Rolf Urbach

Molecular markers for identified neuroblasts in the developing brain of Drosophila

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. We provide neuroblast maps from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, we present a detailed description of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). We show that based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and that it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, our data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS.

The pattern of neuroblast formation, mitotic domains and proneural gene expression during early brain development in Drosophila.

In the Drosophila embryo, studies on CNS development have so far mainly focused on the relatively simply structured ventral nerve cord. In the trunk, proneural genes become expressed in small cell clusters at specific positions of the ventral neuroectoderm. A lateral inhibition process mediated by the neurogenic genes ensures that only one cell within each proneural cluster delaminates as a neural stem cell (neuroblast). Thus, a fixed number of neuroblasts is formed, according to a stereotypical spatiotemporal and segmentally repeated pattern, each subsequently generating a specific cell lineage. Owing to higher complexity and hidden segmental organisation, the mechanisms underlying the development of the brain are much less understood. In order to pave the way towards gaining deeper insight into these mechanisms, we have undertaken a comprehensive survey of early brain development until embryonic stage 11, when all brain neuroblasts have formed. We describe the complete spatiotemporal pattern of formation of about 100 brain neuroblasts on either side building the trito-, deuto- and protocerebrum. Using 4D-microscopy, we have uncovered various modes of neuroblast formation that are related to specific mitotic domains of the procephalic neuroectoderm. Furthermore, a detailed description is provided of the dynamic expression patterns of proneural genes (achaete, scute, lethal of scute, atonal) in the procephalic neuroectoderm and the individual neuroblasts. Finally, we present direct evidence that, in contrast to the trunk, adjacent cells within specific domains of the procephalic neuroectoderm develop as neuroblasts, indicating that mechanisms controlling neuroblast formation differ between head and trunk.

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. Developmental Dynamics 235:861–869, 2006.

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.

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.

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.

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.

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

BackgroundIn 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 conclusionI 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.

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.

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.