Gene expression profiles uncover individual identities of gnathal neuroblasts and serial homologies in the embryonic CNS of Drosophila.

The numbers and types of progeny cells generated by neural stem cells in the developing CNS are adapted to its region-specific functional requirements. In Drosophila, segmental units of the CNS develop from well-defined patterns of neuroblasts. Here we constructed comprehensive neuroblast maps for the three gnathal head segments. Based on the spatiotemporal pattern of neuroblast formation and the expression profiles of 46 marker genes (41 transcription factors), each neuroblast can be uniquely identified. Compared with the thoracic ground state, neuroblast numbers are progressively reduced in labial, maxillary and mandibular segments due to smaller sizes of neuroectodermal anlagen and, partially, to suppression of neuroblast formation and induction of programmed cell death by the Hox gene Deformed Neuroblast patterns are further influenced by segmental modifications in dorsoventral and proneural gene expression. With the previously published neuroblast maps and those presented here for the gnathal region, all neuroectodermal neuroblasts building the CNS of the fly (ventral nerve cord and brain, except optic lobes) are now individually identified (in total 2×567 neuroblasts). This allows, for the first time, a comparison of the characteristics of segmental populations of stem cells and to screen for serially homologous neuroblasts throughout the CNS. We show that approximately half of the deutocerebral and all of the tritocerebral (posterior brain) and gnathal neuroblasts, but none of the protocerebral (anterior brain) neuroblasts, display serial homology to neuroblasts in thoracic/abdominal neuromeres. Modifications in the molecular signature of serially homologous neuroblasts are likely to determine the segment-specific characteristics of their lineages.

The p21-activated kinase Mbt is a component of the apical protein complex in central brain neuroblasts and controls cell proliferation.

The final size of the central nervous system is determined by precisely controlled generation, proliferation and death of neural stem cells. We show here that the Drosophila PAK protein Mushroom bodies tiny (Mbt) is expressed in central brain progenitor cells (neuroblasts) and becomes enriched to the apical cortex of neuroblasts in a cell cycle- and Cdc42-dependent manner. Using mushroom body neuroblasts as a model system, we demonstrate that in the absence of Mbt function, neuroblasts and their progeny are correctly specified and are able to generate different neuron subclasses as in the wild type, but are impaired in their proliferation activity throughout development. In general, loss of Mbt function does not interfere with establishment or maintenance of cell polarity, orientation of the mitotic spindle and organization of the actin or tubulin cytoskeleton in central brain neuroblasts. However, we show that mbt mutant neuroblasts are significantly reduced in cell size during different stages of development, which is most pronounced for mushroom body neuroblasts. This phenotype correlates with reduced mitotic activity throughout development. Additionally, postembryonic neuroblasts are lost prematurely owing to apoptosis. Yet, preventing apoptosis did not rescue the loss of neurons seen in the adult mushroom body of mbt mutants. From these results, we conclude that Mbt is part of a regulatory network that is required for neuroblast growth and thereby allows proper proliferation of neuroblasts throughout development.

Neuroblast pattern and identity in the Drosophila tail region and role of doublesex in the survival of sex-specific precursors.

The central nervous system is composed of segmental units (neuromeres), the size and complexity of which evolved in correspondence to their functional requirements. In Drosophila, neuromeres develop from populations of neural stem cells (neuroblasts) that delaminate from the early embryonic neuroectoderm in a stereotyped spatial and temporal pattern. Pattern units closely resemble the ground state and are rather invariant in thoracic (T1-T3) and anterior abdominal (A1-A7) segments of the embryonic ventral nerve cord. Here, we provide a comprehensive neuroblast map of the terminal abdominal neuromeres A8-A10, which exhibit a progressively derived character. Compared with thoracic and anterior abdominal segments, neuroblast numbers are reduced by 28% in A9 and 66% in A10 and are almost entirely absent in the posterior compartments of these segments. However, all neuroblasts formed exhibit serial homology to their counterparts in more anterior segments and are individually identifiable based on their combinatorial code of marker gene expression, position, delamination time point and the presence of characteristic progeny cells. Furthermore, we traced the embryonic origin and characterised the postembryonic lineages of a set of terminal neuroblasts, which have been previously reported to exhibit sex-specific proliferation behaviour during postembryonic development. We show that the respective sex-specific product of the gene doublesex promotes programmed cell death of these neuroblasts in females, and is needed for their survival, but not proliferation, in males. These data establish the terminal neuromeres as a model for further investigations into the mechanisms controlling segment- and sex-specific patterning in the central nervous system.

