Commissure formation in the embryonic CNS of Drosophila.

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. In later stages, midline glial cells are required to separate anterior and posterior commissures into distinct axon bundles. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, we conducted a saturating ethylmethane sulfonate mutagenesis, screening for mutations which disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. In addition to commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene, two gene functions are required for the initial formation of commissural tracts. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures.     

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.     

Genetic control of Drosophila nerve cord development

The Drosophila ventral nerve cord has been a central model system for studying the molecular genetic mechanisms that control CNS development. Studies show that the generation of neural diversity is a multistep process initiated by the patterning and segmentation of the neuroectoderm. These events act together with the process of lateral inhibition to generate precursor cells (neuroblasts) with specific identities, distinguished by the expression of unique combinations of regulatory genes. The expression of these genes in a given neuroblast restricts the fate of its progeny, by activating specific combinations of downstream genes. These genes in turn specify the identity of any given postmitotic cell, which is evident by its cellular morphology and choice of neurotransmitter.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Antagonistic roles for Ultrabithorax and Antennapedia in regulating segment-specific apoptosis of differentiated motoneurons in the Drosophila embryonic central nervous system

The generation of morphological diversity among segmental units of the nervous system is crucial for correct matching of neurons with their targets and for formation of functional neuromuscular networks. However, the mechanisms leading to segment diversity remain largely unknown. We report here that the Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp) regulate segment-specific survival of differentiated motoneurons in the ventral nerve cord of Drosophila embryos. We show that Ubx is required to activate segment-specific apoptosis in these cells, and that their survival depends on Antp. Expression of the Ubx protein is strongly upregulated in the motoneurons shortly before they undergo apoptosis, and our results indicate that this late upregulation is required to activate reaper-dependent cell death. We further demonstrate that Ubx executes this role by counteracting the function of Antp in promoting cell survival. Thus, two Hox genes contribute to segment patterning and diversity in the embryonic CNS by carrying out opposing roles in the survival of specific differentiated motoneurons.     

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 CNS midline cells and spitz class genes are required for proper patterning of Drosophila ventral neuroectoderm.

The Drosophila embryonic central nervous system (CNS) develops from sets of neuroblasts (NBs) which segregate from the ventral neuroectoderm during early embryogenesis. It is not well established how each individual NB in the neuroectoderm acquires its characteristic identity along the dorsal-ventral axis. Since it is known that CNS midline cells and spitz class genes (pointed, rhomboid, single-minded, spitz and Star) are required for the proper patterning of ventral CNS and epidermis originated from the ventral neuroectoderm, this study was carried out to determine the functional roles of the CNS midline cells and spitz class genes in the fate determination of ventral NBs and formation of mature neurons and their axon pathways. Several molecular markers for the identified NBs, neurons, and axon pathways were employed to examine marker gene expression profile, cell lineage and axon pathway formation in the spitz class mutants. This analysis showed that the CNS midline cells specified by single-minded gene as well as spitz class genes are required for identity determination of a subset of ventral NBs and for formation of mature neurons and their axon pathways. This study suggests that the CNS midline cells and spitz class genes are necessary for proper patterning of the ventral neuroectoderm along the dorsal-ventral axis.     

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.     

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. Here, we use loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. We show that expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process.     

The Tribolium columnar genes reveal conservation and plasticity in neural precursor patterning along the embryonic dorsal-ventral axis

The Drosophila columnar genes are key regulators of neural precursor formation and patterning along the dorsal-ventral axis of the developing CNS and include ventral nerve cord defective (vnd), intermediate nerve cord defective (ind), muscle segment homeodomain (msh), and Epidermal growth factor receptor (Egfr). To investigate the evolution of neural pattern formation, we identified and determined the expression patterns of Tribolium vnd, ind, and msh, and found that they are expressed in the medial, intermediate, and lateral columns of the developing CNS, respectively, in patterns similar, but not identical, to their Drosophila orthologs. The pattern of Egfr activity suggests that the genetic regulatory mechanisms that initiate Tc-vnd expression are similar in Drosophila and Tribolium, whereas those that initiate Tc-ind have diverged. RNAi analyses of gene function show that Tc-vnd and Tc-ind promote the formation of medial and intermediate column neural precursors and that vnd-mediated repression of ind establishes the boundary between the medial and intermediate columns. These data suggest that columnar gene expression and function underlie neural pattern formation in Drosophila, Tribolium, and potentially all insects, but that subtle spatiotemporal differences in expression of these genes may produce species-specific morphological differences.     

