrane glycoproteins that act as homophilic calcium-dependent cell–cell adhesion molecules. In the presence of calcium, a cadherin protein may interact with an identical cadherin on an apposing membrane to mediate adhesion between cells. Cadherin proteins are characterised by multiple repeating extracellular cadherin (EC) domains of approximately 110 amino acids in length that bind three Ca2+ ions and rigidify the molecule to promote cell– cell adhesion (36,37). It is assumed that cis-dimers of classical cadherins form within a particular cell, followed by homophilic transinteraction with cadherins of the apposed membrane. Although trans-interactions may occur between monomers as well (38), the formation of cis-dimers is considered to strengthen this interaction (39,40). Subsequent to their discovery over 30 years ago (41), more than 100 cadherin family members have been described in mammals alone (42,43). Furthermore, cadherin and cadherin-like proteins are found in a broad range of animal species, both uni- and multicellular (42,44,45). A number of criteria can be used to classify the cadherin superfamily into two broad families: the cadherins and the cadherinrelated molecules (42,46). The former grouping contains the © 2015 British Society for Neuroendocrinology

prototypical members of the cadherin superfamily, whereas the latter grouping contains the protocadherins, amongst other cadherinrelated molecules (42,46). Protocadherins constitute the largest subdivision of the cadherin superfamily, with more than 70 members, and show structural similarity to classical cadherins within the EC domains. Indeed, protocadherins were discovered via a degenerate polymerase chain reaction using primers based on the ECs of classical cadherins (47). As a result of the presence of these cell adhesion molecules in both vertebrates and invertebrates, the name ‘protocadherin’ was suggested for these molecules. However, subsequent genomic analysis of lower metazoans suggests that the protocadherin family diverged greatly in the chordate lineage (45,48). Despite shared similarities within EC domains that are characteristic of cadherin superfamily members, protocadherins otherwise differ significantly from classical cadherins. Namely, protocadherins typically contain six or seven EC domains, in contrast to the five EC domains found in classical cadherins, and there is low sequence homology of the EC domains in protocadherins versus classical cadherins (20). Furthermore, protocadherins lack intracellular catenin-binding domains, which are responsible for mediating classical cadherin-directed actin cytoskeleton rearrangement (20). Finally, protocadherins display unique differences from one another, in terms of both genomic organisation and cytoplasmic domain structure.

Genomic organisation of protocadherins Vertebrate protocadherins can be divided into two subgroups based on their genomic organisation: clustered and nonclustered protocadherins. Specifically, in mammals, clustered protocadherins are comprised of three separate gene clusters, designated Pcdha, Pcdhb and Pcdhc, which collectively encode more than 50 protocadherin proteins (20,49,50). In the a- and c-protocadherin clusters, the extracellular and transmembrane domains are encoded by large variable exons unique to each member of the cluster, whereas part of the cytoplasmic domain is encoded by three constant exons that are shared between the members (20,50). Conversely, no such common cytoplasmic domain is present in the Pcdhb cluster; rather, each member is a single-exon gene that has a unique cytoplasmicdomain encoding sequence, with a high degree of homology to one another (51,52). As their name suggests, nonclustered protocadherins do not show the same genomic organisation as clustered protocadherins and are further divided into three subgroups based on the presence or absence of conserved amino acid motifs. The d-protocadherin family contains these repeats, and is further divided into two groups (d1 and d2) based on the presence of a third conserved motif. Furthermore, whereas the d1 subfamily members contain seven EC domains, the d2 subfamily members contain six EC domains (49). By contrast, Pcdh20 and Pcdh12 lack these conserved motifs, and thus are not classified in either of these subfamilies (42,46).

Adhesive properties of protocadherins Early transfection studies of classical cadherins demonstrated that these molecules are involved in homophilic adhesion between cadhJournal of Neuroendocrinology, 2015, 27, 544–555

