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Dev Dyn. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Dev Dyn. 2016 January ; 245(1): 87–95. doi:10.1002/dvdy.24359.
Expression of the Drosophila homeobox gene, Distal-less supports an ancestral role in neural development Jessica S. Plavickia,b, Jayne M. Squirrellc, Kevin W. Eliceiric,d, and Grace Boekhoff-Falka,e,1 aNeuroscience
Training Program, University of Wisconsin, Madison, Wisconsin, United States of
America
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bSchool
of Pharmacy, University of Wisconsin, Madison, Wisconsin, United States of America
cLaboratory
for Optical and Computational Instrumentation, University of Wisconsin, Madison, Wisconsin, United States of America
dDepartment
of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States of America eDepartment
of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, United States of America
Abstract
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Background—Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Results—Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos we show that Dll is expressed in the embryonic, larval and adult CNS and PNS in embryonic and larval neurons, brain and ventral nerve cord (VNC) glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes.
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Conclusions—Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts. Keywords Dll; Dlx; nervous system; sensory system; olfaction
1
To whom correspondence should be addressed: 1111 Highland Ave, Madison, WI 53705, Tel: 608-262-1609, Fax: 608-262-7306, [email protected].
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Introduction
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The Dlx homeodomain transcription factors play essential roles in the development of the vertebrate forebrain and are necessary for the formation of neural and ectodermal components of the vertebrate olfactory system (reviewed in Panganiban and Rubenstein, 2002). Expression of Dll and Dlx genes in both the invertebrate and vertebrate nervous systems led to the hypothesis that the ancestral function of Dll may have been in the nervous system (Panganiban, 1997; Mittmann and Scholtz, 2001) and that additional Dll/Dlx functions were acquired later in evolution. The protostome-deuterostome ancestor (PDA) represents the last common ancestor to invertebrates and vertebrates. In recent revisions to metazoan (animal) phylogeny, the PDA is also the last common ancestor to all bilaterians (Erwin and Davidson, 2002) with the last common ancestor to protostomes and deuterosomes being a complex organism with many of the same features as modern bilaterians (De Robertis and Sasai, 1996; Holland and Holland, 2001; Erwin and Davidson, 2002). Genetic conservation supports the idea that body parts formed by similar developmental regulatory genes represent either evolutionarily conserved structures or the reuse of ancestral gene networks or “toolkits” (Carroll, 2005). In the case of embryonic neural development, homologous genes control proliferation, regionalization and cell fate specification in both invertebrates and vertebrates. These observations have been used to argue for a common evolutionary origin of the protostome and deuterostome brain (reviewed in Arendt and Nubler-Jung, 1999; Reichert and Simeone, 1999; Sprecher and Reichert, 2003; Wigle and Eisenstat, 2008).
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Our previous studies identified Dll as a critical factor in the development of both central and peripheral nervous system structures in the Drosophila larval olfactory system (Plavicki et al., 2012). The Dlx genes play multiple roles in vertebrate olfactory system development including neural progenitor cell specification and migration (reviewed in Panganiban and Rubenstein, 2002). However, they also play a number of more specific roles necessary for the development of the olfactory system. For instance, Dlx5 is expressed in the olfactory placode, olfactory pit and olfactory epithelium and is needed for the development of all three structures (Long et al., 2003). Within the olfactory epithelium, Dlx5 also is necessary for the differentiation of olfactory receptor neurons (ORNs), while within the olfactory bulb, Dlx5 is required for development of glia that ensheath ORN axons (Long et al., 2003).
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Our earlier findings, together with studies by others of Dlx function in vertebrate brain development, suggest that Dll and Dlx genes have conserved functions during nervous system development. However, studies of the Dlx genes have been complicated by genetic redundancy. There are 6 Dlx genes in mice and humans, 4 of which have overlapping expression in the developing brain (reviewed in Panganiban and Rubenstein, 2002). Thus, characterizing Dll expression in the developing invertebrate nervous system not only is essential for understanding Dll function in invertebrate neural development, but also could lend insight into Dlx gene function in vertebrates. We therefore examined Dll expression in the embryonic, larval and adult nervous systems in Drosophila. The manipulations and techniques available in Drosophila make it a powerful model system for the identification of novel genetic relationships and functions difficult to discern in vertebrate models.
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Results and Discussion In vivo imaging of Dll:GFP embryos reveals Dll expressing cells contribute to both the central and peripheral nervous systems
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In order to observe the dynamic movements of Dll expressing cells during embryogenesis, we captured multiphoton movies spanning late germ-band elongation (stage 10) to head involution (stage 17; stages according to (Campos-Ortega and Hartenstein, 1985). To visualize Dll expression, we took advantage of a Gal4 enhancer trap insertion into the Dll locus (P{GawB}Dllmd23; (Calleja et al., 1996) driving nuclear expression of green fluorescent protein (GFP) from a second transgene (P{UAS-GFP.nls}14 (Shiga et al., 1996). Images were collected from dechorionated embryos of genotype: w1118; P{UASGFP.nls}14, P{GawB}Dllmd23/CyO (Dong et al., 2002) at 5 μm intervals every 5 minutes over a period of 8 hours (Figure 1; Supplemental Movie 1). We observed broad Dll expression in the head segments during stages 10–17 (Figure 1A–J). The GFP was not confined to nuclei, but also detectable, albeit weakly, in the cytoplasm and in neuronal processes. These Dll-expressing cells give rise to elements of both the peripheral and central nervous systems (PNS and CNS, respectively). Specifically, the Dll-expressing cells include precursors of multiple larval sense organs and of the brain regions that receive inputs from these sense organs.
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For example, both the larval dorsal organ (DO), which has olfactory and gustatory functions, and the neuroblasts that form the larval antennal lobe in the CNS are derived from the antennal head segment (Schmidt-Ott et al., 1994; Younossi-Hartenstein et al., 1996; Urbach et al., 2003; Younossi-Hartenstein et al., 2006). The antennal lobe consists of glomeruli that carry out the initial processing of olfactory information from the DO and relay that information to higher order brain structures, including the mushroom bodies and the lateral horn (Jefferis et al., 2001; Marin et al., 2002). The larval terminal and ventral organs (TO and VO, respectively), which have gustatory functions, arise from the maxillary segment (Urbach et al., 2003) and project their axons to the subesophageal ganglion (SOG).
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Dll is expressed in the labral, antennal, maxillary and labial head segments and necessary for the development of the non-neural, cuticular components of the labral, antennal (DO), maxillary (TO and VO) and labial sense organs (Cohen and Jurgens, 1989). Loss of Dll function also results in the loss of the Keilin’s organ (KO), a larval mechanosensory structure, associated with the adult thoracic limb primordia (Keilin, 1915; Cohen and Jurgens, 1989). We recently reported that in later stages of embryogenesis Dll is expressed in neurons and their support cells in the DO and TO and that loss of Dll function results in loss of both these neurons and their support cells (Plavicki et al., 2012). Consistent with this, our multiphoton analysis of living embryos permits us to visualize Dll-expressing cells contributing to both neural and non-neural components of the DO, TO, VO and KO (Figure 2; Supplemental Movie 2). By carefully comparing Dll protein expression with GFP expression driven by the P{GawB}Dllmd23, we conclude that the Gal4 driver faithfully recapitulates Dll expression with two exceptions: 1) the embryonic ventral nerve cord glia that express Dll lack P{GawB}Dllmd23 expression; and 2) several cells in the brain that express Dll, also lack P{GawB}Dllmd23 expression.
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Our live imaging indicates that Dll-expressing cells in the antennal and maxillary head segment give rise to both neural and non-neural components of DO, TO and VO. It remains unclear whether Dll-expressing cells in the head segments specifically contribute to the central nervous system regions that receive input from the DO, TO and VO. However, clusters of Dll-expressing cells invaginate from the head segments and migrate to form Dllexpressing regions in the supraesophageal ganglion of the brain (arrowheads in Figure 1 and Supplemental Movie 1). Although Dll was previously shown to be required for DO, TO and VO development and to be expressed within the differentiated organs, this is first time that Dll expression has been followed directly from the precursor stage into the differentiated sense organs. Also, although Dll expression has been reported in primary neuronal clusters in the embryonic Drosophila brain (Sprecher et al., 2007), this is the first demonstration of Dll expression in Drosophila brain precursors (‘stem cells’) prior to their delamination/ specification. Our data, therefore, support the hypothesis that Dll may play critical roles in the specification and/or renewal of neural progenitors as well as in the differentiation of specific neural lineages. Dll expression in cephalic and thoracic sensory organ precursors
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To determine whether the Dll expression observed via live imaging corresponded to precursors that give rise to the sense organs affected in Dll mutants, we examined Dll expression in relation to genes expressed in neural progenitors. Specifically, we examined Dll expression in relation to the zinc-finger transcription factor, senseless (sens), which is expressed in sense organ precursors (SOPs) in the head and thoracic segments (Nolo et al., 2000), and prospero (pros), a homeodomain transcription factor, that is asymmetrically distributed with Numb to confer neural fates (Hirata et al., 1995; Knoblich et al., 1995). We find that Dll is expressed in SOPs in both the cephalic and thoracic segments (Figure 3) as indicated by the co-localization of Dll and Sens in a subset of Sens-expressing SOPs (Figure 3C, F, F′, G and G′). Dll also was co-expressed with Pros in subsets of cells in the head and thorax (Figure 3D, F, F″, G, G″). Previous work has shown that Pros is necessary for neuronal differentiation in both the CNS and PNS (Doe et al., 1991). In the developing larval olfactory system, Pros marks supporting cells, including socket, sheath and shaft cells (Grillenzoni et al., 2007) and is co-expressed in late embryogenesis with Dll (Plavicki et al., 2012). We note that the majority of the Dll-expressing cells in the thoracic segments also express Sens and/or Pros. This suggests that these cells primarily contribute to neural structures such as Keilin’s organ and does not support the current view (e.g. (McKay et al., 2009) that many of the thoracic Dll-expressing cells are leg imaginal disc precursors.
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Dll is expressed in subsets of glia in the ventral nerve cord (VNC) and in neurons and glia in the supraesophageal ganglion Dll expression in the VNC is first observed at embryonic stage 12 and continues in the VNC during the third larval instar (Figure 4A–D, G and I). In contrast to Dll expression in the brain, which includes both neurons and glia, Dll expression in the VNC is exclusively glial. In the embryonic VNC, Dll is expressed in both cell body associated glia (arrows in Figure 4A and B) and dorsal longitudinal glia (arrowheads in Figure 4B and D). Both are subsets of the lateral glia (reviewed in Klaembt et al., 1996). In the larval VNC, Dll is expressed in glial cells that surround the thoracic ganglion (Figure 4I, J, K). Based on their location, it is Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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likely that at least some of these glia migrate into the developing leg during metamorphosis. We also observed Dll expression in subsets of glia within the embryonic and larval brain (Figure 4E–H, J). During embryogenesis, Dll-expressing glia line the preoral commissure (arrowhead in Figure 4F) and also are found in the supraesophageal ganglion (Figure 4F). Dll continues to be expressed in glial cells within the developing larval brain (Figure 4G and H), however we did not detect Dll-expressing glia in the adult CNS (not shown). Dll is expressed in larval and chemosensory neurons
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Dll expression is first observed in chemosensory organs (DO, TO, and VO) during embryogenesis (Figures 2 and 5). During larval and adult stages, Dll expression continues to be detected in chemosensory neurons (Figure 6A–D and not shown) as well as their supporting cells. Chemosensory neurons are clustered together under their respective sense organs and referred to as ganglia. During embryogenesis Dll is expressed in DO, TO and VO ganglia (DOG, TOG and VOG, respectively; Figure 6B and (Plavicki et al., 2012). The majority of DOG neurons respond to olfactory stimuli and send afferent projections to the larval antennal lobe that (LAL) (Gerber and Stocker, 2007). A subset of DO neurons respond to gustatory stimuli and, along with the TO and VO send projections to different regions of the larval brain (Figure 6A). Dll-expressing neurons can be seen projecting to both the LAL and the subesophageal ganglion (SOG; Figure 6A and C).
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During metamorphosis, the CNS and the peripheral components of olfactory system undergo substantial reorganization. The adult Drosophila antenna, which arises from the antennal imaginal disc, houses most adult olfactory neurons. Analogous to the larval organization, the adult ORNs send afferent projections to the adult antennal lobe (AL). ORNs that express the same odorant receptor converge on the same targets within the AL forming structures termed glomeruli (Gao et al., 2000; Vosshall, 2000; Couto et al., 2005). Dll is necessary for specifying antennal fate and loss of Dll function results antennal to leg transformations (Sunkel and Whittle, 1987; Cohen and Jurgens, 1989; Dong et al., 2000). Here, we report that Dll also is expressed in adult ORNs and that projections from Dll-expressing ORNs form glomeruli in the adult AL (Figure 6D). Dll-expressing ORNs also are located in the maxillary palps (data not shown), which like the antenna is derived from the Dll-expressing antennal disc (reviewed in Stocker, 1994). These ORNs also project to the adult AL (reviewed in Stocker, 1994). Gustatory receptor neurons (GRNs) are found in a number places on the adult fly including the labellum, legs, wing margins and female genitalia (reviewed in Vosshall, 2000). The precursors of all of these structures also express Dll. Thus, there appears to be a strong correlation between Dll expression and chemosensation.
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Dll is expressed in the larval and adult optic lobes In addition to being expressed in the developing olfactory system across multiple stages of development, Dll is consistently associated with the development of the visual system. Dll expression is detected in the optic lobes during larval and adult stages, however, in contrast to Dll expression in other sensory systems, expression seems to be limited to brain processing regions and is not detected in retinal neurons or their support cells. The adult optic lobes are divided into distinct morphological and functional domains. From most external, to most internal, these are the lamina, medulla, lobula, and lobula plate (Hofbauer
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and Campos-Ortega, 1990; Meinertzhagen and Hanson, 1993). The larval optic lobe consists of the two proliferative centers, the inner and outer proliferative centers (IPC and OPC respectively) (Hofbauer and Campos-Ortega, 1990). The outer proliferative center (OPC) is crescent shaped neuroepithelium, which is spatially divided along the anterior-posterior axis by the expression of wingless (wg), decapentaplegic (dpp), vsx1, and optix, and gives rise to the medulla (Kaphingst and Kunes, 1994; Egger et al., 2007; Li et al., 2013). wg expression defines the tips of the OPC (tOPC) and has reported to co-localize with Dll expression (Kaphingst and Kunes, 1994). During optic lobe development, Dll along with eyeless (Ey), Sloppy-paired (Slp), and Dichaete (D), are necessary for temporally patterning neuroblasts in the tOPC and generating four neuronal classes that will innervate the adult medulla, lobula, and lobula plate (Bertet et al., 2014). Dll-expressing neuroepithelial cells give rise to the youngest neuroblasts in the tOPC and produce Dll-expressing ganglion mother cells and, ultimately, Dll-expressing class I Spalt major (Salm)/Runt neurons (Bertet et al., 2014). As development persists, Dll expressing neuroblasts begin to express Ey and, as Dll expression wanes, Ey expressing neuroblasts give rise to class 2 sevenup (SVP) neurons (Bertet et al., 2014). Neurons specified during the temporal window of Dll expression are located in the lobula plate cortex (Bertet et al., 2014).
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The cell bodies making up the adult medulla are positioned in either the medulla cortex, which is located between the lamina and medulla neuropil, or the medulla rim, which is situated between the medulla and lobula plate (Morante and Desplan, 2011). Dll is expressed in both the adult medulla cortex and medulla rim (Morante and Desplan, 2008). During larval development of the medulla cortex, Dll is expressed specifically in neurons derived from the oldest neuroblasts, but not in the neuroblasts themselves and does not colocalize with eyeless (ey) or apterous (ap), two broadly expressed markers of cortical cells in the medulla (Morante and Desplan, 2011). Consistent with previous reports, we observed Dll expression in the larval and adult medulla. We also observed Dll expression in laminal neurons in the larval optic lobe in both early and late third instar larvae (Figures 4J and 7A–D, G) and find that Dll is co-expressed with Elav, a marker of differentiated neurons, in a subset of laminal neurons. Dll does not colocalize with Dac, is an early marker of differentiated laminal neurons (Morante and Desplan, 2011). Dll continues to be expressed in the lamina (arrowhead in E) and medulla (arrow in E) during the adult stages (Figure 7E–G). We also observed Dll expression in the central brain in adults (asterisks in Figure 7). We hypothesize that the persistence of Dll expression in adult neurons may indicate a role in the maintenance of specific neuronal fates.
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In summary, we provide a detailed description of Dll expression during embryonic, larval and adult stages of the Drosophila life cycle. We focused on neurons and their supporting cells and demonstrated for the first time that Dll is expressed in the embryonic ectoderm in precursors of the brain. We strengthened the existing link between Dll and chemosensation by establishing that Dll is expressed in most, if not all, olfactory and gustatory organs. In addition, we report that Dll is expressed in only glia of the ventral nerve cord, in both neurons and glia of the larval brain, and in only neurons within the adult brain. We also validated the fidelity of a widely used P{GawB} insertion into the Dll locus. Together, this Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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information provides an important framework for future investigations of specific Dll functions in neural stem cells, neurons, and glia as well as for comparative studies of the role of Dll during development and neural function.
Experimental Procedures Drosophila Stocks The following Drosophila strains were used in this study: Oregon R (Bloomington Drosophila Stock Center); y1 w1 (Bloomington Drosophila Stock Center), Dll01092 (DlllacZ reporter; (Goto and Hayashi, 1997) and w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (Calleja et al., 1996; Shiga et al., 1996; Dong et al., 2002). Immunohistochemistry
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Embryo collection and fixation were performed as described (Langeland, 1999) with staging of embryos according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). The following primary antibodies and dilutions were used: rabbit anti-Dll, 1:100 (Panganiban et al., 1994); 1:200 mouse 22C10, 1:10 (Fujita et al., 1982; Zipursky et al., 1984); rat anti-Elav, 1:10 (O’Neill et al., 1994); mouse anti-Fas2, 1:10 (Developmental Studies Hybridoma Bank) (Hummel et al., 2000); mouse anti-Repo (Alfonso and Jones, 2002); mouse anti-Pros, 1:100 (Campbell et al., 1994); Cy5 anti-HRP, 1:100 (Jackson Immunochem) and guinea pig anti-Sens, 1:5,000 (Nolo et al., 2000). Alexa secondary antibodies (Life Technologies) were used at 1:100. Secondary antibodies used were Alexa Fluor 488 anti-mouse; Alexa Fluor 488 anti-rabbit; Alexa Fluor 488 anti-guinea pig; Alexa Fluor 568 anti-rabbit; Alexa Fluor 633 anti-mouse; Alexa Fluor 633 anti-rabbit; Alexa Fluor 633 streptavidin. Embryos were mounted in Vectashield (Vector Laboratories).
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Confocal Microscopy Confocal images were collected on a Zeiss LSM 510 microscope. Three-dimensional reconstructions were made with Zeiss LSM software, and images were processed by using combinations of Zeiss LSM filtering functions, Image J, and Adobe Photoshop. Optical sections in Z-series collections were collected at 0.4-μm intervals. Multiphoton Microscopy
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Movies were collected using a multiphoton laser scanning microscope (MPLSM) at the Laboratory for Optical and Computational Instrumentation (LOCI) at the University of Wisconsin. This custom-built MPLSM consisted of a titanium sapphire laser (Spectra Physics, Tsunami), set to 900nm excitation wavelength, a photomultiplier tube (ThornEMI-9924B) for detection, and an inverted Nikon Eclipse TE300 microscope coupled to a Bio-Rad MRC-1024 scan assembly. A Nikon 40× oil-immersion lens with a numerical aperture of 1.3 and working distance of 0.2 mm was used for these experiments. Embryos were adhered to coverslips using glue derived from heptane and tape, and covered with a small amount of halocarbon oil. Multiphoton movies spanned embryonic stages 10 to 17. In each movie, a z-series was collected every 5 minutes at an optical step size of 5 μm.
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Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments FUNDING: JP was supported by NIH Training Grant T32 GM-7507 and a grant from the University of Wisconsin Graduate School. This work also was supported by NIH R01 GM59871 (GBF), NIH K99 ES0238 (JSP), and core LOCI support funds (KWE).
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We thank Drs. Margaret McFall-Ngai and Ned Ruby for access to the Zeiss LSM confocal microscope in the University of Wisconsin Department of Medical Microbiology and Immunology that is supported by National Institutes of Health Grant RR12294. We also thank Hugo Bellen for Sens Antibody. The monoclonal, Dac, Elav, Fas2, Pros, Repo, and 22C10 antibodies used in these studies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by the University of Iowa Department of Biological Sciences. J.P. was supported by National Institutes of Health Training Grant T32 GM-7507 and a grant from the University of Wisconsin Graduate School. This work also was supported by National Institutes of Health Grant R01 GM59871 (to G.B.-F.) and an National Institutes of Health Grant K99 ES0238 (J.S.P).
References
Author Manuscript Author Manuscript
Alfonso T, Jones B. gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila. Dev Biol. 2002; 248:369–383. [PubMed: 12167411] Arendt D, Nubler-Jung K. Comparison of early nerve cord development in insects and vertebrates. Development. 1999; 126:2309–2325. [PubMed: 10225991] Bertet C, Li X, Erclik T, Cavey M, Wells B, Desplan C. Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper. Cell. 2014; 158:1173–1186. [PubMed: 25171415] Calleja M, Moreno E, Pelaz S, Morata G. Visualization of gene expression in living adult Drosophila. Science. 1996; 274(5285):252–255. [PubMed: 8824191] Campbell G, Goring H, Lin T, Spana E, Andersson S, Doe CQ, Tomlinson A. RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development. 1994; 120:2957–2966. [PubMed: 7607085] Campos-Ortega, J.; Hartenstein, V. The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag; 1985. p. 227 Carroll, SB. Endless Forms Most Beautiful: The New Science of Evo Devo. New York: W.W. Norton; 2005. p. 368 Cohen SM, Jurgens G. Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO Journal. 1989; 8(7):2045–2055. [PubMed: 16453891] Couto A, Alenius M, Dickson BJ. Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol. 2005; 15:1535–1547. [PubMed: 16139208] De Robertis E, Sasai Y. A common plan for dorsoventral patterning in Bilateria. Nature. 1996; 380:37–40. [PubMed: 8598900] Doe C, Chu-LaGraff Q, Wright D, Scott M. The prospero gene specifies cell fates in the Drosophila central nervous system. Cell. 1991; 65:451–464. [PubMed: 1673362] Dong PDS, Chu J, Panganiban G. Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity. Development. 2000; 127:209–216. [PubMed: 10603339] Dong PDS, Dicks JS, Panganiban G. Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna. Development. 2002; 129:1967–1974. [PubMed: 11934862] Egger B, Boone JQ, Stevens NR, Brand AH, Doe CQ. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Dev. 2007; 2:1. [PubMed: 17207270] Erwin DH, Davidson EH. The last common bilaterian ancestor. Development. 2002; 129:3021–3032. [PubMed: 12070079]
Dev Dyn. Author manuscript; available in PMC 2017 January 01.
Plavicki et al.
Page 9
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Fujita SC, Zipursky SL, Benzer S, Ferrus A, Shotwell SL. Monoclonal antibodies against the Drosophila nervous system. Proc Natl Acad Sci U S A. 1982; 79:7929–7933. [PubMed: 6818557] Gao Q, Yuan B, Chess A. Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat Neurosci. 2000; 3:780–785. [PubMed: 10903570] Gerber B, Stocker RF. The Drosophila larva as a model for studying chemosensation and chemosensory learning: a review. Chem Senses. 2007; 32:65–89. [PubMed: 17071942] Goto S, Hayashi S. Specification of the embryonic limb primordium by graded activity of Decapentaplegic. Development. 1997; 124:125–132. [PubMed: 9006073] Grillenzoni N, de Vaux V, Meuwly J, Vuichard S, Jarman A, Holohan E, Gendre N, Stocker R. Role of proneural genes in the formation of the larval olfactory organ of Drosophila. Development, Genes and Evolution. 2007; 217:209–219. [PubMed: 17260155] Hirata J, Nakagoshi H, Nabeshima Y, Matsuzaki F. Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature. 1995; 377:627–630. [PubMed: 7566173] Hofbauer A, Campos-Ortega JA. Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux’s Archives of Developmental Biology. 1990; 198:264–274. Holland LZ, Holland ND. Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate? J Anat. 2001; 199:85–98. [PubMed: 11523831] Hummel T, Krukkert K, Roos J, Davis G, Klaembt C. Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron. 2000; 26:357–370. [PubMed: 10839355] Jan LY, Jan YN. Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos. Proc Natl Acad Sci U S A. 1982; 79:2700–2704. [PubMed: 6806816] Jefferis GS, Marin EC, Stocker RF, Luo L. Target neuron prespecification in the olfactory map of Drosophila. Nature. 2001; 414:204–208. [PubMed: 11719930] Kaphingst K, Kunes S. Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentaplegic to specify the dorsoventral axis. Cell. 1994; 78:437–448. [PubMed: 8062386] Keilin D. Recherches sur les larves de Dipteres cyclorhaphes. Bulletin Scientifique de la France et de la Belgique. 1915; 49:15–198. Klaembt, C.; Hummel, T.; Menne, T.; Sadlowski, E.; Scholz, H.; Stollewerk, A. Development and function of embryonic central nervous system glial cells in Drosophila. 1996. p. 40-49. Knoblich JA, Jan LY, Jan YN. Asymmetric segregation of numb and prospero during cell division. Nature. 1995; 377:624–627. [PubMed: 7566172] Langeland, J. Imaging Immunolabeled Drosophila Embryos by Confocal Microscopy. In: Paddock, S., editor. Confocal Microscopy Methods and Protocols. Totowa: Humana Press; 1999. p. 167-172. Li X, Erclik T, Bertet C, Chen Z, Voutev R, Venkatesh S, Morante J, Celik A, Desplan C. Temporal patterning of Drosophila medulla neuroblasts controls neural fates. Nature. 2013; 498:456–462. [PubMed: 23783517] Long J, Garel S, Depew M, Tobet S, Rubenstein J. DLX5 regulates development of peripheral and central components of the olfactory system. Journal of Neuroscience. 2003; 23:568–578. [PubMed: 12533617] Marin O, Baker J, Puelles L, Rubenstein JL. Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections. Development. 2002; 129:761–773. [PubMed: 11830575] McKay DJ, Estella C, Mann RS. The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene. Development. 2009; 136:61–71. [PubMed: 19036798] Meinertzhagen, IA.; Hanson, TE. The development of the optic lobe. 1993. p. 1363-1491. Mittmann B, Scholtz G. Distal-less expression in embryos of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of mechanoreceptors, chemoreceptors, and the CNS. Dev Genes Evol. 2001; 211:232–243. [PubMed: 11455438] Morante J, Desplan C. The color-vision circuit in the medulla of Drosophila. Curr Biol. 2008; 18:553– 565. [PubMed: 18403201]
Dev Dyn. Author manuscript; available in PMC 2017 January 01.
Plavicki et al.
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Morante J, Desplan C. Dissection and staining of Drosophila optic lobes at different stages of development. Cold Spring Harb Protoc. 2011; 2011:652–656. [PubMed: 21632779] Nolo R, Abbott LA, Bellen HJ. Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell. 2000; 102:349–362. [PubMed: 10975525] O’Neill EM, Rebay I, Tjian R, Rubin GM. The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell. 1994; 78:137–147. [PubMed: 8033205] Panganiban G, Nagy L, Carroll SB. The role of the Distal-less gene in the development and evolution of insect limbs. Current Biology. 1994; 4:671–675. [PubMed: 7953552] Panganiban G, Rubenstein J. Developmental functions of the Distal-less/Dlx homeobox genes. Development. 2002; 129:4371–4386. [PubMed: 12223397] Panganiban G, Irvine SM, Lowe C, Roehl H, Corley LS, Sherbon B, Grenier J, Fallon JF, Kimble J, Walker M, Wray GA, Swalla BJ, Martindale MQ, Carroll SB. The origin and evolution of animal appendages. Proceedings of the National Academy of Sciences USA. 1997; 94:5162–5166. Plavicki J, Mader S, Pueschel E, Peebles P, Boekhoff-Falk G. Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system. Proc Natl Acad Sci U S A. 2012; 109:1578–1583. [PubMed: 22307614] Reichert H, Simeone A. Conserved usage of gap and homeotic genes in patterning the CNS. Curr Opin Neurobiol. 1999; 9:589–595. [PubMed: 10508733] Schmidt-Ott U, Gonzalez-Gaitan M, Jackle H, Technau GM. Number, identity, and sequence of the Drosophila head segments as revealed by neural elements and their deletion patterns in mutants. Proc Natl Acad Sci U S A. 1994; 91:8363–8367. [PubMed: 7915837] Shiga Y, Tanaka-Matakatsu M, Hayashi S. A nuclear GFP/beta-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Development, Growth and Differentiation. 1996; 38:99–106. Sprecher S, Reichert H, Hartenstein V. Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain. Gene Expression Patterns. 2007; 7:584–595. [PubMed: 17300994] Sprecher SG, Reichert H. The urbilaterian brain: developmental insights into the evolutionary origin of the brain in insects and vertebrates. Arthropod Struct Dev. 2003; 32:141–156. [PubMed: 18089000] Stocker, RF. The organization of the chemosensory system in Drosophila melanogaster: a review. 1994. p. 3-26. Sunkel CE, Whittle JRS. Brista: A gene involved in the specification and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster. Wilhelm Roux’s Archives of Developmental Biology. 1987; 196:124–132. Urbach R, Schnabel R, Technau GM. The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila. Development. 2003; 130:3589–3606. [PubMed: 12835378] Vosshall LB. Olfaction in Drosophila. Curr Opin Neurobiol. 2000; 10:498–503. [PubMed: 10981620] Wigle JT, Eisenstat DD. Homeobox genes in vertebrate forebrain development and disease. Clin Genet. 2008; 73:212–226. [PubMed: 18241223] Younossi-Hartenstein A, Nassif C, Green P, Hartenstein V. Early neurogenesis of the Drosophila brain. J Comp Neurol. 1996; 370:313–329. [PubMed: 8799858] Younossi-Hartenstein A, Nguyen B, Shy D, Hartenstein V. Embryonic origin of the Drosophila brain neuropile. J Comp Neurol. 2006; 497:981–998. [PubMed: 16802336] Zipursky SL, Venkatesh TR, Teplow DB, Benzer S. Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes. Cell. 1984; 36:15–26. [PubMed: 6420071]
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Author Manuscript Figure 1. In vivo multiphoton imaging of Dll expressing cells during embryogenesis
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A–J: Still shots from a multiphoton movie spanning late germ-band elongation (stage 10; A) to head involution (stage 17; J) of an embryo of genotype: w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO in which nuclear GFP expression is driven by a Dll-Gal4 enhancer trap. Dll expression is detected in the antennal and maxillary head segments (asterisks in A– C) that give rise to the olfactory and gustatory organs (asterisks in D–J) as well as presumptive (arrowheads in B–E) and delaminated cells of the brain (arrowheads in F–J). Still images are taken from Supplemental Movie 1 which was initiated at ~6 hours after egg laying and continued until ~14 hours after egg laying. Minutes indicated are relative to start of imaging. Scale bar = 50 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 2. Colocalization of GFP expression regulated by P{GawB}Dllmd23 driver and Dll protein
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A-B″: Dorsal views of stage 16 embryos. A: Still image from Supplemental Movie 2 (frame #97). B: w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO embryo stained for Dll (red) and 22C10 (blue). Monoclonal antibody 22C10 recognizes the microtubule associated protein Futsch and is used to delineate neuronal morphology (Hummel et al., 2000). Dll protein, which is nuclear, is contained within a broader domain of GFP expression. This is expected due to both the perdurance of GFP and GFP localizing to cytoplasm as well as nucleus. We note that while there does not appear to be GFP outside the Dll expression domains, there is some Dll protein expression where we do not detect GFP (e.g. red alone in panel B). GFP and Dll are expressed in the dorsal organ ganglion (arrows), the terminal organ ganglion Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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(arrowheads), and the neurons in the labral sensory organ complex (asterisks). B′: GFP expression relative to 22C10. B″: Dll protein expression relative to 22C10. A–B″: 40× confocal images. Scale bar = 50 μm.
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Figure 3. Dll expression in sensory organ precursors
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A-G: Lateral views of a stage 13 embryo stained for Dll (green), Senseless (Sens; red), and Prospero (Pros; blue). A, E–G″: Single focal planes. B–D: Brightest point projections of a zseries from the boxed region in (A). C: Dll is coexpressed with a subset of Sens-expressing cells in the antennal (an), maxillary (mx), and labial (lb) head segments and the thoracic segments (T1–3). D: Dll is coexpressed with a subset of Pros-expressing cells in the an, mx, and lb head segments and the thoracic segments. E: Boxes 1 and 2 mark the segments shown at higher magnification in F–F″, and G–G″, respectively. In the mx segment, there are many Dll-expressing cells that lack both Pros and Sens, while in the T1 segment almost all of the Dll-expressing cells have Pros and/or Sens expression. F: Dll, Sens and Pros expression in the mx segment. F′: Dll and Sens. F″: Dll and Pros. G: Dll, Sens and Pros expression in T1. G′: Dll and Sens. G″: Dll and Pros. A: 20× confocal image. B–G″: 40× confocal images. Scale bars = 50 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 4. Dll is expressed in subsets of glia in the ventral nerve cord (VNC) and in both neurons and glia in the supraesophageal ganglion
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Lateral (A, B), ventral (C, D), and dorsal (E, F) views of wild type stage 14 embryos stained for Dll (green), the glial marker Repo (red), and the neuronal morphology marker 22C10 (blue). Yellow indicates areas of overlapping Dll and Repo expression. A, B: Dll expression in cell body associated glia (arrows in B) and dorsal longitudinal glia (arrowheads in B). Boxed region in (A) is shown at higher magnification in (B). C, D: Dll expression in dorsal longitudinal glia (arrowheads in D). Boxed region in (C) is shown at higher magnification in (D). E, F: Dll expression in neurons (asterisks in F) and glia (arrows in F) in the supraesophageal ganglion. Boxed region in (E) is shown at higher magnification in (F). G,
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H, I: Dorsal views of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO third instar larval brain stained with anti-Repo (red) and anti-horseradish peroxidase (anti-HRP; blue). The region marked by boxes 1 & 2 are shown at higher magnification in (H) and (I), respectively. Dll continues to be expressed in brain (H) and VNC glia (I) during larval stages. J, K: X-gal staining of an early third instar larval central nervous system from an animal carrying a Dll-lacZ enhancer trap (Dll01092; Goto and Hayashi, 1997). Boxed region in (J) is shown at higher magnification in (K). A, C, E, G: 10× confocal images. B, D, F, H, I: 40× confocal images. J: 10× image. K: 40× image. A, C, E: Scale bars = 100 μm. B, D, F, H, I, K: Scale bars = 25 μm. G, J: Scale bar = 50 μm.
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Figure 5. Dll expression in anterior sense organs of the embryonic peripheral nervous system (PNS)
A–D: Lateral views of a wild type stage 14 embryo stained for Dll (green) and 22C10 (purple). A: Dll is expressed in the labial (LIS; arrow), labral (LRS; open arrowhead) and antennomaxillary sensory complexes (AMC: arrowhead). B: Dll expression in the labial sensory complex. C: Dll expression in the developing cuticular components of the DO and TO. D: Dll expression in the dorsal organ ganglion (DOG) neurons that innervate the dorsal organ (DO in panel C) and in the neurons of the terminal organ ganglion (TOG) that innervate the terminal organ (TO in panel C). E, F: Lateral view of a stage 16 embryo
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stained for Dll (green) and 22C10 (purple). Dll is expressed in both neurons and supporting cells of multiple sense organs in the head, including the DOG, TOG, ventral organ ganglion (VOG), labial organ (LBO). Dll also is expressed in the mechanosensory Keilin’s organs (KO) of the three thoracic segments (T1–T3). The Dll-expressing sense organs in (F) are highlighted in (E). G: Schematic of the large, Dll-expressing chemosensory organs in (E) and (F). DO, TO, and VO are the dorsal organ, terminal organ, and ventral organ, respectively. These organs lie in the larval epidermis and are innervated by neurons with cell bodies in their respective ganglia (DOG, TOG and VOG). From the ganglia, axons project to targets within the larval brain as. Olfactory DOG axons project to the larval antennal lobe (LAL). The LAL relays olfactory information to the mushroom body (MB) and lateral horn (not shown). Gustatory DOG axons, along with gustatory TOG and VOG axons project to the subesophageal ganglion (SOG). A: 10× confocal image. B, C, D, E, F: 40× confocal images. A: Scale bar = 100 μm. B, C, D, F: Scale bars = 50 μm.
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Figure 6. Dll is expressed in larval and adult chemosensory neurons
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A: Dorsal view of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (green) third instar larval brain stained with anti-horseradish peroxidase (HRP; purple). Anti-HRP recognizes Nervana proteins in neuronal membranes and is used to visualize the neuropil (Jan and Jan, 1982). Dll expression is seen in central brain regions. Dll-expressing olfactory receptor neurons (ORNs) in the dorsal organ ganglion (DOG) project to the larval antennal lobe (LAL) whereas Dll-expressing gustatory receptor neurons in the DOG, terminal organ ganglion (TOG) and the ventral organ (VOG) project to the subesophageal ganglion (SOG). B: Co-localization of Dll antibody staining with the neuronal marker Elav (O’Neill et al., 1994) and with GFP driven by P{GawB}Dllmd23 (Calleja et al., 1996). Dll is expressed in TOG neurons. C: Higher magnification view of the LAL boxed in panel A. D: Anterior view of the adult antennal lobes (AL). Both larval and adult Dll expressing ORNs project to their respective antennal lobes. E: Schematic of an adult brain from a frontal view. The mushroom body (MB), lateral horn (LH), and antennal lobe (AL) are indicated. The inset is a schematic of an adult antenna with the olfactory receptor neuron (ORN) cell bodies indicated in red, orange, and yellow. ORNs expressing the same odorant receptor project to a single glomerulus in the AL. A: 10× confocal images. B, C, D: 40× confocal images. A, B, D: Scale bars = 50 μm. C: Scale bar = 12.5 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 7. Dll is expressed in the larval and adult optic lobes
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A: Lateral view of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (green) third instar larval optic lobe stained for Dac (blue) and Elav (red). Dac is an early marker of differentiated laminal neurons, Elav marks laminal neurons in later stages of optic lobe development (Morante and Desplan, 2008; Bertet et al., 2014). B: Higher magnification view of optic lobe shown in (A). C: Dorsal view of a larval third instar brain stained for Dll (green) and Fas2 (red). Fas2 marks a subset of neuronal membranes. Dll is expressed in laminal neurons (box in C) and the medulla (arrows in C). D: High magnification view of boxed area from (C). E: Anterior view of an adult brain stained for Dll (green), Repo (red)
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and anti-HRP (blue). Dll is expressed in neurons in central regions of the adult brain (asterisks) and in the optic lobes (arrow and arrowhead). Arrow indicates Dll-expressing cells in the medulla, while arrowhead indicates Dll-expressing cells in the lamina. F: High magnification view of boxed area in (E). Dll is expressed in adult optic lobe neurons, but not glia. (G) X-gal staining of an adult brain from an animal carrying a reporter insertion into the Dll locus (Dll01092; Goto and Hayashi, 1997). This reporter faithfully recapitulates Dll expression in both the central brain (asterisks) as well as the medulla (arrow) and lamina (arrowhead) of the optic lobes. A, C: 20× confocal images. E: 10× confocal image. B, D, F: 40× confocal images. A, C, E, F, G: Scale bars = 50 μm. B, D: Scale bars = 25 μm.
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Dev Dyn. Author manuscript; available in PMC 2017 January 01. Published in final edited form as: Dev Dyn. 2016 January ; 245(1): 87–95. doi:10.1002/dvdy.24359.
Expression of the Drosophila homeobox gene, Distal-less supports an ancestral role in neural development Jessica S. Plavickia,b, Jayne M. Squirrellc, Kevin W. Eliceiric,d, and Grace Boekhoff-Falka,e,1 aNeuroscience
Training Program, University of Wisconsin, Madison, Wisconsin, United States of
America
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bSchool
of Pharmacy, University of Wisconsin, Madison, Wisconsin, United States of America
cLaboratory
for Optical and Computational Instrumentation, University of Wisconsin, Madison, Wisconsin, United States of America
dDepartment
of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States of America eDepartment
of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin, United States of America
Abstract
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Background—Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Results—Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos we show that Dll is expressed in the embryonic, larval and adult CNS and PNS in embryonic and larval neurons, brain and ventral nerve cord (VNC) glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes.
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Conclusions—Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts. Keywords Dll; Dlx; nervous system; sensory system; olfaction
1
To whom correspondence should be addressed: 1111 Highland Ave, Madison, WI 53705, Tel: 608-262-1609, Fax: 608-262-7306, [email protected].
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Introduction
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The Dlx homeodomain transcription factors play essential roles in the development of the vertebrate forebrain and are necessary for the formation of neural and ectodermal components of the vertebrate olfactory system (reviewed in Panganiban and Rubenstein, 2002). Expression of Dll and Dlx genes in both the invertebrate and vertebrate nervous systems led to the hypothesis that the ancestral function of Dll may have been in the nervous system (Panganiban, 1997; Mittmann and Scholtz, 2001) and that additional Dll/Dlx functions were acquired later in evolution. The protostome-deuterostome ancestor (PDA) represents the last common ancestor to invertebrates and vertebrates. In recent revisions to metazoan (animal) phylogeny, the PDA is also the last common ancestor to all bilaterians (Erwin and Davidson, 2002) with the last common ancestor to protostomes and deuterosomes being a complex organism with many of the same features as modern bilaterians (De Robertis and Sasai, 1996; Holland and Holland, 2001; Erwin and Davidson, 2002). Genetic conservation supports the idea that body parts formed by similar developmental regulatory genes represent either evolutionarily conserved structures or the reuse of ancestral gene networks or “toolkits” (Carroll, 2005). In the case of embryonic neural development, homologous genes control proliferation, regionalization and cell fate specification in both invertebrates and vertebrates. These observations have been used to argue for a common evolutionary origin of the protostome and deuterostome brain (reviewed in Arendt and Nubler-Jung, 1999; Reichert and Simeone, 1999; Sprecher and Reichert, 2003; Wigle and Eisenstat, 2008).
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Our previous studies identified Dll as a critical factor in the development of both central and peripheral nervous system structures in the Drosophila larval olfactory system (Plavicki et al., 2012). The Dlx genes play multiple roles in vertebrate olfactory system development including neural progenitor cell specification and migration (reviewed in Panganiban and Rubenstein, 2002). However, they also play a number of more specific roles necessary for the development of the olfactory system. For instance, Dlx5 is expressed in the olfactory placode, olfactory pit and olfactory epithelium and is needed for the development of all three structures (Long et al., 2003). Within the olfactory epithelium, Dlx5 also is necessary for the differentiation of olfactory receptor neurons (ORNs), while within the olfactory bulb, Dlx5 is required for development of glia that ensheath ORN axons (Long et al., 2003).
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Our earlier findings, together with studies by others of Dlx function in vertebrate brain development, suggest that Dll and Dlx genes have conserved functions during nervous system development. However, studies of the Dlx genes have been complicated by genetic redundancy. There are 6 Dlx genes in mice and humans, 4 of which have overlapping expression in the developing brain (reviewed in Panganiban and Rubenstein, 2002). Thus, characterizing Dll expression in the developing invertebrate nervous system not only is essential for understanding Dll function in invertebrate neural development, but also could lend insight into Dlx gene function in vertebrates. We therefore examined Dll expression in the embryonic, larval and adult nervous systems in Drosophila. The manipulations and techniques available in Drosophila make it a powerful model system for the identification of novel genetic relationships and functions difficult to discern in vertebrate models.
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Results and Discussion In vivo imaging of Dll:GFP embryos reveals Dll expressing cells contribute to both the central and peripheral nervous systems
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In order to observe the dynamic movements of Dll expressing cells during embryogenesis, we captured multiphoton movies spanning late germ-band elongation (stage 10) to head involution (stage 17; stages according to (Campos-Ortega and Hartenstein, 1985). To visualize Dll expression, we took advantage of a Gal4 enhancer trap insertion into the Dll locus (P{GawB}Dllmd23; (Calleja et al., 1996) driving nuclear expression of green fluorescent protein (GFP) from a second transgene (P{UAS-GFP.nls}14 (Shiga et al., 1996). Images were collected from dechorionated embryos of genotype: w1118; P{UASGFP.nls}14, P{GawB}Dllmd23/CyO (Dong et al., 2002) at 5 μm intervals every 5 minutes over a period of 8 hours (Figure 1; Supplemental Movie 1). We observed broad Dll expression in the head segments during stages 10–17 (Figure 1A–J). The GFP was not confined to nuclei, but also detectable, albeit weakly, in the cytoplasm and in neuronal processes. These Dll-expressing cells give rise to elements of both the peripheral and central nervous systems (PNS and CNS, respectively). Specifically, the Dll-expressing cells include precursors of multiple larval sense organs and of the brain regions that receive inputs from these sense organs.
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For example, both the larval dorsal organ (DO), which has olfactory and gustatory functions, and the neuroblasts that form the larval antennal lobe in the CNS are derived from the antennal head segment (Schmidt-Ott et al., 1994; Younossi-Hartenstein et al., 1996; Urbach et al., 2003; Younossi-Hartenstein et al., 2006). The antennal lobe consists of glomeruli that carry out the initial processing of olfactory information from the DO and relay that information to higher order brain structures, including the mushroom bodies and the lateral horn (Jefferis et al., 2001; Marin et al., 2002). The larval terminal and ventral organs (TO and VO, respectively), which have gustatory functions, arise from the maxillary segment (Urbach et al., 2003) and project their axons to the subesophageal ganglion (SOG).
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Dll is expressed in the labral, antennal, maxillary and labial head segments and necessary for the development of the non-neural, cuticular components of the labral, antennal (DO), maxillary (TO and VO) and labial sense organs (Cohen and Jurgens, 1989). Loss of Dll function also results in the loss of the Keilin’s organ (KO), a larval mechanosensory structure, associated with the adult thoracic limb primordia (Keilin, 1915; Cohen and Jurgens, 1989). We recently reported that in later stages of embryogenesis Dll is expressed in neurons and their support cells in the DO and TO and that loss of Dll function results in loss of both these neurons and their support cells (Plavicki et al., 2012). Consistent with this, our multiphoton analysis of living embryos permits us to visualize Dll-expressing cells contributing to both neural and non-neural components of the DO, TO, VO and KO (Figure 2; Supplemental Movie 2). By carefully comparing Dll protein expression with GFP expression driven by the P{GawB}Dllmd23, we conclude that the Gal4 driver faithfully recapitulates Dll expression with two exceptions: 1) the embryonic ventral nerve cord glia that express Dll lack P{GawB}Dllmd23 expression; and 2) several cells in the brain that express Dll, also lack P{GawB}Dllmd23 expression.
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Our live imaging indicates that Dll-expressing cells in the antennal and maxillary head segment give rise to both neural and non-neural components of DO, TO and VO. It remains unclear whether Dll-expressing cells in the head segments specifically contribute to the central nervous system regions that receive input from the DO, TO and VO. However, clusters of Dll-expressing cells invaginate from the head segments and migrate to form Dllexpressing regions in the supraesophageal ganglion of the brain (arrowheads in Figure 1 and Supplemental Movie 1). Although Dll was previously shown to be required for DO, TO and VO development and to be expressed within the differentiated organs, this is first time that Dll expression has been followed directly from the precursor stage into the differentiated sense organs. Also, although Dll expression has been reported in primary neuronal clusters in the embryonic Drosophila brain (Sprecher et al., 2007), this is the first demonstration of Dll expression in Drosophila brain precursors (‘stem cells’) prior to their delamination/ specification. Our data, therefore, support the hypothesis that Dll may play critical roles in the specification and/or renewal of neural progenitors as well as in the differentiation of specific neural lineages. Dll expression in cephalic and thoracic sensory organ precursors
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To determine whether the Dll expression observed via live imaging corresponded to precursors that give rise to the sense organs affected in Dll mutants, we examined Dll expression in relation to genes expressed in neural progenitors. Specifically, we examined Dll expression in relation to the zinc-finger transcription factor, senseless (sens), which is expressed in sense organ precursors (SOPs) in the head and thoracic segments (Nolo et al., 2000), and prospero (pros), a homeodomain transcription factor, that is asymmetrically distributed with Numb to confer neural fates (Hirata et al., 1995; Knoblich et al., 1995). We find that Dll is expressed in SOPs in both the cephalic and thoracic segments (Figure 3) as indicated by the co-localization of Dll and Sens in a subset of Sens-expressing SOPs (Figure 3C, F, F′, G and G′). Dll also was co-expressed with Pros in subsets of cells in the head and thorax (Figure 3D, F, F″, G, G″). Previous work has shown that Pros is necessary for neuronal differentiation in both the CNS and PNS (Doe et al., 1991). In the developing larval olfactory system, Pros marks supporting cells, including socket, sheath and shaft cells (Grillenzoni et al., 2007) and is co-expressed in late embryogenesis with Dll (Plavicki et al., 2012). We note that the majority of the Dll-expressing cells in the thoracic segments also express Sens and/or Pros. This suggests that these cells primarily contribute to neural structures such as Keilin’s organ and does not support the current view (e.g. (McKay et al., 2009) that many of the thoracic Dll-expressing cells are leg imaginal disc precursors.
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Dll is expressed in subsets of glia in the ventral nerve cord (VNC) and in neurons and glia in the supraesophageal ganglion Dll expression in the VNC is first observed at embryonic stage 12 and continues in the VNC during the third larval instar (Figure 4A–D, G and I). In contrast to Dll expression in the brain, which includes both neurons and glia, Dll expression in the VNC is exclusively glial. In the embryonic VNC, Dll is expressed in both cell body associated glia (arrows in Figure 4A and B) and dorsal longitudinal glia (arrowheads in Figure 4B and D). Both are subsets of the lateral glia (reviewed in Klaembt et al., 1996). In the larval VNC, Dll is expressed in glial cells that surround the thoracic ganglion (Figure 4I, J, K). Based on their location, it is Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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likely that at least some of these glia migrate into the developing leg during metamorphosis. We also observed Dll expression in subsets of glia within the embryonic and larval brain (Figure 4E–H, J). During embryogenesis, Dll-expressing glia line the preoral commissure (arrowhead in Figure 4F) and also are found in the supraesophageal ganglion (Figure 4F). Dll continues to be expressed in glial cells within the developing larval brain (Figure 4G and H), however we did not detect Dll-expressing glia in the adult CNS (not shown). Dll is expressed in larval and chemosensory neurons
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Dll expression is first observed in chemosensory organs (DO, TO, and VO) during embryogenesis (Figures 2 and 5). During larval and adult stages, Dll expression continues to be detected in chemosensory neurons (Figure 6A–D and not shown) as well as their supporting cells. Chemosensory neurons are clustered together under their respective sense organs and referred to as ganglia. During embryogenesis Dll is expressed in DO, TO and VO ganglia (DOG, TOG and VOG, respectively; Figure 6B and (Plavicki et al., 2012). The majority of DOG neurons respond to olfactory stimuli and send afferent projections to the larval antennal lobe that (LAL) (Gerber and Stocker, 2007). A subset of DO neurons respond to gustatory stimuli and, along with the TO and VO send projections to different regions of the larval brain (Figure 6A). Dll-expressing neurons can be seen projecting to both the LAL and the subesophageal ganglion (SOG; Figure 6A and C).
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During metamorphosis, the CNS and the peripheral components of olfactory system undergo substantial reorganization. The adult Drosophila antenna, which arises from the antennal imaginal disc, houses most adult olfactory neurons. Analogous to the larval organization, the adult ORNs send afferent projections to the adult antennal lobe (AL). ORNs that express the same odorant receptor converge on the same targets within the AL forming structures termed glomeruli (Gao et al., 2000; Vosshall, 2000; Couto et al., 2005). Dll is necessary for specifying antennal fate and loss of Dll function results antennal to leg transformations (Sunkel and Whittle, 1987; Cohen and Jurgens, 1989; Dong et al., 2000). Here, we report that Dll also is expressed in adult ORNs and that projections from Dll-expressing ORNs form glomeruli in the adult AL (Figure 6D). Dll-expressing ORNs also are located in the maxillary palps (data not shown), which like the antenna is derived from the Dll-expressing antennal disc (reviewed in Stocker, 1994). These ORNs also project to the adult AL (reviewed in Stocker, 1994). Gustatory receptor neurons (GRNs) are found in a number places on the adult fly including the labellum, legs, wing margins and female genitalia (reviewed in Vosshall, 2000). The precursors of all of these structures also express Dll. Thus, there appears to be a strong correlation between Dll expression and chemosensation.
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Dll is expressed in the larval and adult optic lobes In addition to being expressed in the developing olfactory system across multiple stages of development, Dll is consistently associated with the development of the visual system. Dll expression is detected in the optic lobes during larval and adult stages, however, in contrast to Dll expression in other sensory systems, expression seems to be limited to brain processing regions and is not detected in retinal neurons or their support cells. The adult optic lobes are divided into distinct morphological and functional domains. From most external, to most internal, these are the lamina, medulla, lobula, and lobula plate (Hofbauer
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and Campos-Ortega, 1990; Meinertzhagen and Hanson, 1993). The larval optic lobe consists of the two proliferative centers, the inner and outer proliferative centers (IPC and OPC respectively) (Hofbauer and Campos-Ortega, 1990). The outer proliferative center (OPC) is crescent shaped neuroepithelium, which is spatially divided along the anterior-posterior axis by the expression of wingless (wg), decapentaplegic (dpp), vsx1, and optix, and gives rise to the medulla (Kaphingst and Kunes, 1994; Egger et al., 2007; Li et al., 2013). wg expression defines the tips of the OPC (tOPC) and has reported to co-localize with Dll expression (Kaphingst and Kunes, 1994). During optic lobe development, Dll along with eyeless (Ey), Sloppy-paired (Slp), and Dichaete (D), are necessary for temporally patterning neuroblasts in the tOPC and generating four neuronal classes that will innervate the adult medulla, lobula, and lobula plate (Bertet et al., 2014). Dll-expressing neuroepithelial cells give rise to the youngest neuroblasts in the tOPC and produce Dll-expressing ganglion mother cells and, ultimately, Dll-expressing class I Spalt major (Salm)/Runt neurons (Bertet et al., 2014). As development persists, Dll expressing neuroblasts begin to express Ey and, as Dll expression wanes, Ey expressing neuroblasts give rise to class 2 sevenup (SVP) neurons (Bertet et al., 2014). Neurons specified during the temporal window of Dll expression are located in the lobula plate cortex (Bertet et al., 2014).
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The cell bodies making up the adult medulla are positioned in either the medulla cortex, which is located between the lamina and medulla neuropil, or the medulla rim, which is situated between the medulla and lobula plate (Morante and Desplan, 2011). Dll is expressed in both the adult medulla cortex and medulla rim (Morante and Desplan, 2008). During larval development of the medulla cortex, Dll is expressed specifically in neurons derived from the oldest neuroblasts, but not in the neuroblasts themselves and does not colocalize with eyeless (ey) or apterous (ap), two broadly expressed markers of cortical cells in the medulla (Morante and Desplan, 2011). Consistent with previous reports, we observed Dll expression in the larval and adult medulla. We also observed Dll expression in laminal neurons in the larval optic lobe in both early and late third instar larvae (Figures 4J and 7A–D, G) and find that Dll is co-expressed with Elav, a marker of differentiated neurons, in a subset of laminal neurons. Dll does not colocalize with Dac, is an early marker of differentiated laminal neurons (Morante and Desplan, 2011). Dll continues to be expressed in the lamina (arrowhead in E) and medulla (arrow in E) during the adult stages (Figure 7E–G). We also observed Dll expression in the central brain in adults (asterisks in Figure 7). We hypothesize that the persistence of Dll expression in adult neurons may indicate a role in the maintenance of specific neuronal fates.
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In summary, we provide a detailed description of Dll expression during embryonic, larval and adult stages of the Drosophila life cycle. We focused on neurons and their supporting cells and demonstrated for the first time that Dll is expressed in the embryonic ectoderm in precursors of the brain. We strengthened the existing link between Dll and chemosensation by establishing that Dll is expressed in most, if not all, olfactory and gustatory organs. In addition, we report that Dll is expressed in only glia of the ventral nerve cord, in both neurons and glia of the larval brain, and in only neurons within the adult brain. We also validated the fidelity of a widely used P{GawB} insertion into the Dll locus. Together, this Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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information provides an important framework for future investigations of specific Dll functions in neural stem cells, neurons, and glia as well as for comparative studies of the role of Dll during development and neural function.
Experimental Procedures Drosophila Stocks The following Drosophila strains were used in this study: Oregon R (Bloomington Drosophila Stock Center); y1 w1 (Bloomington Drosophila Stock Center), Dll01092 (DlllacZ reporter; (Goto and Hayashi, 1997) and w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (Calleja et al., 1996; Shiga et al., 1996; Dong et al., 2002). Immunohistochemistry
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Embryo collection and fixation were performed as described (Langeland, 1999) with staging of embryos according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). The following primary antibodies and dilutions were used: rabbit anti-Dll, 1:100 (Panganiban et al., 1994); 1:200 mouse 22C10, 1:10 (Fujita et al., 1982; Zipursky et al., 1984); rat anti-Elav, 1:10 (O’Neill et al., 1994); mouse anti-Fas2, 1:10 (Developmental Studies Hybridoma Bank) (Hummel et al., 2000); mouse anti-Repo (Alfonso and Jones, 2002); mouse anti-Pros, 1:100 (Campbell et al., 1994); Cy5 anti-HRP, 1:100 (Jackson Immunochem) and guinea pig anti-Sens, 1:5,000 (Nolo et al., 2000). Alexa secondary antibodies (Life Technologies) were used at 1:100. Secondary antibodies used were Alexa Fluor 488 anti-mouse; Alexa Fluor 488 anti-rabbit; Alexa Fluor 488 anti-guinea pig; Alexa Fluor 568 anti-rabbit; Alexa Fluor 633 anti-mouse; Alexa Fluor 633 anti-rabbit; Alexa Fluor 633 streptavidin. Embryos were mounted in Vectashield (Vector Laboratories).
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Confocal Microscopy Confocal images were collected on a Zeiss LSM 510 microscope. Three-dimensional reconstructions were made with Zeiss LSM software, and images were processed by using combinations of Zeiss LSM filtering functions, Image J, and Adobe Photoshop. Optical sections in Z-series collections were collected at 0.4-μm intervals. Multiphoton Microscopy
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Movies were collected using a multiphoton laser scanning microscope (MPLSM) at the Laboratory for Optical and Computational Instrumentation (LOCI) at the University of Wisconsin. This custom-built MPLSM consisted of a titanium sapphire laser (Spectra Physics, Tsunami), set to 900nm excitation wavelength, a photomultiplier tube (ThornEMI-9924B) for detection, and an inverted Nikon Eclipse TE300 microscope coupled to a Bio-Rad MRC-1024 scan assembly. A Nikon 40× oil-immersion lens with a numerical aperture of 1.3 and working distance of 0.2 mm was used for these experiments. Embryos were adhered to coverslips using glue derived from heptane and tape, and covered with a small amount of halocarbon oil. Multiphoton movies spanned embryonic stages 10 to 17. In each movie, a z-series was collected every 5 minutes at an optical step size of 5 μm.
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Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments FUNDING: JP was supported by NIH Training Grant T32 GM-7507 and a grant from the University of Wisconsin Graduate School. This work also was supported by NIH R01 GM59871 (GBF), NIH K99 ES0238 (JSP), and core LOCI support funds (KWE).
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We thank Drs. Margaret McFall-Ngai and Ned Ruby for access to the Zeiss LSM confocal microscope in the University of Wisconsin Department of Medical Microbiology and Immunology that is supported by National Institutes of Health Grant RR12294. We also thank Hugo Bellen for Sens Antibody. The monoclonal, Dac, Elav, Fas2, Pros, Repo, and 22C10 antibodies used in these studies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by the University of Iowa Department of Biological Sciences. J.P. was supported by National Institutes of Health Training Grant T32 GM-7507 and a grant from the University of Wisconsin Graduate School. This work also was supported by National Institutes of Health Grant R01 GM59871 (to G.B.-F.) and an National Institutes of Health Grant K99 ES0238 (J.S.P).
References
Author Manuscript Author Manuscript
Alfonso T, Jones B. gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila. Dev Biol. 2002; 248:369–383. [PubMed: 12167411] Arendt D, Nubler-Jung K. Comparison of early nerve cord development in insects and vertebrates. Development. 1999; 126:2309–2325. [PubMed: 10225991] Bertet C, Li X, Erclik T, Cavey M, Wells B, Desplan C. Temporal patterning of neuroblasts controls Notch-mediated cell survival through regulation of Hid or Reaper. Cell. 2014; 158:1173–1186. [PubMed: 25171415] Calleja M, Moreno E, Pelaz S, Morata G. Visualization of gene expression in living adult Drosophila. Science. 1996; 274(5285):252–255. [PubMed: 8824191] Campbell G, Goring H, Lin T, Spana E, Andersson S, Doe CQ, Tomlinson A. RK2, a glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Drosophila. Development. 1994; 120:2957–2966. [PubMed: 7607085] Campos-Ortega, J.; Hartenstein, V. The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag; 1985. p. 227 Carroll, SB. Endless Forms Most Beautiful: The New Science of Evo Devo. New York: W.W. Norton; 2005. p. 368 Cohen SM, Jurgens G. Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO Journal. 1989; 8(7):2045–2055. [PubMed: 16453891] Couto A, Alenius M, Dickson BJ. Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol. 2005; 15:1535–1547. [PubMed: 16139208] De Robertis E, Sasai Y. A common plan for dorsoventral patterning in Bilateria. Nature. 1996; 380:37–40. [PubMed: 8598900] Doe C, Chu-LaGraff Q, Wright D, Scott M. The prospero gene specifies cell fates in the Drosophila central nervous system. Cell. 1991; 65:451–464. [PubMed: 1673362] Dong PDS, Chu J, Panganiban G. Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity. Development. 2000; 127:209–216. [PubMed: 10603339] Dong PDS, Dicks JS, Panganiban G. Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna. Development. 2002; 129:1967–1974. [PubMed: 11934862] Egger B, Boone JQ, Stevens NR, Brand AH, Doe CQ. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Dev. 2007; 2:1. [PubMed: 17207270] Erwin DH, Davidson EH. The last common bilaterian ancestor. Development. 2002; 129:3021–3032. [PubMed: 12070079]
Dev Dyn. Author manuscript; available in PMC 2017 January 01.
Plavicki et al.
Page 9
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Fujita SC, Zipursky SL, Benzer S, Ferrus A, Shotwell SL. Monoclonal antibodies against the Drosophila nervous system. Proc Natl Acad Sci U S A. 1982; 79:7929–7933. [PubMed: 6818557] Gao Q, Yuan B, Chess A. Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat Neurosci. 2000; 3:780–785. [PubMed: 10903570] Gerber B, Stocker RF. The Drosophila larva as a model for studying chemosensation and chemosensory learning: a review. Chem Senses. 2007; 32:65–89. [PubMed: 17071942] Goto S, Hayashi S. Specification of the embryonic limb primordium by graded activity of Decapentaplegic. Development. 1997; 124:125–132. [PubMed: 9006073] Grillenzoni N, de Vaux V, Meuwly J, Vuichard S, Jarman A, Holohan E, Gendre N, Stocker R. Role of proneural genes in the formation of the larval olfactory organ of Drosophila. Development, Genes and Evolution. 2007; 217:209–219. [PubMed: 17260155] Hirata J, Nakagoshi H, Nabeshima Y, Matsuzaki F. Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature. 1995; 377:627–630. [PubMed: 7566173] Hofbauer A, Campos-Ortega JA. Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux’s Archives of Developmental Biology. 1990; 198:264–274. Holland LZ, Holland ND. Evolution of neural crest and placodes: amphioxus as a model for the ancestral vertebrate? J Anat. 2001; 199:85–98. [PubMed: 11523831] Hummel T, Krukkert K, Roos J, Davis G, Klaembt C. Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron. 2000; 26:357–370. [PubMed: 10839355] Jan LY, Jan YN. Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos. Proc Natl Acad Sci U S A. 1982; 79:2700–2704. [PubMed: 6806816] Jefferis GS, Marin EC, Stocker RF, Luo L. Target neuron prespecification in the olfactory map of Drosophila. Nature. 2001; 414:204–208. [PubMed: 11719930] Kaphingst K, Kunes S. Pattern formation in the visual centers of the Drosophila brain: wingless acts via decapentaplegic to specify the dorsoventral axis. Cell. 1994; 78:437–448. [PubMed: 8062386] Keilin D. Recherches sur les larves de Dipteres cyclorhaphes. Bulletin Scientifique de la France et de la Belgique. 1915; 49:15–198. Klaembt, C.; Hummel, T.; Menne, T.; Sadlowski, E.; Scholz, H.; Stollewerk, A. Development and function of embryonic central nervous system glial cells in Drosophila. 1996. p. 40-49. Knoblich JA, Jan LY, Jan YN. Asymmetric segregation of numb and prospero during cell division. Nature. 1995; 377:624–627. [PubMed: 7566172] Langeland, J. Imaging Immunolabeled Drosophila Embryos by Confocal Microscopy. In: Paddock, S., editor. Confocal Microscopy Methods and Protocols. Totowa: Humana Press; 1999. p. 167-172. Li X, Erclik T, Bertet C, Chen Z, Voutev R, Venkatesh S, Morante J, Celik A, Desplan C. Temporal patterning of Drosophila medulla neuroblasts controls neural fates. Nature. 2013; 498:456–462. [PubMed: 23783517] Long J, Garel S, Depew M, Tobet S, Rubenstein J. DLX5 regulates development of peripheral and central components of the olfactory system. Journal of Neuroscience. 2003; 23:568–578. [PubMed: 12533617] Marin O, Baker J, Puelles L, Rubenstein JL. Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections. Development. 2002; 129:761–773. [PubMed: 11830575] McKay DJ, Estella C, Mann RS. The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene. Development. 2009; 136:61–71. [PubMed: 19036798] Meinertzhagen, IA.; Hanson, TE. The development of the optic lobe. 1993. p. 1363-1491. Mittmann B, Scholtz G. Distal-less expression in embryos of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of mechanoreceptors, chemoreceptors, and the CNS. Dev Genes Evol. 2001; 211:232–243. [PubMed: 11455438] Morante J, Desplan C. The color-vision circuit in the medulla of Drosophila. Curr Biol. 2008; 18:553– 565. [PubMed: 18403201]
Dev Dyn. Author manuscript; available in PMC 2017 January 01.
Plavicki et al.
Page 10
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Morante J, Desplan C. Dissection and staining of Drosophila optic lobes at different stages of development. Cold Spring Harb Protoc. 2011; 2011:652–656. [PubMed: 21632779] Nolo R, Abbott LA, Bellen HJ. Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell. 2000; 102:349–362. [PubMed: 10975525] O’Neill EM, Rebay I, Tjian R, Rubin GM. The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell. 1994; 78:137–147. [PubMed: 8033205] Panganiban G, Nagy L, Carroll SB. The role of the Distal-less gene in the development and evolution of insect limbs. Current Biology. 1994; 4:671–675. [PubMed: 7953552] Panganiban G, Rubenstein J. Developmental functions of the Distal-less/Dlx homeobox genes. Development. 2002; 129:4371–4386. [PubMed: 12223397] Panganiban G, Irvine SM, Lowe C, Roehl H, Corley LS, Sherbon B, Grenier J, Fallon JF, Kimble J, Walker M, Wray GA, Swalla BJ, Martindale MQ, Carroll SB. The origin and evolution of animal appendages. Proceedings of the National Academy of Sciences USA. 1997; 94:5162–5166. Plavicki J, Mader S, Pueschel E, Peebles P, Boekhoff-Falk G. Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system. Proc Natl Acad Sci U S A. 2012; 109:1578–1583. [PubMed: 22307614] Reichert H, Simeone A. Conserved usage of gap and homeotic genes in patterning the CNS. Curr Opin Neurobiol. 1999; 9:589–595. [PubMed: 10508733] Schmidt-Ott U, Gonzalez-Gaitan M, Jackle H, Technau GM. Number, identity, and sequence of the Drosophila head segments as revealed by neural elements and their deletion patterns in mutants. Proc Natl Acad Sci U S A. 1994; 91:8363–8367. [PubMed: 7915837] Shiga Y, Tanaka-Matakatsu M, Hayashi S. A nuclear GFP/beta-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Development, Growth and Differentiation. 1996; 38:99–106. Sprecher S, Reichert H, Hartenstein V. Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain. Gene Expression Patterns. 2007; 7:584–595. [PubMed: 17300994] Sprecher SG, Reichert H. The urbilaterian brain: developmental insights into the evolutionary origin of the brain in insects and vertebrates. Arthropod Struct Dev. 2003; 32:141–156. [PubMed: 18089000] Stocker, RF. The organization of the chemosensory system in Drosophila melanogaster: a review. 1994. p. 3-26. Sunkel CE, Whittle JRS. Brista: A gene involved in the specification and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster. Wilhelm Roux’s Archives of Developmental Biology. 1987; 196:124–132. Urbach R, Schnabel R, Technau GM. The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila. Development. 2003; 130:3589–3606. [PubMed: 12835378] Vosshall LB. Olfaction in Drosophila. Curr Opin Neurobiol. 2000; 10:498–503. [PubMed: 10981620] Wigle JT, Eisenstat DD. Homeobox genes in vertebrate forebrain development and disease. Clin Genet. 2008; 73:212–226. [PubMed: 18241223] Younossi-Hartenstein A, Nassif C, Green P, Hartenstein V. Early neurogenesis of the Drosophila brain. J Comp Neurol. 1996; 370:313–329. [PubMed: 8799858] Younossi-Hartenstein A, Nguyen B, Shy D, Hartenstein V. Embryonic origin of the Drosophila brain neuropile. J Comp Neurol. 2006; 497:981–998. [PubMed: 16802336] Zipursky SL, Venkatesh TR, Teplow DB, Benzer S. Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes. Cell. 1984; 36:15–26. [PubMed: 6420071]
Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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Author Manuscript Figure 1. In vivo multiphoton imaging of Dll expressing cells during embryogenesis
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A–J: Still shots from a multiphoton movie spanning late germ-band elongation (stage 10; A) to head involution (stage 17; J) of an embryo of genotype: w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO in which nuclear GFP expression is driven by a Dll-Gal4 enhancer trap. Dll expression is detected in the antennal and maxillary head segments (asterisks in A– C) that give rise to the olfactory and gustatory organs (asterisks in D–J) as well as presumptive (arrowheads in B–E) and delaminated cells of the brain (arrowheads in F–J). Still images are taken from Supplemental Movie 1 which was initiated at ~6 hours after egg laying and continued until ~14 hours after egg laying. Minutes indicated are relative to start of imaging. Scale bar = 50 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 2. Colocalization of GFP expression regulated by P{GawB}Dllmd23 driver and Dll protein
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A-B″: Dorsal views of stage 16 embryos. A: Still image from Supplemental Movie 2 (frame #97). B: w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO embryo stained for Dll (red) and 22C10 (blue). Monoclonal antibody 22C10 recognizes the microtubule associated protein Futsch and is used to delineate neuronal morphology (Hummel et al., 2000). Dll protein, which is nuclear, is contained within a broader domain of GFP expression. This is expected due to both the perdurance of GFP and GFP localizing to cytoplasm as well as nucleus. We note that while there does not appear to be GFP outside the Dll expression domains, there is some Dll protein expression where we do not detect GFP (e.g. red alone in panel B). GFP and Dll are expressed in the dorsal organ ganglion (arrows), the terminal organ ganglion Dev Dyn. Author manuscript; available in PMC 2017 January 01.
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(arrowheads), and the neurons in the labral sensory organ complex (asterisks). B′: GFP expression relative to 22C10. B″: Dll protein expression relative to 22C10. A–B″: 40× confocal images. Scale bar = 50 μm.
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Figure 3. Dll expression in sensory organ precursors
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A-G: Lateral views of a stage 13 embryo stained for Dll (green), Senseless (Sens; red), and Prospero (Pros; blue). A, E–G″: Single focal planes. B–D: Brightest point projections of a zseries from the boxed region in (A). C: Dll is coexpressed with a subset of Sens-expressing cells in the antennal (an), maxillary (mx), and labial (lb) head segments and the thoracic segments (T1–3). D: Dll is coexpressed with a subset of Pros-expressing cells in the an, mx, and lb head segments and the thoracic segments. E: Boxes 1 and 2 mark the segments shown at higher magnification in F–F″, and G–G″, respectively. In the mx segment, there are many Dll-expressing cells that lack both Pros and Sens, while in the T1 segment almost all of the Dll-expressing cells have Pros and/or Sens expression. F: Dll, Sens and Pros expression in the mx segment. F′: Dll and Sens. F″: Dll and Pros. G: Dll, Sens and Pros expression in T1. G′: Dll and Sens. G″: Dll and Pros. A: 20× confocal image. B–G″: 40× confocal images. Scale bars = 50 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 4. Dll is expressed in subsets of glia in the ventral nerve cord (VNC) and in both neurons and glia in the supraesophageal ganglion
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Lateral (A, B), ventral (C, D), and dorsal (E, F) views of wild type stage 14 embryos stained for Dll (green), the glial marker Repo (red), and the neuronal morphology marker 22C10 (blue). Yellow indicates areas of overlapping Dll and Repo expression. A, B: Dll expression in cell body associated glia (arrows in B) and dorsal longitudinal glia (arrowheads in B). Boxed region in (A) is shown at higher magnification in (B). C, D: Dll expression in dorsal longitudinal glia (arrowheads in D). Boxed region in (C) is shown at higher magnification in (D). E, F: Dll expression in neurons (asterisks in F) and glia (arrows in F) in the supraesophageal ganglion. Boxed region in (E) is shown at higher magnification in (F). G,
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H, I: Dorsal views of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO third instar larval brain stained with anti-Repo (red) and anti-horseradish peroxidase (anti-HRP; blue). The region marked by boxes 1 & 2 are shown at higher magnification in (H) and (I), respectively. Dll continues to be expressed in brain (H) and VNC glia (I) during larval stages. J, K: X-gal staining of an early third instar larval central nervous system from an animal carrying a Dll-lacZ enhancer trap (Dll01092; Goto and Hayashi, 1997). Boxed region in (J) is shown at higher magnification in (K). A, C, E, G: 10× confocal images. B, D, F, H, I: 40× confocal images. J: 10× image. K: 40× image. A, C, E: Scale bars = 100 μm. B, D, F, H, I, K: Scale bars = 25 μm. G, J: Scale bar = 50 μm.
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Figure 5. Dll expression in anterior sense organs of the embryonic peripheral nervous system (PNS)
A–D: Lateral views of a wild type stage 14 embryo stained for Dll (green) and 22C10 (purple). A: Dll is expressed in the labial (LIS; arrow), labral (LRS; open arrowhead) and antennomaxillary sensory complexes (AMC: arrowhead). B: Dll expression in the labial sensory complex. C: Dll expression in the developing cuticular components of the DO and TO. D: Dll expression in the dorsal organ ganglion (DOG) neurons that innervate the dorsal organ (DO in panel C) and in the neurons of the terminal organ ganglion (TOG) that innervate the terminal organ (TO in panel C). E, F: Lateral view of a stage 16 embryo
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stained for Dll (green) and 22C10 (purple). Dll is expressed in both neurons and supporting cells of multiple sense organs in the head, including the DOG, TOG, ventral organ ganglion (VOG), labial organ (LBO). Dll also is expressed in the mechanosensory Keilin’s organs (KO) of the three thoracic segments (T1–T3). The Dll-expressing sense organs in (F) are highlighted in (E). G: Schematic of the large, Dll-expressing chemosensory organs in (E) and (F). DO, TO, and VO are the dorsal organ, terminal organ, and ventral organ, respectively. These organs lie in the larval epidermis and are innervated by neurons with cell bodies in their respective ganglia (DOG, TOG and VOG). From the ganglia, axons project to targets within the larval brain as. Olfactory DOG axons project to the larval antennal lobe (LAL). The LAL relays olfactory information to the mushroom body (MB) and lateral horn (not shown). Gustatory DOG axons, along with gustatory TOG and VOG axons project to the subesophageal ganglion (SOG). A: 10× confocal image. B, C, D, E, F: 40× confocal images. A: Scale bar = 100 μm. B, C, D, F: Scale bars = 50 μm.
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Figure 6. Dll is expressed in larval and adult chemosensory neurons
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A: Dorsal view of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (green) third instar larval brain stained with anti-horseradish peroxidase (HRP; purple). Anti-HRP recognizes Nervana proteins in neuronal membranes and is used to visualize the neuropil (Jan and Jan, 1982). Dll expression is seen in central brain regions. Dll-expressing olfactory receptor neurons (ORNs) in the dorsal organ ganglion (DOG) project to the larval antennal lobe (LAL) whereas Dll-expressing gustatory receptor neurons in the DOG, terminal organ ganglion (TOG) and the ventral organ (VOG) project to the subesophageal ganglion (SOG). B: Co-localization of Dll antibody staining with the neuronal marker Elav (O’Neill et al., 1994) and with GFP driven by P{GawB}Dllmd23 (Calleja et al., 1996). Dll is expressed in TOG neurons. C: Higher magnification view of the LAL boxed in panel A. D: Anterior view of the adult antennal lobes (AL). Both larval and adult Dll expressing ORNs project to their respective antennal lobes. E: Schematic of an adult brain from a frontal view. The mushroom body (MB), lateral horn (LH), and antennal lobe (AL) are indicated. The inset is a schematic of an adult antenna with the olfactory receptor neuron (ORN) cell bodies indicated in red, orange, and yellow. ORNs expressing the same odorant receptor project to a single glomerulus in the AL. A: 10× confocal images. B, C, D: 40× confocal images. A, B, D: Scale bars = 50 μm. C: Scale bar = 12.5 μm.
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Author Manuscript Author Manuscript Author Manuscript Figure 7. Dll is expressed in the larval and adult optic lobes
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A: Lateral view of a w1118; P{UAS-GFP.nls}14 P{GawB}Dllmd23/CyO (green) third instar larval optic lobe stained for Dac (blue) and Elav (red). Dac is an early marker of differentiated laminal neurons, Elav marks laminal neurons in later stages of optic lobe development (Morante and Desplan, 2008; Bertet et al., 2014). B: Higher magnification view of optic lobe shown in (A). C: Dorsal view of a larval third instar brain stained for Dll (green) and Fas2 (red). Fas2 marks a subset of neuronal membranes. Dll is expressed in laminal neurons (box in C) and the medulla (arrows in C). D: High magnification view of boxed area from (C). E: Anterior view of an adult brain stained for Dll (green), Repo (red)
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and anti-HRP (blue). Dll is expressed in neurons in central regions of the adult brain (asterisks) and in the optic lobes (arrow and arrowhead). Arrow indicates Dll-expressing cells in the medulla, while arrowhead indicates Dll-expressing cells in the lamina. F: High magnification view of boxed area in (E). Dll is expressed in adult optic lobe neurons, but not glia. (G) X-gal staining of an adult brain from an animal carrying a reporter insertion into the Dll locus (Dll01092; Goto and Hayashi, 1997). This reporter faithfully recapitulates Dll expression in both the central brain (asterisks) as well as the medulla (arrow) and lamina (arrowhead) of the optic lobes. A, C: 20× confocal images. E: 10× confocal image. B, D, F: 40× confocal images. A, C, E, F, G: Scale bars = 50 μm. B, D: Scale bars = 25 μm.
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