Formation of the head-trunk boundary in the animal body plan: an evolutionary perspective

Gene expression analyses and anatomical studies suggest that the body plans of protostomes and deuterostomes are phylogenetically related. In the central nervous system (CNS), arthropods and vertebrates (as well as their closest related phyla the urochordates and cephalochordates) share a nerve cord with rostral specification: the cerebral neuromeres in Drosophila, cerebral sensory vesicle of ascidians and lancelets and the large brain of craniates. Homologous genes, in particular of the otd/Otx and Hox families, are at play in these species to specify the anterior and posterior CNS territories, respectively. In contrast, homologies in the establishment of boundary regions like those separating head and trunk structures in arthropods or mid- and hindbrain domains in chordates are still unclear. We compare in these species the formation, properties and molecular characteristics of these boundaries during embryonic development. We also discuss recent findings suggesting that insects and vertebrates might have co-opted factors of related families to control the formation of these boundary regions, the evolution of which would then appear dramatically different from that of the anterior and posterior CNS domains.     

The midline glial cells are required for regionalization of commissural axons in the embryonic CNS of Drosophila

 The ventral nerve cord of arthropods is characterised by the organisation of major axon tracts in a ladder-like pattern. The individual neuromeres are connected by longitudinal connectives whereas the contra-lateral connections are brought about through segmental commissures. In each neuromere of the embryonic central nervous system (CNS) of Drosophila an anterior and a posterior commissure is found. The development of these commissures requires a set of neurone-glia interactions at the midline. Here we show that both the anterior as well as the posterior commissures are subdivided into three axon-containing regions. Electron microscopy of the ventral nerve cord of mutations affecting CNS midline cells indicates that the midline glial cells are required for this subdivision. In addition the midline glial cells appear required for a crossing of commissural growth cones perpendicular to the longitudinal tracts, since in mutants with defective midline glial cells commissural axons frequently cross the midline at aberrant angles. Received: 6 July 1997 / Accepted: 27 August 1997     

Insights into the urbilaterian brain: conserved genetic patterning mechanisms in insect and vertebrate brain development

Recent molecular genetic analyses of Drosophila melanogaster and mouse central nervous system (CNS) development revealed strikingly similar genetic patterning mechanisms in the formation of the insect and vertebrate brain. Thus, in both insects and vertebrates, the correct regionalization and neuronal identity of the anterior brain anlage is controlled by the cephalic gap genes otd/Otx and ems/Emx, whereas members of the Hox genes are involved in patterning of the posterior brain. A third intermediate domain on the anteroposterior axis of the vertebrate and insect brain is characterized by the expression of the Pax2/5/8 orthologues, suggesting that the tripartite ground plans of the protostome and deuterostome brains share a common evolutionary origin. Furthermore, cross-phylum rescue experiments demonstrate that insect and mammalian members of the otd/Otx and ems/Emx gene families can functionally replace each other in embryonic brain patterning. Homologous genes involved in dorsoventral regionalization of the CNS in vertebrates and insects show remarkably similar patterning and orientation with respect to the neurogenic region (ventral in insects and dorsal in vertebrates). This supports the notion that a dorsoventral body axis inversion occurred after the separation of protostome and deuterostome lineages in evolution. Taken together, these findings demonstrate conserved genetic patterning mechanisms in insect and vertebrate brain development and suggest a monophyletic origin of the brain in protostome and deuterostome bilaterians.     

Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.