Origin of Drosophila mushroom body neuroblasts and generation of divergent embryonic lineages.

Key to understanding the mechanisms that underlie the specification of divergent cell types in the brain is knowledge about the neurectodermal origin and lineages of their stem cells. Here, we focus on the origin and embryonic development of the four neuroblasts (NBs) per hemisphere in Drosophila that give rise to the mushroom bodies (MBs), which are central brain structures essential for olfactory learning and memory. We show that these MBNBs originate from a single field of proneural gene expression within a specific mitotic domain of procephalic neuroectoderm, and that Notch signaling is not needed for their formation. Subsequently, each MBNB occupies a distinct position in the developing MB cortex and expresses a specific combination of transcription factors by which they are individually identifiable in the brain NB map. During embryonic development each MBNB generates an individual cell lineage comprising different numbers of neurons, including intrinsic γ-neurons and various types of non-intrinsic neurons that do not contribute to the MB neuropil. This contrasts with the postembryonic phase of MBNB development during which they have been shown to produce identical populations of intrinsic neurons. We show that different neuron types are produced in a lineage-specific temporal order and that neuron numbers are regulated by differential mitotic activity of the MBNBs. Finally, we demonstrate that γ-neuron axonal outgrowth and spatiotemporal innervation of the MB lobes follows a lineage-specific mode. The MBNBs are the first stem cells of the Drosophila CNS for which the origin and complete cell lineages have been determined.

Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain.

In central nervous system development, the identity of neural stem cells (neuroblasts) critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. Here, we uncover a novel gene regulatory network underlying DV patterning in the Drosophila brain, and show that the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). We show that Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and that Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis.

Role of en and novel interactions between msh, ind, and vnd in dorsoventral patterning of the Drosophila brain and ventral nerve cord.

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. Here, we extend our knowledge on dorsoventral (DV) patterning of the Drosophila brain and uncover novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). We show that cross-repression between Ind and Msh stabilizes the border between intermediate and dorsal tritocerebrum and deutocerebrum, and that both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunc, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, we find that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord.

Nonfluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

Analysis of Complete Neuroblast Cell Lineages in the Drosophila Embryonic Brain via DiI Labeling.

Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of an NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate individual cells within an NB clone, which reveals information about the temporal succession in which daughter cells are generated. Finally, we describe how to combine clonal DiI labeling with fluorescent antibody staining that permits relating protein expression to individual cells within a labeled NB lineage. These protocols make it feasible to uncover precise lineage relationships between a brain NB and its daughter cells, and to assign gene expression to individual clonal cells. Such lineage-based information is a critical key for understanding the cellular and molecular mechanisms that underlie specification of cell fates in spatial and temporal dimension in the embryonic brain.

Dorsoventral patterning of the brain: a comparative approach.

Development of the central nervous system (CNS) involves the transformation of a two-dimensional epithelial sheet of uniform ectodermal cells, the neuroectoderm, into a highly complex three-dimensional structure consisting of a huge variety of different neural cell types. Characteristic numbers of each cell type become arranged in reproducible spatial patterns, which is a prerequisite for the establishment of specific functional contacts. Specification of cell fate and regional patterning critical depends on positional information conferred to neural stem cells early in the neuroectoderm. This chapter compares recent findings on mechanisms that control the specification of cell fates along the dorsoventral axis during embryonic development of the CNS in Drosophila andvertebrates. Despite the clear structural differences in the organization of the CNS in arthropods and vertebrates, corresponding domains within the developing brain and truncal nervous system express a conserved set of columnar genes (msh/Msx, ind/Gsh, vnd/Nkx) involved in dorsoventral regionalization. In both Drosophila and mouse the expression of these genes exhibits distinct differences between the cephalic and truncal part of the CNS. Remarkably, not only the expression of columnar genes shows striking parallels between both species, but to some extent also their genetic interactions, suggesting an evolutionary conservation of key regulators ofdorsoventral patterning in the brain in terms of expression and function.

Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain.

The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. Here, we investigate the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. We find that localized EGFR signalling in the brain neuroectoderm relies on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, we reveal a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, we show that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, we demonstrate that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.

Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain.

The Drosophila mushroom bodies, centers of olfactory learning and memory in the fly 'forebrain', develop from a set of neural stem cells (neuroblasts) that generate a large number of Kenyon cells (KCs) during sustained cell divisions from embryonic to late pupal stage. We show that retinal homeobox (rx), encoding for an evolutionarily conserved transcription factor, is required for proper development of the mushroom bodies. Throughout development rx is expressed in mushroom body neuroblasts (MBNBs), their ganglion mother cells (MB-GMCs) and young KCs. In the absence of rx function, MBNBs form correctly but exhibit a reduction in cell size and mitotic activity, whereas overexpression of rx increases growth of MBNBs. These data suggest that Rx is involved in the control of MBNB growth and proliferation. Rx also promotes cell cycling of MB-GMCs. Moreover, we show that Rx is important for the survival of MBNBs and Kenyon cells which undergo premature cell death in the absence of rx function. Simultaneous blocking of cell death restores the normal set of MBNBs and part of the KCs, demonstrating that both, impaired proliferation and premature cell death (of MBNBs and KCs) account for the observed defects in mushroom body development. We then show that Rx controls proliferation within the MBNB clones independently of Tailless (Tll) and Prospero (Pros), and does not regulate the expression of other key regulators of MB development, Eyeless (Ey) and Dachshund (Dac). Our data support that the role of Rx in forebrain development is conserved between vertebrates and fly.

Non-fluorescent RNA in situ hybridization combined with antibody staining to visualize multiple gene expression patterns in the embryonic brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for whole mount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

Analysis of complete neuroblast cell lineages in the Drosophila embryonic brain via DiI labeling.

Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of a NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate individual cells within a NB clone, which reveals information about the temporal succession in which daughter cells are generated. Finally, we describe how to combine clonal DiI labeling with fluorescent antibody staining that permits relating protein expression to individual cells within a labeled NB lineage. These protocols make it feasible to uncover precise lineage relationships between a brain NB and its daughter cells, and to assign gene expression to individual clonal cells. Such lineage-based information is a critical key for understanding the cellular and molecular mechanisms that underlie specification of cell fates in spatial and temporal dimension in the embryonic brain.

Six3 demarcates the anterior-most developing brain region in bilaterian animals.

