Dscam1 Has Diverse Neuron Type-Specific Functions in the Developing Drosophila CNS.

Two key features endow Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) with the potential to provide a ubiquitous code for neuronal arbor self-avoidance. First, Dscam1 contains three large cassettes of alternative exons, so that stochastic alternative splicing yields 19,008 Dscam1 isoforms with different Ig ectodomains. Second, each neuron expresses a different subset of Dscam1 isoforms, and isoform-specific homophilic binding causes repulsion. This results in even spacing of self-arbors, while processes of other neurons can intermingle and share the same synaptic partners. In principle, this Dscam1 code could ensure arbor spacing of all neurons in Drosophila This model is strongly supported by studies on dendrite spacing in the peripheral nervous system and studies on axonal branch segregation during brain development. However, the situation is less clear for central neuron dendrites, the major substrate for synaptic input in the CNS. We systematically tested the role of Dscam1 for dendrite growth and spacing in eight different types of identified central neurons. Knockdown of Dscam1 causes severe dendritic clumping and length reductions in efferent glutamatergic and aminergic neurons. The primary cause for these dendritic phenotypes could be impaired self-avoidance, a growth defect, or both. In peptidergic efferent neurons, many central arbors are not formed, arguing for a growth defect. By contrast, knockdown of Dscam1 does not affect dendrite growth or spacing in any of the five different types of interneurons tested. Axon arbor patterning is not affected in any neuron type tested. We conclude that Dscam1 mediates diverse, neuron type-specific functions during central neuron arbor differentiation.

Progressive derivation of serially homologous neuroblast lineages in the gnathal CNS of Drosophila.

Along the anterior-posterior axis the central nervous system is subdivided into segmental units (neuromeres) the composition of which is adapted to their region-specific functional requirements. In Drosophila melanogaster each neuromere is formed by a specific set of identified neural stem cells (neuroblasts, NBs). In the thoracic and anterior abdominal region of the embryonic ventral nerve cord segmental sets of NBs resemble the ground state (2nd thoracic segment, which does not require input of homeotic genes), and serial (segmental) homologs generate similar types of lineages. The three gnathal head segments form a transitional zone between the brain and the ventral nerve cord. It has been shown recently that although all NBs of this zone are serial homologs of NBs in more posterior segments, they progressively differ from the ground state in anterior direction (labial > maxillary > mandibular segment) with regard to numbers and expression profiles. To study the consequences of their derived characters we traced the embryonic lineages of gnathal NBs using the Flybow and DiI-labelling techniques. For a number of clonal types serial homology is rather clearly reflected by their morphology (location and projection patterns) and cell specific markers, despite of reproducible segment-specific differences. However, many lineages, particularly in the mandibular segment, show a degree of derivation that impedes their assignment to ground state serial homologs. These findings demonstrate that differences in gene expression profiles of gnathal NBs go along with anteriorly directed progressive derivation in the composition of their lineages. Furthermore, lineage sizes decrease from labial to mandibular segments, which in concert with decreasing NB-numbers lead to reduced volumes of gnathal neuromeres, most significantly in the mandibular segment.

Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster.

The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS) of Drosophila, neural stem cells (neuroblasts) exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC) detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

Composition of a neuromere and its segmental diversification under the control of Hox genes in the embryonic CNS of Drosophila.

Studies performed at the level of single, identified cells in the fruitfly Drosophila have decisively contributed to our understanding of the mechanisms underlying the development and function of the nervous system. This review highlights some of the work based on single-cell analyses in the embryonic/larval CNS that sheds light on the principles underlying formation and organization of an entire segmental unit and its divergence along the anterior/posterior body axis.

Labeling of single cells in the central nervous system of Drosophila melanogaster.

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

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.

Morphological characterization of the entire interneuron population reveals principles of neuromere organization in the ventral nerve cord of Drosophila.

Decisive contributions to our understanding of the mechanisms underlying the development of the nervous system have been made by studies performed at the level of single, identified cells in the fruit fly Drosophila. While all the motor neurons and glial cells in thoracic and abdominal segments of the Drosophila embryo have been individually identified, few of the interneurons, which comprise the vast majority of cells in the CNS, have been characterized at this level. We have applied a single cell labeling technique to carry out a detailed morphological characterization of the entire population of interneurons in abdominal segments A1-A7. Based on the definition of a set of spatial parameters specifying axonal projection patterns and cell body positions, we have identified 270 individual cell types as the complete hemisegmental set of interneurons and placed these in an interactive database. As well as facilitating analyses of developmental processes, this comprehensive set of data sheds light on the principles underlying the formation and organization of an entire segmental unit of the CNS.