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erin proteins on apposing cell membranes, probably through the formation of trans-interactions that are mediated by the most distal EC domain (i.e. EC1) (53). Selective adhesion is observed between cells transfected with the same type of cadherin (54–56). Similarly, in cultures of cells transfected with multiple cadherins, those expressing a given cadherin show selective adhesion to cells expressing the same cadherin subtype, at the same time as segregating from cells expressing different cadherins (57–60). When applied to different tissues, cells expressing a given cadherin associate preferentially with tissue also expressing that same cadherin (59). This affinity for homophilic adhesion does not appear to be absolute; for example, heterophilic adhesion may occur between cells expressing R-cadherin and those expressing N-cadherin (61,62), or between different combinations of other cadherins (63). However, these cadherins often show greater affinity for cadherins of the same type than for their heterophilic binding partners (62,63). Taken together, these results illustrate a mechanism by which cadherins can regulate cell sorting and morphogenesis during development. The notion that cadherins are involved in early morphogenic processes is well supported. During development, cadherin genes show both distinct spatial and temporal expression patterns. N-cadherin expression starts during gastrulation, and is maintained in neural and muscle tissues (44). This protein is especially important for the invagination of the neural tube (64), and mutation of CDH2 (encoding N-cadherin) in both mice (65) and zebrafish (66) results in aberrant neural tube development. Additionally, conditional disruption of CDH2 in the mouse cortex leads to disorganisation of the cortical layers (67). Conversely, R-cadherin shows expression in forebrain and bone (44) and is especially important in the formation of the laminar structure of the retina, as well as outgrowth of retinotectal projections (68). Interestingly, N-cadherin is also involved in laminar development of the retina (69,70), although it is unclear whether heterophilic adhesion plays any role in this. As with classical cadherins, transfection studies performed using protocadherins have demonstrated that these proteins also mediate homophilic adhesion (27,47,71–75). However, this adhesive function appears to be weak relative to classical cadherins. This difference in adhesive character may be a result of variations in the extracellular and/or cytoplasmic domains of protocadherins from those of cadherins. Evidence for the latter comes from the observation that adhesion strength is improved when the cytoplasmic domain of cprotocadherin C3 is replaced with that of E-cadherin (72). The intracellular domain of cadherins is necessary for interaction with catenins, and thereby with the cytoskeleton (76). Furthermore, examination of the extracellular domain of protocadherins has revealed the lack of a hydrophobic pocket that is required for homophilic adhesion in classical cadherins (77). This difference may result in the observed differences in homophilic adhesion affinity (49). Whether or not this weak binding affinity reflects the binding affinity in vivo is uncertain, especially considering that processing by metalloproteases can enhance the homophilic binding activity of c-protocadherin C3 (73). In addition to differences in structure, protocadherins may also differ from classical cadherins in terms of their cellular adhesion Journal of Neuroendocrinology, 2015, 27, 544–555

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behaviour. Although evidence of trans-heterophilic interaction between cadherins exists, trans-interaction between c-protocadherins appears to be strictly homophilic in nature (78). However, similar to cadherins, heterophilic cis-interaction between protocadherins may also occur (79,80). Previous investigations suggest that c-protocadherins may mediate cell adhesion through the formation of cis-tetramers, either homophilic or heterophilic (78). These tetramers may then engage in homophilic interactions with tetramers of the same composition (78). These cis-interactions appear to be promiscuous and, thus, given the 22 c-protocadherins, over 230 000 possible tetramers are possible. Cis-interaction between members of the different clustered protocadherin subfamilies would vastly increase the number of possible tetramers (78,79). However, this model is speculative, and has recently been challenged by the finding that cells expressing the same set of clustered protocadherins, regardless of either one or five protocadherins being expressed, show cell aggregation, and also that one mismatched protein can interfere with this aggregation (81). Furthermore, if assembly of such cis-tetramers is promiscuous and stochastic, then many of these tetramers will be of the wrong composition required for interaction, and therefore, the correct tetramer will be present at a greatly reduced copy number (81). Regardless, these models suggest that protocadherin diversity may allow for highly specific interactions to occur. Thus, given the high expression of protocadherins within nuclei of the developing hypothalamus (34,82), it is tempting to speculate that the wealth of protocadherin combinations influences the sorting of hypothalamic neurones into nuclei and their subcompartments.

Signalling by protocadherins The observation that the intracellular domains of protocadherins diverge greatly between subfamilies suggests variation in protocadherin-mediated intracellular signalling. Indeed, activation of intracellular signalling cascades has been demonstrated in both clustered and nonclustered protocadherins. Clustered protocadherins from the a- and b-protocadherin subfamilies have been shown to form heteromeric complexes with important signalling proteins, including receptor tyrosine kinases (RTKs) and phosphatases (83). In particular, the RTK RET is present in such complexes in differentiated neuroblastomas, and can both stabilise and phosphorylate protocadherins. Glial cell line-derived neurotrophic factor may serve as a signal to trigger this phosphorylation (83). Furthermore, both aand c-protocadherins have been shown to interact with and negatively regulate the tyrosine kinases Pyk2 and FAK (84). This interaction may be important for regulating dendrite arborisation and spine morphology via a Rho-GTPase-dependent mechanism (85,86). Proteomic analysis has uncovered a number of other clustered protocadherin intracellular binding partners, including kinases, cytoskeletal proteins and other cell adhesion molecules (80). Signalling mediated by nonclustered protocadherins has also been observed. For example, a protein phosphatase 1a-interacting domain is conserved in d1-protocadherin members but not in d2 (22,23). As with clustered protocadherins, this pathway has been implicated in dendritic spine morphology. Furthermore, Pcdh8, a © 2015 British Society for Neuroendocrinology

