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.     

Gene expression patterns in primary neuronal clusters of the Drosophila embryonic brain

The brain of Drosophila is formed by approximately 100 lineages, each lineage being derived from a stem cell-like neuroblast that segregates from the procephalic neurectoderm of the early embryo. A neuroblast map has been established in great detail for the early embryo, and a suite of molecular markers has been defined for all neuroblasts included in this map [Urbach, R., Technau, G.M. (2003a) Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development 130, 3621-3637]. However, the expression of these markers was not followed into later embryonic or larval stages, mainly due to the fact that anatomical landmarks to which expression patterns could be related had not been defined. Such markers, in the form of stereotyped clusters of neurons whose axons project along cohesive bundles ("primary axon bundles" or "PABs") are now available [Younossi-Hartenstein, A., Nguyen, B., Shy, D., Hartenstein, V. 2006. Embryonic origin of the Drosophila brain neuropile. J. Comp. Neurol. 497, 981-998]. In the present study we have mapped the expression of molecular markers in relationship to primary neuronal clusters and their PABs. The markers we analyzed include many of the genes involved in patterning of the brain along the anteroposterior axis (cephalic gap genes, segment polarity genes) and dorso-ventral axis (columnar patterning genes), as well as genes expressed in the dorsal protocerebrum and visual system (early eye genes). Our analysis represents an important step along the way to identify neuronal lineages of the mature brain with genes expressed in the early embryo in discrete neuroblasts. Furthermore, the analysis helped us to reconstruct the morphogenetic movements that transform the two-dimensional neuroblast layer of the early embryo into the three-dimensional larval brain and provides the basis for deeper understanding of how the embryonic brain develops.     

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.     

Convergence of dorsal, dpp, and egfr signaling pathways subdivides the drosophila neuroectoderm into three dorsal-ventral columns

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). Here we investigate the origins of DV patterning within the Drosophila CNS. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm-ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh)-which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. Here we describe the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. We confirm that the vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report we show that vnd is not regulated by Dpp signaling. We show that ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and that abnormally high Dpp can repress ind expression. Finally, we show that the msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. We conclude that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS.     

Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen

During Drosophila neuroblast lineage development, temporally ordered transitions in neuroblast gene expression have been shown to accompany the changing repertoire of functionally diverse cells generated by neuroblasts. To broaden our understanding of the biological significance of these ordered transitions in neuroblast gene expression and the events that regulate them, additional genes have been sought that participate in the timing and execution of these temporally controlled events. To identify dynamically expressed neural precursor genes, we have performed a differential cDNA hybridization screen on a stage specific embryonic head cDNA library, followed by whole-mount embryo in situ hybridizations. Described here are the embryonic expression profiles of 57 developmentally regulated neural precursor genes. Information about 2389 additional genes identified in this screen, including 1614 uncharacterized genes, is available on-line at 'BrainGenes: a search for Drosophila neural precursor genes' (http://sdb.bio.purdue.edu/fly/brain/ahome.htm).     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.     

A single cell analysis of engrailed expression in the early embryonic brain of the grasshopper Schistocerca gregaria: ontogeny and identity of the secondary headspot cells

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Local inhibition of Drosophila homeobox-containing neural dorsoventral patterning genes by Dpp

An important step in Drosophila neurogenesis is to establish the neural dorsoventral (DV) patterning. Here we describe how dpp loss-of- and gain-of-function mutation affects the homeobox-containing neural DV patterning genes expressed in the ventral neuroectoderm. Ventral nervous system defective (vnd), intermediate neuroblast defective (ind), muscle-specific homeobox (msh), and orthodenticle (otd) genes participate in development of the central nervous system and peripheral nervous system, and encode homeodomain proteins. otd and msh genes were ectopically expressed in dpp loss-of-function mutation, but vnd and ind were not affected. However, when dpp was ectopically expressed in the ventral neuroectoderm by rho-GAL4/UAS-dpp system, it caused the repression of vnd, and msh expressions in ventral and dorsal columns of the neuroectoderm, respectively, but not that of ind. The later expression pattern of otd was also restricted by Dpp. The expression pattern of msh, vnd and otd in dpp loss-of-function and gain-of-function mutation indicates that Dpp activity does not reach to the ventral midline and it works locally to establish the dorsal boundary of the ventral neuroectoderm.     