Spatio-temporal pattern of cells expressing the clock genes period and timeless and the lineages of period expressing neurons in the embryonic CNS of Drosophila melanogaster.

The initial steps towards the generation of cell diversity in the central nervous system of the fruitfly Drosophila melanogaster take place during early phases of embryonic development when a stereotypic population of neural progenitor cells (neuroblasts and midline precursors) is formed in a precise spatial and temporal pattern, and subsequently expresses a particular sequence of genes. The clarification of the positional, temporal and molecular features of the individual progenitor cells in the nerve cord and brain as well as of their specific types of neuronal and/or glial progeny cells forms an essential basis to understand the mechanisms controlling their development. The present study contributes to this effort by tracing the expression of period and timeless, two genes that encode transcription factors with a key role in the molecular mechanism of the biological clock. Using a combination of genetic markers and immunocytochemistry with antibodies specific for period and timeless we define the number, location, origin and lineage of period cells in the nerve cord throughout embryogenesis. We also provide the first description of the expression of timeless in the embryonic central nervous system. We found a major transformation in the number and types of cells that express period and timeless takes place between embryonic and larval life.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression.

In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines we have clarified the lineages of the lateral glia in the embryonic ventral nerve cord and linked each glial cell to a specific stem cell. For the lineage of the longitudinal glioblast we show that it consists of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. We demonstrate this by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells.

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo.

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. Here we studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, we show that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, we uncovered the origin of each of the peripheral glial cells and linked them to identified central and peripheral neural stem cells.

Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding.

Cell-shape changes during development require a precise coupling of the cytoskeleton with proteins situated in the plasma membrane. Important elements controlling the shape of cells are the Spectrin proteins that are expressed as a subcortical cytoskeletal meshwork linking specific membrane receptors with F-actin fibers. Here, we demonstrate that Drosophila karussell mutations affect beta-spectrin and lead to distinct axonal patterning defects in the embryonic CNS. karussell mutants display a slit-sensitive axonal phenotype characterized by axonal looping in stage-13 embryos. Further analyses of individual, labeled neuroblast lineages revealed abnormally structured growth cones in these animals. Cell-type-specific rescue experiments demonstrate that beta-Spectrin is required autonomously and non-autonomously in cortical neurons to allow normal axonal patterning. Within the cell, beta-Spectrin is associated with alpha-Spectrin. We show that expression of the two genes is tightly regulated by post-translational mechanisms. Loss of beta-Spectrin significantly reduces levels of neuronal alpha-Spectrin expression, whereas gain of beta-Spectrin leads to an increase in alpha-Spectrin protein expression. Because the loss of alpha-spectrin does not result in an embryonic nervous system phenotype, beta-Spectrin appears to act at least partially independent of alpha-Spectrin to control axonal patterning.

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.

The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells.

In Drosophila, neurons and glial cells are produced by neural precursor cells called neuroblasts (NBs), which can be individually identified. Each NB generates a characteristic cell lineage specified by a precise spatiotemporal control of gene expression within the NB and its progeny. Here we show that the homeobox genes ladybird early and ladybird late are expressed in subsets of cells deriving from neuroblasts NB 5-3 and NB 5-6 and are essential for their correct development. Our analysis revealed that ladybird in Drosophila, like their vertebrate orthologous Lbx1 genes, play an important role in cell fate specification processes. Among those cells that express ladybird are NB 5-6-derived glial cells. In ladybird loss-of-function mutants, the NB 5-6-derived exit glial cells are absent while overexpression of these genes leads to supernumerary glial cells of this type. Furthermore, aberrant glial cell positioning and aberrant spacing of axonal fascicles in the nerve roots observed in embryos with altered ladybird function suggest that the ladybird genes might also control directed cell movements and cell-cell interactions within the developing Drosophila ventral nerve cord.

FlyMove--a new way to look at development of Drosophila.

Development of any organism requires a complex interplay of genes to orchestrate the many movements needed to build up an embryo. Previously, work on Drosophila melanogaster has provided important insights that are often applicable in other systems. But developmental processes, which take place in space and time, are difficult to convey in textbooks. Here, we introduce FlyMove (http://flymove.uni-muenster.de), a new database combining movies, animated schemata, interactive "modules" and pictures that will greatly facilitate the understanding of Drosophila development.