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erin proteins on apposing cell membranes, probably through the formation of trans-interactions that are mediated by the most distal EC domain (i.e. EC1) (53). Selective adhesion is observed between cells transfected with the same type of cadherin (54–56). Similarly, in cultures of cells transfected with multiple cadherins, those expressing a given cadherin show selective adhesion to cells expressing the same cadherin subtype, at the same time as segregating from cells expressing different cadherins (57–60). When applied to different tissues, cells expressing a given cadherin associate preferentially with tissue also expressing that same cadherin (59). This affinity for homophilic adhesion does not appear to be absolute; for example, heterophilic adhesion may occur between cells expressing R-cadherin and those expressing N-cadherin (61,62), or between different combinations of other cadherins (63). However, these cadherins often show greater affinity for cadherins of the same type than for their heterophilic binding partners (62,63). Taken together, these results illustrate a mechanism by which cadherins can regulate cell sorting and morphogenesis during development. The notion that cadherins are involved in early morphogenic processes is well supported. During development, cadherin genes show both distinct spatial and temporal expression patterns. N-cadherin expression starts during gastrulation, and is maintained in neural and muscle tissues (44). This protein is especially important for the invagination of the neural tube (64), and mutation of CDH2 (encoding N-cadherin) in both mice (65) and zebrafish (66) results in aberrant neural tube development. Additionally, conditional disruption of CDH2 in the mouse cortex leads to disorganisation of the cortical layers (67). Conversely, R-cadherin shows expression in forebrain and bone (44) and is especially important in the formation of the laminar structure of the retina, as well as outgrowth of retinotectal projections (68). Interestingly, N-cadherin is also involved in laminar development of the retina (69,70), although it is unclear whether heterophilic adhesion plays any role in this. As with classical cadherins, transfection studies performed using protocadherins have demonstrated that these proteins also mediate homophilic adhesion (27,47,71–75). However, this adhesive function appears to be weak relative to classical cadherins. This difference in adhesive character may be a result of variations in the extracellular and/or cytoplasmic domains of protocadherins from those of cadherins. Evidence for the latter comes from the observation that adhesion strength is improved when the cytoplasmic domain of cprotocadherin C3 is replaced with that of E-cadherin (72). The intracellular domain of cadherins is necessary for interaction with catenins, and thereby with the cytoskeleton (76). Furthermore, examination of the extracellular domain of protocadherins has revealed the lack of a hydrophobic pocket that is required for homophilic adhesion in classical cadherins (77). This difference may result in the observed differences in homophilic adhesion affinity (49). Whether or not this weak binding affinity reflects the binding affinity in vivo is uncertain, especially considering that processing by metalloproteases can enhance the homophilic binding activity of c-protocadherin C3 (73). In addition to differences in structure, protocadherins may also differ from classical cadherins in terms of their cellular adhesion Journal of Neuroendocrinology, 2015, 27, 544–555

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behaviour. Although evidence of trans-heterophilic interaction between cadherins exists, trans-interaction between c-protocadherins appears to be strictly homophilic in nature (78). However, similar to cadherins, heterophilic cis-interaction between protocadherins may also occur (79,80). Previous investigations suggest that c-protocadherins may mediate cell adhesion through the formation of cis-tetramers, either homophilic or heterophilic (78). These tetramers may then engage in homophilic interactions with tetramers of the same composition (78). These cis-interactions appear to be promiscuous and, thus, given the 22 c-protocadherins, over 230 000 possible tetramers are possible. Cis-interaction between members of the different clustered protocadherin subfamilies would vastly increase the number of possible tetramers (78,79). However, this model is speculative, and has recently been challenged by the finding that cells expressing the same set of clustered protocadherins, regardless of either one or five protocadherins being expressed, show cell aggregation, and also that one mismatched protein can interfere with this aggregation (81). Furthermore, if assembly of such cis-tetramers is promiscuous and stochastic, then many of these tetramers will be of the wrong composition required for interaction, and therefore, the correct tetramer will be present at a greatly reduced copy number (81). Regardless, these models suggest that protocadherin diversity may allow for highly specific interactions to occur. Thus, given the high expression of protocadherins within nuclei of the developing hypothalamus (34,82), it is tempting to speculate that the wealth of protocadherin combinations influences the sorting of hypothalamic neurones into nuclei and their subcompartments.

Signalling by protocadherins The observation that the intracellular domains of protocadherins diverge greatly between subfamilies suggests variation in protocadherin-mediated intracellular signalling. Indeed, activation of intracellular signalling cascades has been demonstrated in both clustered and nonclustered protocadherins. Clustered protocadherins from the a- and b-protocadherin subfamilies have been shown to form heteromeric complexes with important signalling proteins, including receptor tyrosine kinases (RTKs) and phosphatases (83). In particular, the RTK RET is present in such complexes in differentiated neuroblastomas, and can both stabilise and phosphorylate protocadherins. Glial cell line-derived neurotrophic factor may serve as a signal to trigger this phosphorylation (83). Furthermore, both aand c-protocadherins have been shown to interact with and negatively regulate the tyrosine kinases Pyk2 and FAK (84). This interaction may be important for regulating dendrite arborisation and spine morphology via a Rho-GTPase-dependent mechanism (85,86). Proteomic analysis has uncovered a number of other clustered protocadherin intracellular binding partners, including kinases, cytoskeletal proteins and other cell adhesion molecules (80). Signalling mediated by nonclustered protocadherins has also been observed. For example, a protein phosphatase 1a-interacting domain is conserved in d1-protocadherin members but not in d2 (22,23). As with clustered protocadherins, this pathway has been implicated in dendritic spine morphology. Furthermore, Pcdh8, a © 2015 British Society for Neuroendocrinology

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nate isoforms (118). Because c-protocadherins are involved in circuit assembly, this result implies that the loss in synaptic density is partly a result of cell death (118). Deletion of the entire c-protocadherin cluster also leads to a loss of dendritic self-avoidance (21). During dendrite outgrowth, neurites from the same neurone repel each other, leading to a dendritic field in which branches from the same neurone do not cross over one another (17,18). Such a process requires dendrites to be able to recognise self from nonself, and thus necessitates a system for generating single-cell diversity. Mouse starburst amacrine cells lacking c-protocadherins fail to undergo dendrite self-avoidance, as demonstrated by the formation of dendrite bundles (21). Interestingly, in Drosophila, this phenomenon is mediated by Dscam1, which, similar to clustered protocadherins, has the capacity to produce a vast number of isoforms through alternative splicing and demonstrate homophilic adhesion properties (17). In both of these systems, homophilic interaction may lead to an intracellular signal that mediates repulsion (17,21). In addition to functioning in synapse formation and dendrite outgrowth, c-protocadherins have also been implicated in the function of the neuroendocrine system. Clustered protocadherins show high expression in the hypothalamus, and thus may be fundamental in the development of its specific neuronal circuitry (119). Conditional knockout mice lacking c-protocadherins in rat insulin promoter (RIP)-expressing neurones or pro-opiomelanocortin (POMC)-expressing neurones show hyperphagy and obesity (119). Furthermore, mice deficient in RIP-specific neurone c-protocadherins showed increased apoptosis in the hypothalamus, despite no changes in the gross morphology of the hypothalamus. Because these two cell populations do not overlap, this effect may be mediated by cell–cell interaction, which is supported by the observation that POMC neurone-specific c-protocadherin knockout animals show reductions in excitatory synapse density (119). Thus, c-protocadherins also appear to regulate neuronal survival and circuit assembly in the hypothalamus. Functional studies of a-protocadherins have also been conducted using animals expressing truncated a-protocadherins. Unlike mice lacking the c-protocadherin cluster, mice deficient in a functional a-protocadherin cluster are viable and fertile. Instead, these animals have revealed a role for a-protocadherins in neurite outgrowth. Loss of a-protocadherins results in a failure of olfactory sensory neurone axons to coalesce into olfactory bulb glomeruli, which manifests as multiple small glomeruli, rather than fewer, larger glomeruli (120). This deficit persists into adulthood (120). Further support for the role of a-protocadherins in axonal development is illustrated by the finding that Pcdha hypomorphic mutant mice show changes in serotonergic projections, with some brain areas showing increased axon terminal density, and others showing decreased density (121). Changes in the behaviour of such animals can also be observed because decreased a-protocadherin protein levels leads to increased fear conditioning response, as well as spatial memory deficits (122). However, these results are confounded by the fact that production of small amounts (albeit very low) of truncated protein (120) may lead to the observed phenotypes. A true loss-of-function strain is needed to differentiate these possibilJournal of Neuroendocrinology, 2015, 27, 544–555

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ities. Interestingly, many single nucleotide polymorphisms of the aprotocadherin cluster have been associated with autism (123). By contrast to the a- and c-protocadherin clusters, little data exists regarding the function of the b-protocadherin cluster, although what is available suggests that b-protocadherins show similar broad expression profiles as were observed for a- and cprotocadherins. For example, expression of Pcdhb16 is widely detected throughout the brain, and especially in the neocortex, hippocampus, olfactory bulb and cerebellum, and expression is localised to synapses (104,105). Predictably, this expression is restricted to a subset of cerebellar neurones, which are distinct from those expressing Pcdhb22. Given the similarity between the expression of b-protocadherins and that of the other clustered protocadherins, it is likely that combinatorial expression of b-protocadherins could also contribute to single neurone diversity, and thus to processes such as synapse formation and neurite self-avoidance (105). Indeed, recent research has demonstrated combinatorial expression of bprotocadherins in Purkinje cells (104), although functional studies of b-protocadherins are necessary to fully clarify the role of this co-expression.

The role of nonclustered protocadherins By contrast to the broad expression patterns of clustered protocadherins (103), nonclustered protocadherins show spatially restricted expression in the brain (23,34) (Table 1). This latter organisation mirrors that of classical cadherins (35,95,124). Within the brain, nonclustered protocadherins show expression in both specific nuclei and laminae. For example, Pcdh10 shows high expression in thalamic nuclei, as well as the olfactory cortex (24,27). Furthermore, Pcdh10 expression is temporally regulated, whereby its expression in the thalamus decreases between postnatal and adult stages (27). Similarly, Pcdh10 is highly expressed in the developing rat suprachiasmatic nucleus, along with Pcdh9 and Pcdh19, indicating an unappreciated role for these gene in the development of this hypothalamic nucleus (22). Finally, Vanhalst et al. (34) and Kim et al. (29) have demonstrated unique expression profiles for nonclustered protocadherins in the mature and developing ventromedial hypothalamic nucleus, respectively, providing further evidence that this subfamily of proteins is probably involved in development of the hypothalamus. The hypothalamic expression patterns of nonclustered protocadherins are summarised in Table 1 for a variety of model organisms. Further studies have also revealed distinct and overlapping expression patterns for many d-protocadherins. In the developing basal ganglia, classical cadherins and nonclustered protocadherins show partial overlap between their expression patterns (90). Similarly, similar patterns of partial overlap between classical cadherins and nonclustered protocadherins have been observed in the developing mouse, ferret and chick cerebella (93,97), as well as in the developing ferret retina (125). Perhaps the most striking example of this co-expression is the presence of classical cadherins and d-protocadherin transcripts in the developing ferret primary visual cortex (92). At each stage of development, the distinct cortical layers can be differentiated based on the particular complement of cadherins © 2015 British Society for Neuroendocrinology

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Table 1. Summary of Hypothalamic Expression Pattern for Nonclustered Protocadherins in Different Model Organisms.