BACKGROUND: The heads of annelids (earthworms, polychaetes, and others) and arthropods (insects, myriapods, spiders, and others) and the arthropod-related onychophorans (velvet worms) show similar brain architecture and for this reason have long been considered homologous. However, this view is challenged by the 'new phylogeny' placing arthropods and annelids into distinct superphyla, Ecdysozoa and Lophotrochozoa, together with many other phyla lacking elaborate heads or brains. To compare the organisation of annelid and arthropod heads and brains at the molecular level, we investigated head regionalisation genes in various groups. Regionalisation genes subdivide developing animals into molecular regions and can be used to align head regions between remote animal phyla. RESULTS: We find that in the marine annelid Platynereis dumerilii, expression of the homeobox gene six3 defines the apical region of the larval body, peripherally overlapping the equatorial otx+ expression. The six3+ and otx+ regions thus define the developing head in anterior-to-posterior sequence. In another annelid, the earthworm Pristina, as well as in the onychophoran Euperipatoides, the centipede Strigamia and the insects Tribolium and Drosophila, a six3/optix+ region likewise demarcates the tip of the developing animal, followed by a more posterior otx/otd+ region. Identification of six3+ head neuroectoderm in Drosophila reveals that this region gives rise to median neurosecretory brain parts, as is also the case in annelids. In insects, onychophorans and Platynereis, the otx+ region instead harbours the eye anlagen, which thus occupy a more posterior position. CONCLUSIONS: These observations indicate that the annelid, onychophoran and arthropod head develops from a conserved anterior-posterior sequence of six3+ and otx+ regions. The six3+ anterior pole of the arthropod head and brain accordingly lies in an anterior-median embryonic region and, in consequence, the optic lobes do not represent the tip of the neuraxis. These results support the hypothesis that the last common ancestor of annelids and arthropods already possessed neurosecretory centres in the most anterior region of the brain. In light of its broad evolutionary conservation in protostomes and, as previously shown, in deuterostomes, the six3-otx head patterning system may be universal to bilaterian animals.

A procephalic territory in Drosophila exhibiting similarities and dissimilarities compared to the vertebrate midbrain/hindbrain boundary region.

BACKGROUND: In vertebrates, the primordium of the brain is subdivided by the expression of Otx genes (forebrain/anterior midbrain), Hox genes (posterior hindbrain), and the genes Pax2, Pax5 and Pax8 (intervening region). The latter includes the midbrain/hindbrain boundary (MHB), which acts as a key organizer during brain patterning. Recent studies in Drosophila revealed that orthologous sets of genes are expressed in a similar tripartite pattern in the late embryonic brain, which suggested correspondence between the Drosophila deutocerebral/tritocerebral boundary region and the vertebrate MHB. To gain more insight into the evolution of brain regions, and particularly the MHB, I examined the expression of a comprehensive array of MHB-specific gene orthologs in the procephalic neuroectoderm and in individually identified neuroblasts during early embryonic stages 8-11, at which the segmental organization of the brain is most clearly displayed. RESULTS AND CONCLUSION: I show that the early embryonic brain exhibits an anterior Otx/otd domain and a posterior Hox1/lab domain, but that Pax2/5/8 orthologs are not expressed in the neuroectoderm and neuroblasts of the intervening territory. Furthermore, the expression domains of Otx/otd and Gbx/unpg exhibit a small common interface within the anterior deutocerebrum. In contrast to vertebrates, Fgf8-related genes are not expressed posterior to the otd/unpg interface. However, at the otd/unpg interface the early expression of other MHB-specific genes (including btd, wg, en), and of dorsoventral patterning genes, closely resembles the situation at the vertebrate MHB. Altogether, these results suggest the existence of an ancestral territory within the primordium of the deutocerebrum and adjacent protocerebrum, which might be the evolutionary equivalent of the region of the vertebrate MHB. However, lack of expression of Pax2/5/8 and Fgf8-related genes, and significant differences in the expression onset of other key regulators at the otd/unpg interface, imply that genetic interactions crucial for the vertebrate organizer activity are absent in the early embryonic brain of Drosophila.

Generation of cell diversity and segmental pattern in the embryonic central nervous system of Drosophila.

Development of the central nervous system (CNS) involves the transformation of a two-dimensional epithelial sheet of uniform ectodermal cells, the neuroectoderm, into a highly complex three-dimensional structure consisting of a huge variety of different neural cell types. Characteristic numbers of each cell type become arranged in reproducible spatial patterns, which is a prerequisite for the establishment of specific functional contacts. The fruitfly Drosophila is a suitable model to approach the mechanisms controlling the generation of cell diversity and pattern in the developing CNS, as it allows linking of gene function to individually identifiable cells. This review addresses aspects of the formation and specification of neural stem cells (neuroblasts) in Drosophila in the light of recent studies on their segmental diversification.

Segment-specific requirements for dorsoventral patterning genes during early brain development in Drosophila.

