© 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 1290-1301 doi:10.1242/dev.133546

STEM CELLS AND REGENERATION TECHNIQUES AND RESOURCES ARTICLE Gene expression profiles uncover individual identities of gnathal neuroblasts and serial homologies in the embryonic CNS of Drosophila Rolf Urbach*,‡, David Jussen* and Gerhard M. Technau

ABSTRACT hemisegment acquires a unique identity that is reflected in the The numbers and types of progeny cells generated by neural stem typical developmental time point and position of its delamination cells in the developing CNS are adapted to its region-specific from the neuroectoderm, the combinatorial code of genes it functional requirements. In Drosophila, segmental units of the CNS expresses (Hartenstein and Campos-Ortega, 1984; Doe, 1992; develop from well-defined patterns of neuroblasts. Here we Urbach and Technau, 2003b), and the production of a specific cell constructed comprehensive neuroblast maps for the three gnathal lineage (Bossing et al., 1996; Schmidt et al., 1997; Schmid et al., head segments. Based on the spatiotemporal pattern of neuroblast 1999). The developmental patterns and identities of embryonic NBs formation and the expression profiles of 46 marker genes (41 have been described in detailed maps for the brain (Younossi- transcription factors), each neuroblast can be uniquely identified. Hartenstein et al., 1996; Urbach et al., 2003; Urbach and Technau, Compared with the thoracic ground state, neuroblast numbers are 2003a,b) and the thoracic (T1-T3) and anterior abdominal progressively reduced in labial, maxillary and mandibular segments neuromeres (A1-A7) of the ventral nerve cord (VNC) (Doe, 1992; due to smaller sizes of neuroectodermal anlagen and, partially, to Broadus et al., 1995). In contrast to the brain, neuromeres T1-A7 ∼ suppression of neuroblast formation and induction of programmed originate from a rather stereotypic array of 30 NBs per cell death by the Hox gene Deformed. Neuroblast patterns are further hemisegment. In each hemisegment, the Cartesian grid-like influenced by segmental modifications in dorsoventral and proneural expression of anteroposterior (AP) and dorsoventral (DV) gene expression. With the previously published neuroblast maps and patterning genes is virtually identical. Hence, NBs developing ‘ ’ those presented here for the gnathal region, all neuroectodermal from the same quadrants in different segments represent serial neuroblasts building the CNS of the fly (ventral nerve cord and brain, homologs, as they acquire a similar identity (reviewed by Skeath and except optic lobes) are now individually identified (in total 2×567 Thor, 2003; Technau et al., 2006). Recently, NB patterns were also neuroblasts). This allows, for the first time, a comparison of the established for the derived terminal abdominal neuromeres (A8- characteristics of segmental populations of stem cells and to screen A10), and were shown to consist of segment-specifically reduced for serially homologous neuroblasts throughout the CNS. We show sets of serial homologous NBs (Birkholz et al., 2013a). that approximately half of the deutocerebral and all of the tritocerebral Accordingly, the gnathal neuromeres, which constitute the (posterior brain) and gnathal neuroblasts, but none of the subesophageal zone (between brain and truncal neuromeres) protocerebral (anterior brain) neuroblasts, display serial homology comprise the last region of the fly CNS in which patterns and to neuroblasts in thoracic/abdominal neuromeres. Modifications in the identities of NBs have not been resolved so far. The labial (LB), molecular signature of serially homologous neuroblasts are likely to maxillary (MX) and mandibular (MN) neuromeres (from posterior determine the segment-specific characteristics of their lineages. to anterior) develop in the gnathal part of the head, in which segmental units are more evident compared with the pregnathal KEY WORDS: Central nervous system, Neuroblasts, Segmental head from which the embryonic brain arises (Schmidt-Ott and patterning, Drosophila brain, Gene expression profile, Deformed Technau, 1992; Urbach and Technau, 2003a). During ongoing development, these neuromeres fuse to become the subesophageal INTRODUCTION zone at the posteriormost site of the adult brain (Ito et al., 2014), a The development of the central nervous system (CNS) in region implicated in feeding and the taste response (e.g. Scott et al., Drosophila begins with the formation of a stereotyped population 2001; Gendre et al., 2004; Melcher and Pankratz, 2005). of neural stem cells, termed neuroblasts (NBs), which delaminate In this study, we have undertaken a comprehensive survey of from the neuroectoderm in a precise spatiotemporal pattern. the early development of the gnathal neuromeres. We traced the Positional cues within the neuroectoderm provided by products of spatiotemporal pattern of formation of the gnathal NBs. Within the early regulatory genes control the identity of embryonic NBs final NB pattern by stage 11, numbers are progressively diminished (e.g. reviewed by Skeath and Thor, 2003). Each NB within a in LB, MX and MN (i.e. in posterior-anterior order), as compared with thoracic neuromeres. This is mainly due to smaller segmental

Institute of Genetics, University of Mainz, Mainz D-55099, Germany. sizes of gnathal neuroectodermal anlagen, modifications in *These authors contributed equally to this work patterning gene expression, and by the activity of Deformed

‡ (Dfd), which supresses NB formation. Moreover, we provide Author for correspondence ([email protected]) comprehensive maps of the ∼76 gnathal NBs (within the three This is an Open Access article distributed under the terms of the Creative Commons Attribution hemisegments, plus three unpaired midline NBs), which reveal the License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. expression patterns of 46 different marker genes (encoding 41 transcription factors), that are specifically expressed in particular

Received 2 December 2015; Accepted 22 February 2016 NB subsets and form combinatorial codes specifying each NB DEVELOPMENT

1290 STEM CELLS AND REGENERATION Development (2016) 143, 1290-1301 doi:10.1242/dev.133546 individually. Thus, detailed spatiotemporal and molecular maps (Table S1). Thus, we identified 46 marker genes as expressed in all now exist for the entire population of NBs giving rise to the brain [asense (ase), wor, dpn] or in specific subsets of gnathal NBs (except for the optic lobes) and VNC of the fly (in total 2×567 NBs (including two marker genes for typical lineage components) as of neuroectodermal origin). The completed map makes it possible, shown in detail in Fig. 2 (see also Figs S2.1-S2.6) and summarized for the first time, to compare the patterns and molecular in Fig. 3. Among these we provide novel markers for NB subsets: characteristics of segmental populations of NBs throughout the knirps (kni), buttonhead (btd), Dbx, Centaurin gamma 1A CNS and to identify serial homologies. Our data demonstrate that (CenG1A), charybde (chrb), collier (col; knot – FlyBase) and almost all gnathal NBs are serially homologous to NBs in more giant (gt). All of the ∼76 NBs of the three gnathal hemisegments posterior segments, and provide support that most NBs in the (plus three unpaired MNBs) are individually identifiable by their tritocerebrum, and about half of the NBs in the deutocerebrum, characteristic developmental time point, neuroectodermal origin show serial homology to NBs in the VNC. (along AP and DV axes) (Fig. 1, Table S2) and by their unique This study provides a basis for investigating, at the level of expression profiles (Figs 2 and 3, Figs S2.1-S2.6). identified NBs and their lineages, the mechanisms that underlie the structural and functional diversification of the segmental CNS units. Serial homology of gnathal and thoracic/abdominal NBs It will also facilitate comparisons of the patterns and molecular As 32 of the 46 marker genes (indicated with an asterisk in Table S1) profiles of neural stem cells among different species in the context are expressed in segmentally repeated subsets of thoracic and of evolutionary investigations. abdominal NBs, we used the expression of these ‘segmentally conserved’ marker genes as indicators for serial homology of gnathal RESULTS NBs, in addition to the position and time point of their formation Spatiotemporal pattern of NB formation in the gnathal head (Fig. 1, Table S2). Compared with T1 (resembling the ground state) we segments found these characteristics to be strongly conserved in corresponding We traced the pattern of NBs in the gnathal segments in comparison to NBs in LB and MX and, despite more pronounced modifications, T1 in flat preparations of fixed Drosophila embryos during stages 8- largely in the MN. Only in a few cases was the serial homology of 12. This developmental period was subdivided into six stages, most of mandibular NBs ambiguous. Forexample, NB5-5 (lacking in MX and which match those previously reported in the truncal CNS LB) expresses the typical markers gooseberry (gsb)-lacZ, wingless (Hartenstein et al., 1987; Doe, 1992; Broadus et al., 1995; Birkholz (wg)andsloppy paired 1 (slp1), but lacks expression of unplugged et al., 2013a) and brain (Urbach et al., 2003). NBs were identified by (unpg)-lacZ, huckebein (hkb)-lacZ and svp-lacZ, and atypically size, subectodermal position and expression of the stem cell markers expresses chrb, Gt, Dachshund (Dac) and Ladybird early (Lbe) deadpan (dpn)andworniu (wor). The final NB pattern is established (Figs 2 and 3, Figs S2.1, S2.2 and S2.4). Further, we identified ∼3 by late stage 11. At that stage, dpn and wor are expressed not only in NBs per mandibular hemisegment that express an MP2-like marker NBs but also in gnathal sensory organ precursors (SOPs), some of gene profile of Prospero (Pros), Achaete (Ac), Hunchback (Hb) and which lie in close vicinity to dorsal NBs. The SOP-specific marker Ventral nervous system defective (Vnd), but lack the MP2-specific cousin of atonal (cato) (Goulding et al., 2000) allowed us to markers Odd and Aj96-lacZ (lacZAJ96) (Fig. 3C-E, Fig. S2.6A-C). unambiguously discriminate NBs from SOPs (Fig. S1). By means of Pros is nuclear from their time of formation and is not localized the stereotypic position and time at which each NB develops within cortically during the largely symmetric division (Fig. S2.6D,E), as is the neuroectoderm, and expression of the five marker genes engrailed characteristic for MP2 (Spana and Doe, 1995). Considering their (en; indicating posterior NBs and segmental boundaries) (DiNardo molecular profile and division behavior and the fact that they all et al., 1985; Doe, 1992), intermediate neuroblasts defective (ind; emerge from the same ac/scute (sc)-coexpressing proneural domain indicating intermediate NBs) (Weiss et al., 1998), odd skipped (odd), (see below), they might represent ‘duplicated’ serial homologs of the seven up (svp)-lacZ (Broadus et al., 1995) and empty spiracles (ems) MP2s found in truncal segments. (all expressed in specific NB subsets) we were able to identify Lack of marker gene expression indicated the segment-specific individual gnathal NBs in the developing NB pattern (Fig. 1). The absence of particular gnathal NBs (compared with T1), as shown for spatiotemporal pattern of gnathal NB development is largely invariant some examples in the following (Fig. 3, Fig. S2 and as detailed in (among specimen). Whereas the formation of NBs in LB and MX Table S2). Lack of the expression profile Dpn+/Pox neuro (Poxn)+/ resembles that of truncal segments, it is significantly delayed in MN, Eagle (Eg)+/hkb-lacZ+ revealed the absence of NB2-4, and lack of particularly in anterior positions. As compared with T1 (32 NBs per the Dpn+/mirror (mirr)-lacZ+/En–/Runt (Run)+ profile revealed the hemisegment), the final number of NBs is reduced in all gnathal absence of NB2-3, both in all gnathal segments. Lack of the Dpn+/ segments, decreasing from LB to MN: aside the unpaired median NB Eyeless (Ey)+/Ind+/Dbx+/Run+ profile demonstrated loss of NB3-2, (MNB), we found ∼28 NBs in LB, ∼26 NBs in MX, and 22 NBs in and lack of Dpn+/Eg+/Ems+/svp-lacZ–/Run+ loss of NB3-3, both MN per hemisegment (Fig. 1F). specifically in MN. Our data show that segmental modifications in the NB pattern are due to dorsal NBs missing preferentially in the Each gnathal NB expresses a specific combinatorial code of anterior compartment of the gnathal segments. Accordingly, NBs 2- marker genes 3, 2-4 and 5-5 are lacking in LB, and NBs 2-3, 2-4, 2-5, 5-4, 5-5 and In order to further characterize individual gnathal NBs in our map MP2 are lacking in MX. NBs 1-3, 2-3, 2-4, 2-5, 3-2, 3-3, 5-4 and the we investigated the expression of 64 NB marker genes (using longitudinal glioblast (LGB) are missing in MN, but so are the antibodies, in situ probes or lacZ lines; see Table S1), most of which ventral NBs 1-1, 1-2, 5-1 and, exceptionally, the posterior NB6-4. have previously been used to map NBs in the thorax, abdomen and There seems to be no correlation between NBs lacking in gnathal brain (Doe, 1992; Broadus et al., 1995; Younossi-Hartenstein et al., segments and their time point of formation in T1. 1996; Urbach and Technau, 2003b; Birkholz et al., 2013a). In summary, although MD shows the most significant reduction Eighteen of these marker genes, expressed in subsets of brain in NB numbers we identified a potential NB5-5 (missing in MX NBs (Urbach and Technau, 2003b; Urbach, 2007; Kunz et al., 2012; and LB) and ∼3 MP2-like NBs instead of one in the other data not shown), were not expressed in gnathal (or thoracic) NBs hemineuromeres (except MX, where MP2 is missing). Based on DEVELOPMENT

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Fig. 1. Spatiotemporal development of the NB pattern in the gnathal segments. (A-F) Semi-schematic representations of ventral views of the left half of the gnathal segments (MN, mandibular; MX, maxillary; LB, labial) and the prothoracic segment (T1), as framed in the inset in A. Typical NB arrangement is shown at (A) late stage 8 (lst8), (B) stage 9 (st9), (C) early stage 10 (est10), (D) early stage 11 (est11), (E) late stage 11 (lst11) and (F) early stage 12 (est12). The position, formation time point and expression of five marker genes (see color code) allows the identification of individual NBs. NB formation is significantly delayed in MN, similar to observations made in tritocerebrum (TC; NBs light gray) (Urbach et al., 2003). Existence of NB1-3 is unclear in MX and LB (see E and Fig. S2.5). (F) Dorsal labial NBs become separated by salivary gland anlagen (SGA). is, intercalary en stripe; MNB, median neuroblast; ML, ventral midline; LGB, longitudinal glioblast; TA, tracheal anlagen (see also following figures). their individual expression profiles, all NBs in LB and MX and (out of 22) NBs in MN (Fig. 3F). This indicates that profiles of almost all NBs in MN have serially homologous counterparts in the segmentally expressed genes in serially homologous NBs thoracic/abdominal segments. progressively differ from LB to MN. We identified further genes to be exclusively (col, gt, chrb) or preferentially (dac, CenG1A) Gene expression profiles in serially homologous NBs are expressed in particular subsets of mandibular NBs (Fig. 2E,F, progressively modified from LB to MN Fig. 3E, Figs S2.1 and S2.4). Considering these factors altogether, Next, we estimated the extent to which gene expression is modified the expression profile of almost all mandibular NBs is altered in individual gnathal NBs by considering those 32 ‘conserved’ compared with corresponding NBs in other VNC neuromeres. Thus, marker genes (indicated with an asterisk in Table S1) that are the identity of many gnathal, and in particular mandibular, NBs has expressed in segmentally repeated subsets of thoracic/abdominal undergone segmental modifications. NBs (Table S2). We often observed that, compared with T1, expression of genes is lacking or they are ‘ectopically’ expressed: in Identification of serially homologous NBs in the neuromeres total, the expression of eight genes was altered in a subset of 11 (out of trunk and brain of 29) NBs in LB, the expression of ten genes in a subset of 14 (out We further investigated the extent to which individual NBs in the of 26) NBs in MX, and the expression of 18 genes in a subset of 17 brain and trunk share specific developmental and molecular DEVELOPMENT

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Fig. 2. Mapping and identification of NBs in gnathal neuromeres. (A-G) Composite confocal images of flat preparations (ventral view) of late stage 11 embryos stained for different combinations of molecular markers as indicated. Subsets of NBs identified by marker staining(s) and position are labeled. (A′,A″,B′,B″,C′,C″,D′,D″,G′, G″) Left side. (A-A″) Ind+ NB3-2 is lacking in MN. (B-B″) Lbe is atypically expressed in mandibular NB5-5. (C-C″) Run is found in increasingly smaller NB subsets from LB to MN. Ey is expressed in a reduced NB subset in MN. (D-D″) mirr-lacZ is detected in reduced NB subsets in gnathal segments, and Ems in MN. (E,E′)Col is exclusively expressed in the four anteriormost mandibular NBs/ hemisegment. (F,F′)Dacis exclusively expressed in MN, in four to five anterior NBs/hemisegment. (G-G″) Mid is expressed in a reduced NB subset in MN. Repo+ glial cells derive from NB6-4, 7-4 and LGB.

traits, which may support serial homology (Urbach and Technau, individual thoracic and gnathal NBs with those in the two 2004). Although the expression of many marker genes has posterior brain neuromeres, i.e. the tritocerebrum and already been described in brain NBs (Urbach and Technau, deutocerebrum, remarkable parallels were observed 2003b), further markers were analyzed in brain NBs that are (summarized in Fig. 4 and Table S2). Accordingly, we could segmentally expressed in truncal NBs [Nkx6 (HGTX – FlyBase), attribute ten out of 13 NBs in the tritocerebrum to corresponding Dbx, H15-lacZ, Midline (Mid), btd, Ind] (Table S2; and data not NBs in the thoracic and, usually, gnathal neuromeres. Each of the shown) to allow a comprehensive comparison of their molecular remaining three NBs (Td3, Tv4,5) shares features with two or signatures. When comparing the developmental time point, three thoracic NBs, but could not be unambiguously assigned neuroectodermal origin and specific molecular signature of (see Table S2). Although all NBs in the tritocerebrum develop DEVELOPMENT

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Fig. 3. NB maps summarizing the expression of AP and DV patterning genes, temporal genes and other NB marker genes in gnathal neuromeres compared with the prothoracic neuromere. (A-E) Expression of subgroups of marker genes (as indicated by color code) in the full complement of gnathal NBs (left side) at late stage 11; sublineage markers for specific NBs in D are indicated by outer circles. (F) Gray columns represent the total number of NBs in the respective segments. Colored columns indicate numbers of NBs in which expression of the indicated genes is ‘ectopically′ detected (red) or lacking (blue), compared with T1. For example, in MN 14 NBs exhibit ‘ectopic’ expression of one or more of those genes (out of the ten indicated), whereas 13 NBs lack expression of one or more other genes (out of the 11 indicated). In some NBs, expression of certain genes is ‘ectopically’ detected and that of others is lacking (indicated by overlapping columns). from neuroectodermal positions similar to those of their truncal Applying these criteria to individual NBs in the deutocerebrum, homologs, most of them develop significantly later, comparable ∼9 out of 21 NBs exhibit strong parallels with specific NBs in the to the corresponding NBs in MN. thoracic and gnathal neuromeres. Usually, the gene expression DEVELOPMENT

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sublineage markers [e.g. Even skipped (Eve), eveRRK-Gal4, CQ- Gal4] (Fig. 3D, Fig. S2.5). Eve and eveRRK-Gal4 are continuously coexpressed in the NB4-2-derived RP2 motoneuron in thoracic/ abdominal segments (Bossing et al., 1996). In MN, however, we did not detect Eve/eveRRK>GFP+ cells at any stage, indicating that mandibular NB4-2 does not form RP2. By contrast, in LB and MX, we observed that RP2 is often lacking at stage 15, although consistently present at earlier stages (Fig. S2.5). In cell death- deficient Df(3L)H99 embryos (White et al., 1994), Eve+ RP2 is restored to 100% in MX and LB at stage 15 (n=22 hemisegments), indicating that RP2 normally undergoes programmed cell death (PCD) in both segments (Fig. S2.5, Table S3). Thus, the gnathal NB4-2 lineages reveal segment-specific diversification of RP2, which does not develop in MN and undergoes PCD in MX and LB.

PCD does not significantly account for the reduction in NB numbers in gnathal segments To investigate whether PCD regulates segment-specific differences in the gnathal NB pattern we performed antibody staining against Death caspase-1 (Dcp-1), an early hallmark of cell death. During the period of NB formation, until early stage 12, Dcp-1 signal was largely absent from the labial neuroectoderm, and detected particularly in neuroectoderm of the posterior mandibular and anterior maxillary compartment; it was not detectable in gnathal NBs (Fig. 5K). This suggests that PCD does not account for the reduced NB numbers in LB. To investigate whether NB formation is affected by PCD occurring in the posterior MN and anterior MX, we analyzed the NB patterns in Df(3L)H99 embryos at late stage 11. The spatial arrangement and total number of Dpn+ NBs in all mutant gnathal segments did not Fig. 4. Serial homologous NBs in neuromeres of the trunk and brain. Assignment of potentially serially homologous NBs (same color) in obviously differ from wild type (Fig. 5H). Nevertheless, using more neuromeres of brain (TC, tritocerebrum; DC, deutocerebrum; PC, specific markers, in 15% of hemisegments (n=52) an ectopic Eg/En- protocerebrum), gnathal segments (MN, mandibular; MX, maxillary; LB, labial) coexpressing NB was observed at the position of NB6-4 in mutant and prothorax (T1; resembling the ground state), based on developmental time MN (Fig. 5N,N′). Other NBs were never restored in gnathal segments point, neuroectodermal origin (in AP and DV axes) and the specific molecular of Df(3L)H99 mutants. These data suggest that, with the exception of signature of individual NBs (as summarized in Table S2). A few NBs in TC and mandibular NB6-4, PCD does not significantly account for the DC show serial homology to two NBs in neuromeres of ventral nerve cord (VNC). The brain NB map is according to Urbach et al. (2003). reduction in NB numbers in gnathal segments. profiles of deutocerebral NBs show greater similarity to those of Deformed suppresses the formation of specific NBs in corresponding NBs in truncal neuromeres than to those in the gnathal segments tritocerebrum. In some cases, strong parallels were obvious between Because the homeotic gene Deformed (Dfd) is expressed in MN and an NB in the deutocerebrum (Dd3,4,6) and in the truncal neuromeres, MX (Fig. 5I,J) (McGinnes et al., 1998), we tested its role in shaping but a corresponding NB was missing in the tritocerebrum. the gnathal NB pattern. In Dfd16 mutant embryos, we found a slightly Conversely, for some NBs in the tritocerebrum (Td5,7, Tv3) that increased number of Dpn+ NBs in MX (Fig. 5H). Using more specific share similarities with specific NBs in the trunk, corresponding NBs NB markers (e.g. Eg, Ey, En, Ind, Lbe, Vnd, Wg) we often detected could not be identified in the deutocerebrum (Fig. 4). an ectopic (Wg+) NB at the position of NB5-4 (81% of These data demonstrate close correspondence between individual hemisegments, n=26; Fig. 5E-G′) and, in one case (n=48 NBs in the posterior brain (trito- and deutocerebrum) and trunk, hemisegments), an ectopic Eg+ NB at the position of NB2-4 supporting the assumption that these NBs are serially homologous. (Fig. 5C) in the mutant MX. Furthermore, in the mutant MN, an NBs in MX represent a subset of those in LB, and those in MN largely ectopic Eg/En-coexpressing NB at the position of NB6-4 was found represent a subset of those in MX. Interestingly, NBs in the in 20% of hemisegments (n=40; Fig. 5A,B). However, NBs were tritocerebrum again seem to represent a subset of NBs in MN. never restored in the anterior mandibular compartment, where, in the Although our data support the existence of serially homologous NBs wild type, the reduction of the NB pattern is most substantial. Thus, in also in the deutocerebrum, less than half of the deutocerebral NBs and this region Dfd does not seem to play a role in suppressing NB none of the ∼70 protocerebral NBs appear to have counterparts in formation, in accordance with our observation that during the period more posterior segments. Thus, neuromeres following T1 anteriorly of NB formation Dfd is already largely downregulated in the exhibit an increasing degree of derivation from the ground state. neuroectoderm of the anterior mandibular compartment (Fig. 5I). In rare cases, an ectopic Eg+ NB at the position of NB2-4 was also Gnathal NB4-2 lineages reveal segment-specific detected in LB of Dfd16 mutants (2/48 hemisegments; Fig. 5D). Since modifications with regard to RP2 development in wild type we detected Dfd protein in the neuroectoderm from Segmental modifications in gene expression profiles of gnathal NBs which labial NB2-4 seems to develop (Fig. 5I, inset), this suggests are reflected by their cell lineages, as judged from the expression of that Dfd acts cell-autonomously to suppress labial NB2-4 formation. DEVELOPMENT

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Fig. 5. Roles of Dfd, programmed cell death and the number of neuroectodermal progenitors in regulating NB numbers in gnathal segments. (A-D) Gnathal (MN, MX, LB) and prothoracic (T1) left hemisegments in wild type (wt) (A) or Dfd16 mutants (B-D). White dotted lines outline ectopic Eg+ NBs. (E,F) Lbe+ and Ey+ NBs are indicated. Note the ectopic Dpn+ NB5-4-like cell (e5-4) in F. (G,G′) Ectopic NB5-4 expresses row 5-specific Wg (G) and is positioned dorsally to Ind+ NB5-3 (G′). (H) Number of Dpn+ NBs in gnathal and prothoracic hemisegments in wild type (MN, 20.8±1.5; MX, 24.6±1.0; LB, 26.9±0.7; T1, 28.8± 0.5; n=14 each), Df(3L)H99 (H99; MN, 21.2±1.1; MX, 25.7±1.4; LB, 27.5±2.0; T1, 29.2±1.4; n=17 each) and Dfd16 (MN, 21.9±0.9; MX, 27.8±1.8; n=16 each). ND, not determined (Dfd expression largely restricted to MN and MX). (I,J) Dfd expression in outer neuroectoderm (I) and within the NB layer (J). (I) Note the low level of Dfd in the neuroectoderm of MN compared with MX. Inset shows Dfd in anterodorsal labial neuroectodermal cells (arrow). (K-N′) At early stage 12 Dcp-1 signal is reduced in Dfd16 mutant mandibular and maxillary neuroectoderm. Solid white lines indicate the dorsal border of neuromeres. (M) The average number of Dcp-1+ neuroectodermal (NE) cells is diminished in Dfd16 MN and MX. wt, late stage 11 (lst11): MN, 2.6±1.8; MX, 5.1±3.1 (n=14 each). Dfd16 lst11: MN, 0.4±1.6; MX, 0.8±1.2 (n=28 each). wt, early stage 12 (est12): MN, 7.0±5.1; MX, 15.5±5.7 (n=29 each). Dfd16 est12: MN, 2.5±2.0; MX, 5.5±4.5 (n=32 each). (N,N′) Note the ectopic mandibular NB6-4 (e6-4) in Df(3L)H99. (O) Stage 9. Red lines indicate segmental borders, blue lines dorsal neuroectodermal border, black line the ventral midline. In LB, four spatial quadrants are indicated: ad, anterodorsal; av, anteroventral; pd, posterodorsal; pv, posteroventral. (P) Neuroectodermal cell numbers in gnathal and prothoracic hemisegments and in each quadrant of each hemisegment (color coded). n=7-11 (all quadrants). (A-G′,I-L,N,N′) Composite confocal images. (H,M,P) Data are mean±s.d. **P<0.01; ***P<0.0001; ns, not significant; two-tailed t-test.

As we observed an ectopic mandibular NB6-4 both in Df(3L)H99 formation of the mandibular NB6-4. By contrast, the maxillary and Dfd16 mutants, we tested in Dfd16 mutants whether this is due to NB5-4, which was often restored in Dfd16 mutants (see above), was a decrease in PCD in the mandibular neuroectoderm. We observed a never found in Df(3L)H99 mutants (n=24 hemisegments). These substantially reduced number of Dcp-1+ cells in MN and MX findings suggest that Dfd does not exclusively act via PCD to restrict neuroectoderm (Fig. 5K-M), including the En-expressing dorsal NB formation. neuroectoderm from which the ectopic mandibular NB6-4 develops: in wild type, Dcp-1 was detected in 31% of The number of neuroectodermal progenitors differs between hemisegments (n=64), versus 8% of hemisegments (n=26) in gnathal segments Dfd16 mutants (Fig. 5K, inset). Thus, Dfd positively regulates PCD Since PCD does not significantly contribute to segment-specific in neuroectodermal progenitor cells and, thereby, suppresses restriction of NB numbers in the gnathal segments, we next DEVELOPMENT

1296 STEM CELLS AND REGENERATION Development (2016) 143, 1290-1301 doi:10.1242/dev.133546 examined whether it is related to the number of neuroectodermal progenitors from which those NBs develop. Upon staining against Msh (Drop – FlyBase) and En, neuroectodermal cells were counted in all four spatial quadrants of each gnathal and prothoracic hemineuromere at early stage 9, when expression of En (in posterior cells) and Msh (in dorsal cells) is sufficiently established, and before onset of PCD (Fig. 5O). Compared with T1, the total cell number was reduced in MX (by ∼15%) and MN (by ∼43%), but not in LB. Whereas in MX cell numbers were diminished only in the anterior quadrants, in MN a reduction was found in all four quadrants, but most significantly in the anterodorsal quadrant (by ∼64%; Fig. 5P). These findings match the NB pattern of both segments, where NBs preferentially in anterodorsal positions do not form. Therefore, early determination of smaller segmental sizes of gnathal neuroectodermal anlagen appears to mainly define the numbers of delaminating NBs. The various regions within segmental neuroectodermal anlagen (e.g. the anterior compartment of MN) are differentially affected by size reduction.

Modifications in DV gene expression establish an expanded domain of proneural gene expression to allow for rapid formation of NBs in the MN Although NB formation is completed by late stage 11, we find that many NBs of the MN (∼60%), particularly in the anterior compartment, are delayed compared with the other gnathal segments (Fig. 1). Accordingly, most mandibular NBs develop in a fairly narrow time window and, as shown above, from a comparatively small neuroectoderm. To see if this peculiarity is reflected in the activity of proneural genes we investigated the expression of ac, sc and lethal of scute (l’sc; l(1)sc – FlyBase) in the neuroectoderm. The early spatiotemporal expression of these genes in LB, MX and the posterior compartment of MN is similar to the situation in truncal segments, where each NB develops from a small proneural cluster of neuroectodermal cells (Fig. 6F-K) (Jiménez and Campos-Ortega, 1990). In the anterior compartment of MN, however, proneural gene expression starts slightly later, in accordance with the postponed development of many anterior NBs (Fig. 1). Strikingly, during stages 9 to 11, ac and sc are continuously expressed in this compartment at high levels in an Fig. 6. Proneural gene expression in gnathal segments and its segment- ind msh oversized, neuroectodermal stripe (Fig. 6F-I), from which (by specific modification by the activity of DV genes ( , ). (A) Stage 8, showing delayed onset of ac expression in MN. (B,C) During stage 9/10 ac is stages 10/11) eight to nine NBs develop in a narrow time window; expressed in a large domain in MN. (D) Expression of sc is similar to that of ac. these include part of row 3, 4, 5 NBs and the MP2-like NBs (E,F) At stage 11 l’sc expression is downregulated in MX and LB, but is (Fig. 6A-C). Expression of l’sc begins slightly later than that of prominent in anterior MN. Black lines (A,B,D,E) indicate the segmental border ac/sc and, until stage 11, when it is already downregulated in MX of MN. (G-L) Stage 10. Black dashed lines indicate segmental borders and and LB, it is expressed within a large domain covering the MN white dotted frames indicate corresponding regions of intermediate ’ neuroectoderm. Ectopic ind expression in intermediate mandibular (Fig. 6J,K). Unlike ac and sc, l sc is also expressed in the most 68 anterior neuroectoderm of MN, suggesting that development of the neuroectoderm in msh (H) or Ngt40>ind (K) embryos represses ac (J,K). ’ (L) Summary of G-K (see main text for details). (M,N) Composite confocal most anterior NBs (i.e. row 2) depends on L sc activity. images of Dpn and En labeling in wild-type (M) and Ngt40>ind (N) embryos at In contrast to posterior segments, in which ac and sc late stage 11. Upon ind overexpression many row 3, 4, 5 NBs and MP2s are expressed in dorsal and ventral neuroectoderm (Skeath et al., (arrows), which would normally develop from the mandibular ac expression 1994), in the anterior MN expression of both genes also domain (see Fig. S2.6A′,B′), are lacking. covers intermediate neuroectoderm, suggesting a segment-specific regulation by DV genes. It has been shown that Ind is a direct n=28) (Fig. 6M,O). Additionally, upon overexpression of ind (using repressor of ac in truncal segments (Zhao et al., 2007a) and that Msh the maternal driver Ngt40-Gal4), ac expression was significantly is a repressor of ind expression (Zhao et al., 2007b; Seibert et al., reduced in the neuroectoderm (82% of hemisegments, n=40; 2009). We found that, specifically in the anterior MN, msh is also Fig. 6P,Q), often followed by a variable loss of Dpn+ NBs in rows 3, expressed early in the intermediate neuroectoderm where it keeps 4, 5 and the MP2s (50% of hemisegments, n=40; Fig. 6R,S). Thus, ind suppressed and, thereby, enables expression of ac (Fig. 6L,N). segmental modifications in DV gene expression establish an Accordingly, in msh68 mutants ind is derepressed (83% of expanded domain of ac expression to enable the rapid and hemisegments, n=36) and ac is repressed in the anterior- consecutive formation of many NBs from the comparatively small intermediate mandibular neuroectoderm (70% of hemisegments, neuroectoderm in the anterior MN. DEVELOPMENT

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Fig. 7. Comparison of the NB pattern in gnathal, thoracic and abdominal segments. The scheme illustrates NBs that are formed (colored) or missing (X) in the left hemineuromere of mandibular (MN), maxillary (MX) or labial (LB) segment and terminal abdominal segments (A8, A9, A10; according to Birkholz et al., 2013a), as compared with the ground state pattern (according to Doe, 1992), which is repeated in all segments from T1 to A7.

DISCUSSION thorax, the RP2 motoneuron undergoes PCD in the late embryonic NB maps of the gnathal segments complete the LB and MX, whereas it seems not to be formed in MN. characterization of segmental patterns and individual Concomitantly, the NB4-2-specific marker gene code reveals identities for all NBs building the CNS of the fly differences in each gnathal segment in terms of Dfd, Sex combs Whereas substantial knowledge has accumulated regarding the reduced (Scr) and cap-n-collar (cnc) expression; further, CenG1A is characteristics of neural stem cells and the generation of cell tagma-specifically expressed in gnathal NB4-2, and cas and chrb diversity in the truncal (thoracic/abdominal) CNS, relatively little is only in mandibular NB4-2. These divergently expressed factors may known about the developing gnathal neuromeres, which represent a represent candidates that confer the segment-specific characteristics peculiar transitional tissue in that they form the most anterior part of to their lineages. Modifications in the expression profile are found the larval VNC which becomes associated with the adult brain. In among many serially homologous gnathal NBs, but particularly in this study we describe the spatiotemporal pattern of NB formation in mandibular NBs. Therefore, we expect lineage divergences to be the gnathal segments (stages 8 to 11) and provide the first most pronounced in MN. comprehensive maps of 46 marker genes expressed in subsets of The completed NB map sets the stage for systematic cell lineage NBs by late stage 11. At that stage the segmental units of head and analysis. By comparison with the known lineages of the thoracic trunk are most clearly displayed, and the final pattern of NBs is and anterior abdominal neuromeres, such an analysis will uncover established. With the NB maps presented here for the gnathal the degree of conservation of serially homologous lineages in region, characterization of the entire population of neural stem cells derived segments. It will also allow an investigation of the impact of building the VNC and brain (except the optic lobes) of the fly is differences in the molecular signature of their stem cells on the completed (in total 2×567 NBs), each of these cells now being presence or absence of particular progeny cells. individually identified (Doe, 1992; Urbach et al., 2003; Birkholz et al., 2013a; this study, see Figs 4 and 7); this number, however, Regulation of the segment-specific patterns of gnathal NBs does not include the mesectodermal midline progenitors (Bossing Hox genes play a crucial role in segmental identity and patterning, in and Technau, 1994; Bossing and Brand, 2006; Wheeler et al., part by regulating PCD (reviewed by Rogulja-Ortmann and 2006), which remain to be determined in the gnathal and pregnathal Technau, 2008; Reichert and Bello, 2010). In gnathal segments, segments (except MNB, this study). PCD has been reported to occur at segmental boundaries (Nassif Remarkably, almost all identified gnathal NBs represent serial et al., 1998). It is required for the maintenance of a normal boundary homologs of NBs in the thoracic/abdominal segments, and some of between maxillary and mandibular lobes and is induced by Dfd, them of NBs in the brain (as discussed below), which is reflected by which activates the pro-apoptosis gene reaper (Lohmann et al., the neuroectodermal position and time point of their formation, by 2002). Here, we have shown that Dfd suppresses the formation of the NB-specific code of identity genes (see Table S2) and, as shown particular gnathal NBs in different ways: in case of mandibular for some of the gnathal NBs, by the production of typical cell lineage NB6-4 by inducing localized PCD in neuroectodermal progenitors components. As shown in previous studies, some serially that constitute the NB6-4 proneural cluster; in case of maxillary homologous thoracic/abdominal NBs produce modified segment- NB5-4 in a PCD-independent manner, possibly by repressing NB- specific lineages due to differential specification of NBs and their promoting genes (i.e. proneural genes). So far we do not know if Scr progeny, differences in proliferation and/or PCD (for reviews, see plays a role in suppressing NB formation in the labial segment. Rogulja-Ortmann and Technau, 2008; Technau et al., 2006). For the Recently, the Hox genes Abdominal B and caudal have been

NB4-2 lineage, we show that, departing from the situation in the reported to suppress the development of specific NBs in the terminal DEVELOPMENT

1298 STEM CELLS AND REGENERATION Development (2016) 143, 1290-1301 doi:10.1242/dev.133546 abdominal segment in a PCD-independent manner (Birkholz et al., A10. Conversely, seven NBs can be consistently identified in all of 2013b). these segments (NBs 2-1, 2-2, 3-4, 3-5, 5-2, 5-3, 5-6), and thus are Only a few NBs are restored in Dfd16 mutants (NB6-4 in MN, more resistant to segmental modifications in NB patterns. NBs 2-4, 5-4 in MX, NB2-4 in LB). These do not include NBs in the Nevertheless, several of these ‘conserved’ NBs generate lineages strongly reduced anterior compartment of MN. Since Dfd protein is that have been shown to differ among gnathal, thoracic and downregulated until stage 11 specifically in the neuroectoderm of abdominal segments with regard to the number and/or particular this compartment (Fig. 5I) (see also McGinnis et al., 1998), it might types of progeny cells, and there are indications that these not affect the formation of the comparatively late-developing differences primarily affect the later parts of their lineages anterior NBs. As PCD also plays only a minor role, it is likely that (Bossing et al., 1996; Schmidt et al., 1997; Schmid et al., 1999; NB numbers are mainly determined by the smaller size of gnathal Baumgardt et al., 2009; Karlsson et al., 2010). Thus, the early parts segmental anlagen (for blastodermal fate map see Hartenstein et al., of their lineages might form repetitive units of the neuronal network, 1985), which include lower numbers of neuroectodermal the specific functions of which might be required in all truncal and progenitors, especially in the anterior MN, where the decrease in gnathal neuromeres. NBs is most apparent. Different sizes of the gnathal neuroectodermal anlagen are assumed to be defined by early Linking postembryonic lineages to embryonic NBs in the patterning genes acting along the DV (e.g. dpp, sog, dorsal) and AP gnathal CNS (e.g. gap genes hb, btd, gt) axes (Cohen and Jürgens, 1990; Reinitz After a period of mitotic quiescence, a specific subset of embryonic and Levine, 1990; Stathopoulos et al., 2002; Mizutani et al., 2005). NBs resumes proliferation during larval development to produce Finally, we cannot exclude the possibility that homeotic genes (i.e. adult-specific neurons (reviewed by Maurange and Gould, 2005). In Dfd, Scr) are also involved, although the size of the early gnathal thoracic and abdominal segments, embryonic NBs are reactivated in a neuroectoderm seems unaltered in Dfd mutants. segment-specific pattern (Truman and Bate, 1988), which is already Despite the substantial reduction in the number of determined by Hox genes in the embryonic neuroectoderm (Prokop neuroectodermal progenitor cells in the anterior MN, many NBs et al., 1998). In the gnathal segments, a substantial number (>80%) of develop within a short time window from an oversized proneural embryonic NBs disappears during late embryonic and larval domain that constantly expresses high levels of ac and sc. development, most of them due to PCD. Only ∼14 NBs and their Considering its extent and the relatively high number (eight to corresponding lineages (13 paired, 1 unpaired) have been detected in nine) NBs emerging from this proneural domain, we assume that the gnathal region of the late larva, most of which develop in LB these NBs originate from adjacent neuroectodermal progenitor cells, (Kuert et al., 2014). Each of the eight to nine postembryonic lineages similar to the situation in the central brain (Urbach et al., 2003; Kunz in LB could already be linked to a specific embryonic NB, which et al., 2012). This contrasts with the situation in MX, LB and truncal demonstrated that in LB and abdominal segment A1 (with a likewise segments, where proneural clusters are small and where only one cell reduced set of reactivated NBs) they emerge from a similar set of per cluster adopts an NB fate. Thus, spatial and temporal patterns of serially homologous embryonic NBs (Birkholz et al., 2015). Serial proneural gene expression, and presumably the modes of NB homologs of most of these NBs also exist in embryonic MX and MN formation, differ between gnathal segments. We show that the (e.g. NBs 4-2, 5-2, 6-1, 6-2, 7-1, 7-4, MNB), which are likely to oversized ac domain in MN is established by a segment-specific include the precursors of the remaining (approximately six) alteration in DV gene expression. It is likely that other factors are postembryonic lineages described by Kuert et al. (2014) for the also involved in the regionalization and maintenance of ac and sc gnathal region. As four of those lineages express En (Kuert et al., expression. For example, the cephalic gap gene btd (Younossi- 2014), they presumably stem from embryonic En-expressing NBs in Hartenstein et al., 1997), gt (expressed similarly to ac in anterior rows 6 and 7 (NBs 6-1, 6-2, 7-1, 7-4). MN; Fig. S2.1D), cnc or col (both specifically expressed in MN; The postembryonic lineages in the VNC of the larva can be McGinnis et al., 1998; Crozatier et al., 1999) are potential regulators. individually identified based on their morphological characteristics (Truman et al., 2004, 2010) and specific codes of gene expression Segmental deviation from the NB ground state pattern – (e.g. Lacin et al., 2014; Li et al., 2014), and all of them have been comparison of gnathal and terminal abdominal segments linked to identified embryonic NBs (Birkholz et al., 2015). Notably, The pattern of embryonic NBs in the thoracic (T1-T3) and anterior sets of transcription factors found to be expressed in specific abdominal (A1-A7) segments resembles the ground state (T2) postembryonic lineages (Lacin et al., 2014) often differ from those (Lewis, 1978). Segmental divergence from this ground pattern is expressed in their parent embryonic NBs, revealing highly dynamic obvious in all gnathal (this study) and terminal abdominal (A8-A10) expression of these factors. (Birkholz et al., 2013a) segments, as summarized in Fig. 7. In the gnathal segments the population of NBs is progressively diminished Serially homologous NBs in neuromeres of the abdomen/ in the anterior direction, i.e. from LB to MN (∼3, ∼5 and 12 NBs are thorax, gnathal and pregnathal head missing in LB, MX and MN, respectively). In the abdominal tail In a previous report we gave a first example for a serially homologous region it is progressively diminished in the posterior direction, i.e. NB in neuromeres of trunk (abdomen/thorax) and brain (Urbach from A8 to A10 (one, nine and 21 NBs are missing in A8, A9 and et al., 2003). Here we elaborated on these investigations by A10, respectively). Notably, in the terminal abdominal segments comparing the characteristics of all NBs in neuromeres of the (A9, A10) NBs are lacking preferentially in the posterior thorax, gnathal and pregnathal head, the latter giving rise to the brain compartment (Birkholz et al., 2013a), whereas in the gnathal (trito-, deuto- and protocerebrum). Remarkably, almost all NBs in region NBs are missing preferentially in the anterior compartment of the tritocerebrum seem to represent serial homologs of NBs in the each segment. Nevertheless, some NBs appear to be preferred gnathal and thoracic/abdominal segments. Furthermore, the victims of segmental modification in gnathal as well as in terminal population of tritocerebral NBs is likely to represent a subset of abdominal segments: NB2-3 is missing in all gnathal segments and NBs found in MN and, in turn, those in MN, MX and LB represent a in A8-A10, and NB2-4 is missing in all gnathal segments and A9, subset of those in the next posterior segment (MX, LB and T1), DEVELOPMENT

1299 STEM CELLS AND REGENERATION Development (2016) 143, 1290-1301 doi:10.1242/dev.133546 respectively. This suggests that intersegmental modification of the supplementary Materials and Methods. Non-fluorescent stainings were NB pattern in the three gnathal and the tritocerebral neuromeres documented on a Zeiss Axioplan microscope, while fluorescent confocal involves a progressive reduction compared with the thoracic ground images were acquired on a Leica TCS SP5 II microscope. Images were state. Also, approximately half of the deutocerebral NBs display processed with ImageJ (NIH), Adobe Photoshop and Adobe Illustrator. potential serial homology to NBs in the gnathal and thoracic Acknowledgements neuromeres, but not all of them have a counterpart in the We thank Dagmar Volland for technical assistance and are very grateful to Ralf tritocerebrum. Notably, serial homology is not overt in the Pflanz, Uwe Walldorf, Ward Odenwald, Tonia von Ohlen, Joachim Urban, James population of (∼70) protocerebral NBs that constitute the anterior Skeath, Chris Doe, John Reinitz, Tiffany Cook, Christian Klämbt, Andrew Jarman, and largest part of the brain. This is in agreement with recent data Michelè Crozatier, Jürgen Knoblich, Manfred Frasch, Robert Holmgren, Ethan Bier, Krzysztof Jagla, Laura Nilson, Matthew Scott, Ze’ev Paroush, Takako Isshiki, showing that expression patterns of postembryonic lineages in the Markus Noll, Fernando Jimenez, Harald Vaessin, Yu Cai, Benjamin Altenhein, VNC are not consistently linked to the expression of lineages in the Matthias Landgraf, the Bloomington Stock Center, and the Developmental Studies central brain (Li et al., 2014). Together, the appearance of serially Hybridoma Bank (University of Iowa) for providing flies, antibodies and cDNA. homologous NBs seems to mirror the order in which segmental patterning of the neuroectoderm, and the formation and specification Competing interests of NBs, is genetically conserved: conformity to the thoracic ground The authors declare no competing or financial interests. state is highest in LB and progressively diverges from MX to the Author contributions protocerebrum (this study; Crozatier et al., 1999; Seibert et al., 2009; R.U. and G.M.T. conceived the study; R.U. designed experiments with input from reviewed by Urbach and Technau, 2004, 2008). The existence of D.J. and G.M.T.; D.J. performed the majority of experiments; R.U. analyzed the NBs in the brain showing serial homology to those in the VNC is majority of data; R.U. and D.J. prepared the figures; R.U. and G.M.T. wrote the astounding considering that NB formation is significantly delayed in manuscript. MN and tritocerebrum (Fig. 1) (Urbach et al., 2003), and that Funding segmental patterning of the neuroectoderm as well as the formation This work was supported by grants from the Deutsche Forschungsgemeinschaft of NBs is regulated by different mechanisms in the brain (Hirth et al., [UR163/2-2, UR163/3-3 to R.U. and TE130/10-2, TE130/9-3 to G.M.T.]. Deposited 1995; Younossi-Hartenstein et al., 1997; Seibert et al., 2009; Urbach in PMC for immediate release. and Technau, 2004). These intersegmental comparisons not only uncover serial Supplementary information Supplementary information available online at homologies among NBs based on similarities, but also segmental http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.133546/-/DC1 modifications in their expression profiles. Since nearly all of the marker genes considered in this study encode transcription factors, References these modifications are likely to be responsible for the establishment Baumgardt, M., Karlsson, D., Terriente, J., Dıaz-Benjumea,́ F. J. and Thor, S. of segment-specific characteristics of serially homologous NB (2009). Neuronal subtype specification within a lineage by opposing temporal feed-forward loops. Cell 139, 969-982. lineages. Thus, the NB map forms a basis for comprehensive Birkholz, O., Rickert, C., Berger, C., Urbach, R. and Technau, G. M. (2013a). analyses of the lineages building the CNS of the fly and presents Neuroblast pattern and identity in the Drosophila tail region and role of doublesex candidate factors controlling their region-specific composition. in the survival of sex-specific precursors. Development 140, 1830-1842. Birkholz, O., Vef, O., Rogulja-Ortmann, A., Berger, C. and Technau, G. M. Some of these factors are presumably part of the genetic network (2013b). Abdominal-B and caudal inhibit the formation of specific neuroblasts in that provides instructions for the establishment of neural circuits the Drosophila tail region. Development 140, 3552-3564. involved in the processing of feeding and taste response. This work Birkholz, O., Rickert, C., Nowak, J., Coban, I. C. and Technau, G. M. (2015). Bridging might therefore also help to decipher the genetic mechanisms that the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster. Biol. Open 4, 420-434. direct gnathal-specific behavior. Bossing, T. and Brand, A. H. (2006). Determination of cell fate along the For the first time the entire complement of NBs in acomplex animal anteroposterior axis of the Drosophila ventral midline. Development 133, has been mapped. 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