P, postnatal day; POA, preoptic area; SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; AH, anterior hypothalamic nucleus; DMH, dorsomedial nucleus of the hypothalamus; VMH, ventromedial nucleus of the hypothalamus; ARC, arcuate nucleus of the hypothalamus; MB, mammillary bodies; LH, lateral hypothalamus; PH, posterior hypothalamus; Hyp: hypothalamic expression but spatial detail is lacking. Level of expression is indicated as assessed by the original studies’ authors: +++ strong; ++ moderate; + weak; negative; ~ expression indicated by not qualified.

expressed. Such results give rise to the notion that these cadherins/ protocadherins might generate unique adhesive surfaces, which may be involved in compartmentalisation and/or formation of specific neuronal circuitry in the brain (90,92). Because the ventromedial hypothalamus also co-expresses a combination of cadherins and nonclustered protocadherins in restricted patterns within the nuclei (29,34), and given that hypothalamic nuclei in general are exquisitely subcompartmentalised with sharp boarders (Fig. 1), we speculate that cadherins and protocadherins may work in concert to drive cell sorting of individual hypothalamic neurones into distinct compartments during coalescing of the nucleus. © 2015 British Society for Neuroendocrinology

Similar to their clustered counterparts, nonclustered protocadherins may also be involved in the generation of single neurone diversity (91). Neurones in the somatosensory cortex combinatorially express classical cadherins and nonclustered protocadherins, and also express more than one of these cadherins, thus allowing for differentiation of neurones in the cortex. A number of expression patterns can be observed, from complete overlap in expression to no overlap at all (91). If true, these findings greatly expand upon the molecular diversity that is afforded by the clustered protocadherins alone. However, it is not yet known whether this strategy is utilised by other parts of the cerebral cortex, or even in other brain Journal of Neuroendocrinology, 2015, 27, 544–555

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structures. Similarly, the mechanism by which cells might generate combinatorial expression of classical cadherins and nonclustered protocadherins also remains unknown. Given the disparity in brain structures that express nonclustered protocadherins, it should not be surprising that genetic manipulation of these genes leads to a variety of distinct phenotypes. Genetic elimination of Pcdh10 leads to dysfunctional growth of thalamocortical and corticofugal projections through the ventral telencephalon (126,127). This appears to be the result of a failure in the growth of striatal axons, which typically show expression of Pcdh10. These axons have been proposed to function as guides for projections crossing the ventral telencephalon (126,127). Furthermore, a role in Xenopus axonal initiation and elongation has been demonstrated for NF-protocadherin (NFPC), which is homologous to Pcdh7 (128). This d-protocadherin shows expression in developing retinal cells and axons, and these NFPC-null animals display dysfunctional neurite initiation and outgrowth in retinal ganglion cells. For example, such cells may lack an axon, possess a shortened axon or lack all processes. Similar phenotypes result from template-activating factor 1 (TAF1)-null animals, indicating that TAF1 acts downstream of NFPC, and suggesting that this protocadherin mediates axonal initiation and elongation through intracellular signalling (128). An important role for intracellular signalling in protocadherin function has also been demonstrated for Pcdh17 (129). Knockout of this gene in mice leads to disrupted axon elongation of amygdala neurones in vivo, and deficient axonal fasciculation (i.e. growth of an axon along another axon) in vitro. Interaction of the PCDH17 intracellular domain with actin regulating molecules suggests a role for this in protein in cell motility (129). Interestingly, Pcdh17 has also been linked to synapse formation (28).

Possible roles for protocadherins in CNS development Despite a wealth of expression data, and a handful of functional studies, a clear generalised role for protocadherins in neural development has yet not been obtained and perhaps it is not feasible to expect a generalised role for such a complex protein family. Indeed, a given protocadherin may be expressed in multiple brain areas, sometimes in combination with other cadherins, at different times during development. Similarly, the precise function of that protocadherin may differ both spatially and temporally. As a result of this complexity, fully clarifying the function of the protocadherin family will require great effort. However, certain roles for protocadherins in CNS development can be postulated. Given their spatially and temporally distinct expression profiles, as well as homophilic binding properties, protocadherins may be involved in cell sorting during neuronal migration. Roles in cell sorting have been suggested for both classical cadherins and protocadherins (44). Classical cadherins are expressed in a tissue-specific manner and have been shown to mediate cell sorting in vitro as a result of differential cell adhesion (58). In vivo studies also support this notion: motor pools of the developing spinal cord can be defined by specific expression of classical cadherins, and expression of null Cdh20 leads to an intermixing between these motor neurone pools (130). Similarly, Xenopus paraxial and axial protocadherin, Journal of Neuroendocrinology, 2015, 27, 544–555

551

which are tentative homologues of Pcdh8 and Pcdh1, respectively, are necessary for separating cells into paraxial and axial mesoderm (131), although paraxial protocadherin appears to do so through modulation of C-cadherin (132). A review of the role of classical cadherins in brain morphogenesis is provided elsewhere (98). Thus, it is possible that protocadherins also play a role in cell sorting during brain development. This function has already been postulated for classical cadherins in the development of brain nuclei (98,99) and can perhaps be extended to protocadherins. If this is the case, then migrating neurones should show expression of a given subset of cadherins, and should coalesce into defined structures based on those cadherins (98, 99). Protocadherins may provide greater specificity in this process, given the diversity of the subfamily and their potential to mediate specific interactions, alone or in combination with other protocadherins. The vast number of protocadherin interactions could not only specify the development of given hypothalamic nuclei, but also subdomains within those nuclei. The well-defined and restricted spatial expression of nonclustered protocadherins may indicate a key role for these proteins in establishing borders throughout the brain. In addition to defining brain compartments, the specificity afforded by protocadherins may also allow for the formation of precise neuronal connectivity. Protocadherins have been shown to be involved in the processes of dendritic self-avoidance and axon fasciculation. Furthermore, cells expressing a given set of protocadherins may form synapses with cells also expressing those protocadherins. Again, such a role has already been postulated for cadherins (98,99) and is supported by the findings that many cadherin proteins at least partially localise to synapses and that some neuronal networks emerge from structures expressing the same cadherin (Fig. 2). Synaptic localisation of some protocadherins, including clustered protocadherins, has already been demonstrated, although no direct evidence supports a synaptic role as of yet. Thus, given the broad but stochastic expression of clustered protocadherins, and the high connectivity of the cortex, these genes may be instrumental in formation of cortical neuronal networks. Future research is needed to clarify whether protocadherins also guide hypothalamic circuitry. Furthermore, the unique combination of protocadherins involved in the two processes of compartmentalisation and neural network formation may not be mutually exclusive. Indeed, because these two events are temporally distinct, protocadherins involved in cellular migration and sorting may later be recruited in the process of circuit assembly. Alternatively, the specific subset of cadherins expressed in a particular cell may change across development. Such a change in expression is observed in cadherins during morphogenic events, such as neurulation, or during epithelial–mesenchymal transitions, and allows the cell to change its adhesive properties. It is possible that the mechanisms through which cell sorting and circuit assembly occur may require protocadherin-mediated cell–cell adhesion and/or intracellular signalling. Again, these two processes are not distinct from one another. Indeed, as described above, homophilic interaction between apposed Pcdh8 molecules can then trigger intracellular signalling. Such uncertainties regarding the precise mode of action necessitate both spatially- and temporally-detailed © 2015 British Society for Neuroendocrinology

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expression analyses of protocadherins and, even more importantly, functional studies. Finally, understanding the mechanisms underlying neuronal migration and sorting, and the formation of specific neuronal connectivity has profound implications for developmental neurobiology. Although much is known about the development of layered structures of the brain, the development of nuclear structures remains unclear (133). Indeed, understanding protocadherin biology in the developing hypothalamus may serve as an inroad to deducing the cell sorting mechanisms that must be employed to so elegantly cluster similar neuronal phenotypes within an apparently unorganised cluster of neurones. Furthermore, given the wealth of studies showing that the developing hypothalamus is particularly sensitive to environmental stressors (e.g. maternal obesity, stress, environmental contaminants), it will be fascinating to examine whether these cell-sorting mechanisms are sensitive to these external challenges and responsible for translating the environment into perturbations in neuronal organisation and/or circuitry. The impetus for such research is high, especially considering recent findings demonstrating a high occurrence of rare variants for 55 protocadherin genes in obese individuals (134).

Conclusions Protocadherins are highly expressed in the developing brain, and thus may mediate a number of developmental processes through both homophilic adhesion and intracellular signalling. Both clustered and nonclustered protocadherins appear to play a role in this process, albeit the functional differences between the two groups are currently unclear. Based on their expression patterns, as well as limited functional data, we speculate that protocadherins may be especially important in the formation and connectivity of specific brain structures, including hypothalamic nuclei. Thus, protocadherins may represent a vital but underappreciated molecular family in developmental neurobiology and, in particular, developmental neuroendocrinology. Received 11 January 2015, revised 26 March 2015, accepted 27 March 2015

References 1 Pearson CA, Placzek M. Development of the medial hypothalamus: forming a functional hypothalamic-neurohypophyseal interface. Curr Top Dev Biol 2013; 106: 49–88. 2 Rubenstein JL, Shimamura K, Martinez S, Puelles L. Regionalization of the prosencephalic neural plate. Annu Rev Neurosci 1998; 21: 445–477. 3 Hashimoto-Torii K, Motoyama J, Hui CC, Kuroiwa A, Nakafuku M, Shimamura K. Differential activities of Sonic hedgehog mediated by Gli transcription factors define distinct neuronal subtypes in the dorsal thalamus. Mech Dev 2003; 120: 1097–1111. 4 Hirata T, Nakazawa M, Muraoka O, Nakayama R, Suda Y, Hibi M. Zincfinger genes Fez and Fez-like function in the establishment of diencephalon subdivisions. Development 2006; 133: 3993–4004.

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5 Kiecker C, Lumsden A. Hedgehog signaling from the ZLI regulates diencephalic regional identity. Nat Neurosci 2004; 7: 1242–1249. 6 Manning L, Ohyama K, Saeger B, Hatano O, Wilson SA, Logan M, Placzek M. Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation. Dev Cell 2006; 11: 873–885. 7 Ohyama K, Das R, Placzek M. Temporal progression of hypothalamic patterning by a dual action of BMP. Development 2008; 135: 3325– 3331. 8 Scholpp S, Wolf O, Brand M, Lumsden A. Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon. Development 2006; 133: 855–864. 9 Szabo NE, Zhao T, Cankaya M, Theil T, Zhou X, Alvarez-Bolado G. Role of neuroepithelial Sonic hedgehog in hypothalamic patterning. J Neurosci 2009; 29: 6989–7002. 10 Vieira C, Garda AL, Shimamura K, Martinez S. Thalamic development induced by Shh in the chick embryo. Dev Biol 2005; 284: 351–363. 11 Caqueret A, Boucher F, Michaud JL. Laminar organization of the early developing anterior hypothalamus. Dev Biol 2006; 298: 95–106. 12 Altman J, Bayer SA. The development of the rat hypothalamus. Adv Anat Embryol Cell Biol 1986; 100: 1–178. 13 Ifft JD. An autoradiographic study of the time of final division of neurons in rat hypothalamic nuclei. J Comp Neurol 1972; 144: 193–204. 14 van Eerdenburg FJ, Rakic P. Early neurogenesis in the anterior hypothalamus of the rhesus monkey. Brain Res Dev Brain Res 1994; 79: 290–296. 15 Ikeda Y, Takeda Y, Shikayama T, Mukai T, Hisano S, Morohashi KI. Comparative localization of Dax-1 and Ad4BP/SF-1 during development of the hypothalamic-pituitary-gonadal axis suggests their closely related and distinct functions. Dev Dyn 2001; 220: 363–376. 16 Pelling M, Anthwal N, McNay D, Gradwohl G, Leiter AB, Guillemot F, Ang S-L. Differential requirements for neurogenin 3 in the development of POMC and NPY neurons in the hypothalamus. Dev Biol 2011; 349: 406–416. 17 Matthews BJ, Kim ME, Flanagan JJ, Hattori D, Clemens JC, Zipursky SL, Grueber WB. Dendrite self-avoidance is controlled by Dscam. Cell 2007; 129: 593–604. 18 Grueber WB, Sagasti A. Self-avoidance and tiling: mechanisms of dendrite and axon spacing. Cold Spring Harb Perspect Biol 2010; 2: a001750. 19 Zipursky SL, Grueber WB. The molecular basis of self-avoidance. Annu Rev Neurosci 2013; 36: 547–568. 20 Chen WV, Maniatis T. Clustered protocadherins. Development 2013; 140: 3297–3302. 21 Lefebvre JL, Kostadinov D, Chen WV, Maniatis T, Sanes JR. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 2012; 488: 517–521. 22 Kim S-Y, Yasuda S, Tanaka H, Yamagata K, Kim H. Non-clustered protocadherin. Cell Adh Migr 2011; 5: 97–105. 23 Redies C, Vanhalst K, van Roy F. d-protocadherins: unique structures and functions. Cell Mol Life Sci 2005; 62: 2840–2852. 24 Aoki E, Kimura R, Suzuki ST, Hirano S. Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain. Neuroscience 2003; 117: 593–614. 25 Asahina H, Masuba A, Hirano S, Yuri K. Distribution of protocadherin 9 protein in the developing mouse nervous system. Neuroscience 2012; 225: 88–104. 26 Blevins CJ, Emond MR, Biswas S, Jontes JD. Differential expression, alternative splicing, and adhesive properties of the zebrafish delta1protocadherins. Neuroscience 2011; 199: 523–534. 27 Hirano S, Yan Q, Suzuki ST. Expression of a novel protocadherin, OLprotocadherin, in a subset of functional systems of the developing mouse brain. J Neurosci 1999; 19: 995–1005.

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structures. Similarly, the mechanism by which cells might generate combinatorial expression of classical cadherins and nonclustered protocadherins also remains unknown. Given the disparity in brain structures that express nonclustered protocadherins, it should not be surprising that genetic manipulation of these genes leads to a variety of distinct phenotypes. Genetic elimination of Pcdh10 leads to dysfunctional growth of thalamocortical and corticofugal projections through the ventral telencephalon (126,127). This appears to be the result of a failure in the growth of striatal axons, which typically show expression of Pcdh10. These axons have been proposed to function as guides for projections crossing the ventral telencephalon (126,127). Furthermore, a role in Xenopus axonal initiation and elongation has been demonstrated for NF-protocadherin (NFPC), which is homologous to Pcdh7 (128). This d-protocadherin shows expression in developing retinal cells and axons, and these NFPC-null animals display dysfunctional neurite initiation and outgrowth in retinal ganglion cells. For example, such cells may lack an axon, possess a shortened axon or lack all processes. Similar phenotypes result from template-activating factor 1 (TAF1)-null animals, indicating that TAF1 acts downstream of NFPC, and suggesting that this protocadherin mediates axonal initiation and elongation through intracellular signalling (128). An important role for intracellular signalling in protocadherin function has also been demonstrated for Pcdh17 (129). Knockout of this gene in mice leads to disrupted axon elongation of amygdala neurones in vivo, and deficient axonal fasciculation (i.e. growth of an axon along another axon) in vitro. Interaction of the PCDH17 intracellular domain with actin regulating molecules suggests a role for this in protein in cell motility (129). Interestingly, Pcdh17 has also been linked to synapse formation (28).

Possible roles for protocadherins in CNS development Despite a wealth of expression data, and a handful of functional studies, a clear generalised role for protocadherins in neural development has yet not been obtained and perhaps it is not feasible to expect a generalised role for such a complex protein family. Indeed, a given protocadherin may be expressed in multiple brain areas, sometimes in combination with other cadherins, at different times during development. Similarly, the precise function of that protocadherin may differ both spatially and temporally. As a result of this complexity, fully clarifying the function of the protocadherin family will require great effort. However, certain roles for protocadherins in CNS development can be postulated. Given their spatially and temporally distinct expression profiles, as well as homophilic binding properties, protocadherins may be involved in cell sorting during neuronal migration. Roles in cell sorting have been suggested for both classical cadherins and protocadherins (44). Classical cadherins are expressed in a tissue-specific manner and have been shown to mediate cell sorting in vitro as a result of differential cell adhesion (58). In vivo studies also support this notion: motor pools of the developing spinal cord can be defined by specific expression of classical cadherins, and expression of null Cdh20 leads to an intermixing between these motor neurone pools (130). Similarly, Xenopus paraxial and axial protocadherin, Journal of Neuroendocrinology, 2015, 27, 544–555

551

which are tentative homologues of Pcdh8 and Pcdh1, respectively, are necessary for separating cells into paraxial and axial mesoderm (131), although paraxial protocadherin appears to do so through modulation of C-cadherin (132). A review of the role of classical cadherins in brain morphogenesis is provided elsewhere (98). Thus, it is possible that protocadherins also play a role in cell sorting during brain development. This function has already been postulated for classical cadherins in the development of brain nuclei (98,99) and can perhaps be extended to protocadherins. If this is the case, then migrating neurones should show expression of a given subset of cadherins, and should coalesce into defined structures based on those cadherins (98, 99). Protocadherins may provide greater specificity in this process, given the diversity of the subfamily and their potential to mediate specific interactions, alone or in combination with other protocadherins. The vast number of protocadherin interactions could not only specify the development of given hypothalamic nuclei, but also subdomains within those nuclei. The well-defined and restricted spatial expression of nonclustered protocadherins may indicate a key role for these proteins in establishing borders throughout the brain. In addition to defining brain compartments, the specificity afforded by protocadherins may also allow for the formation of precise neuronal connectivity. Protocadherins have been shown to be involved in the processes of dendritic self-avoidance and axon fasciculation. Furthermore, cells expressing a given set of protocadherins may form synapses with cells also expressing those protocadherins. Again, such a role has already been postulated for cadherins (98,99) and is supported by the findings that many cadherin proteins at least partially localise to synapses and that some neuronal networks emerge from structures expressing the same cadherin (Fig. 2). Synaptic localisation of some protocadherins, including clustered protocadherins, has already been demonstrated, although no direct evidence supports a synaptic role as of yet. Thus, given the broad but stochastic expression of clustered protocadherins, and the high connectivity of the cortex, these genes may be instrumental in formation of cortical neuronal networks. Future research is needed to clarify whether protocadherins also guide hypothalamic circuitry. Furthermore, the unique combination of protocadherins involved in the two processes of compartmentalisation and neural network formation may not be mutually exclusive. Indeed, because these two events are temporally distinct, protocadherins involved in cellular migration and sorting may later be recruited in the process of circuit assembly. Alternatively, the specific subset of cadherins expressed in a particular cell may change across development. Such a change in expression is observed in cadherins during morphogenic events, such as neurulation, or during epithelial–mesenchymal transitions, and allows the cell to change its adhesive properties. It is possible that the mechanisms through which cell sorting and circuit assembly occur may require protocadherin-mediated cell–cell adhesion and/or intracellular signalling. Again, these two processes are not distinct from one another. Indeed, as described above, homophilic interaction between apposed Pcdh8 molecules can then trigger intracellular signalling. Such uncertainties regarding the precise mode of action necessitate both spatially- and temporally-detailed © 2015 British Society for Neuroendocrinology

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