At the nexus between pattern formation and cell-type specification: the generation of individual neuroblast fates in the Drosophila embryonic central nervous system.

The specification of specific and often unique fates to individual cells as a function of their position within a developing organism is a fundamental process during the development of multicellular organisms. The development of the Drosophila embryonic central nervous system serves as an excellent model system in which to clarify the developmental mechanisms that link pattern formation to cell-type specification. The Drosophila embryonic central nervous system develops from a set of neural stem cells termed neuroblasts. Neuroblasts arise from the ectoderm in an invariant pattern, and each neuroblast acquires a unique fate based on its position within this pattern. Two groups of genes recently have been demonstrated to govern the individual fate specification of neuroblasts. One group, the segment polarity genes, enables neuroblasts that develop in different anteroposterior positions to acquire different fates. The second group, referred to as the columnar genes, ensures that neuroblasts that develop in different dorsoventral domains assume different fates. When integrated, the activities of the segment polarity and columnar genes create a Cartesian coordinate system that bestows unique fates to individual neuroblasts as a function of their position of formation within the ectoderm. BioEssays 1999;21:922-931.     

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. Here, we investigate the role of PcG genes in postembryonic development of the Drosophila CNS. We use mosaic-based MARCM techniques to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. We find that proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. Our findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development.     

Early steps in building the insect brain: neuroblast formation and segmental patterning in the developing brain of different insect species

In insects, morphological, molecular and genetic studies have provided a detailed insight into the ontogenetic processes that shape the ventral nerve cord. On the other hand, owing to its complexity and less obvious segmental composition, the knowledge about the development of the brain is still fragmentary. A promising approach towards gaining insight into fundamental processes underlying brain development is the comparison of embryonic brain development among different insect species. However, so far such comparative analyses are scarce. In this review, we summarize and compare data on the early steps in brain formation in different hemi- and holometabolous insects. We show that basic aspects of the spatial and temporal development of the embryonic brain neuroblast pattern are conserved among insects. Furthermore, we compare the number and proliferation patterns of neuroblasts related to major neuropil structures such as mushroom bodies, central complex, and antennal lobe. Finally, comparing the expression patterns of engrailed in different species, and considering new data from Drosophila melanogaster, we discuss the segmental organization of the insect brain.     

Unique establishment of procephalic head segments is supported by the identification of cis-regulatory elements driving segment-specific segment polarity gene expression in Drosophila

Anterior head segmentation is governed by different regulatory mechanisms than those that control trunk segmentation in Drosophila. For segment polarity genes, both initial mode of activation as well as cross-regulatory interactions among them differ from the typical genetic circuitry in the trunk and are unique for each of the procephalic segments. In order to better understand the segment-specific gene network responsible for the procephalic expression of the earliest active segment polarity genes wingless and hedgehog, we started to identify and analyze cis-regulatory DNA elements of these genes. For hedgehog, we could identify a cis-regulatory element, ic-CRE, that mediates expression specifically in the posterior part of the intercalary segment and requires promoter-specific interaction for its function. The intercalary stripe is the last part of the metameric hedgehog expression pattern that appears during embryonic development, which probably reflects the late and distinct establishment of this segment. The identification of a cis-regulatory element that is specific for one head segment supports the mutant-based observation that the expression of segment polarity genes is governed by a unique gene network in each of the procephalic segments. This provides further indication that the anterior-most head segments represent primary segments, which are set up independently, in contrast to the secondary segments of the trunk, which resemble true repetitive units.