An initial step in the development of the Drosophila central nervous system is the delamination of a stereotype population of neural stem cells (neuroblasts, NBs) from the neuroectoderm. Expression of the columnar genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh) subdivides the truncal neuroectoderm (primordium of the ventral nerve cord) into a ventral, intermediate and dorsal longitudinal domain, and has been shown to play a key role in the formation and/or specification of corresponding NBs. In the procephalic neuroectoderm (pNE, primordium of the brain), expression of columnar genes is highly complex and dynamic, and their functions during brain development are still unknown. We have investigated the role of these genes (with special emphasis on the Nkx2-type homeobox gene vnd) in early embryonic development of the brain. We show at the level of individually identified cells that vnd controls the formation of ventral brain NBs and is required, and to some extent sufficient, for the specification of ventral and intermediate pNE and deriving NBs. However, we uncovered significant differences in the expression of and regulatory interactions between vnd, ind and msh among brain segments, and in comparison to the ventral nerve cord. Whereas in the trunk Vnd negatively regulates ind, Vnd does not repress ind (but does repress msh) in the ventral pNE and NBs. Instead, in the deutocerebral region, Vnd is required for the expression of ind. We also show that, in the anterior brain (protocerebrum), normal production of early glial cells is independent from msh and vnd, in contrast to the posterior brain (deuto- and tritocerebrum) and to the ventral nerve cord.

The columnar gene vnd is required for tritocerebral neuromere formation during embryonic brain development of Drosophila.

In Drosophila, evolutionarily conserved transcription factors are required for the specification of neural lineages along the anteroposterior and dorsoventral axes, such as Hox genes for anteroposterior and columnar genes for dorsoventral patterning. In this report, we analyse the role of the columnar patterning gene ventral nervous system defective (vnd) in embryonic brain development. Expression of vnd is observed in specific subsets of cells in all brain neuromeres. Loss-of-function analysis focussed on the tritocerebrum shows that inactivation of vnd results in regionalized axonal patterning defects, which are comparable with the brain phenotype caused by mutation of the Hox gene labial (lab). However, in contrast to lab activity in specifying tritocerebral neuronal identity, vnd is required for the formation and specification of tritocerebral neural lineages. Thus, in early vnd mutant embryos, the Tv1-Tv5 neuroblasts, which normally express lab, do not form. Later in embryogenesis, vnd mutants show an extensive loss of lab-expressing cells because of increased apoptotic activity, resulting in a gap-like brain phenotype that is characterized by an almost complete absence of the tritocerebral neuromere. Correspondingly, genetic block of apoptosis in vnd mutant embryos partially restores tritocerebral cells as well as axon tracts. Taken together, our results indicate that vnd is required for the genesis and proper identity specification of tritocerebral neural lineages during embryonic brain development of Drosophila.

Neuroblast formation and patterning during early brain development in Drosophila.

The Drosophila embryo provides a useful model system to study the mechanisms that lead to pattern and cell diversity in the central nervous system (CNS). The Drosophila CNS, which encompasses the brain and the ventral nerve cord, develops from a bilaterally symmetrical neuroectoderm, which gives rise to neural stem cells, called neuroblasts. The structure of the embryonic ventral nerve cord is relatively simple, consisting of a sequence of repeated segmental units (neuromeres), and the mechanisms controlling the formation and specification of the neuroblasts that form these neuromeres are quite well understood. Owing to the much higher complexity and hidden segmental organization of the brain, our understanding of its development is still rudimentary. Recent investigations on the expression and function of proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes have provided new insight into the principles of neuroblast formation and patterning during embryonic development of the fly brain. Comparisons with the same processes in the trunk help us to understand what makes the brain different from the ventral nerve cord. Several parallels in early brain patterning between the fly and the vertebrate systems have become evident.

Segment polarity and DV patterning gene expression reveals segmental organization of the Drosophila brain.

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, we carried out a comprehensive analysis of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, we provide evidence indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deutocerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed.