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.     

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.     

Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete–scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete–scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods – the absence of neuroblast segregation and proneural clusters – might be used to support or reject the possible groupings of paraphyletic crustaceans.     

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.     

The Midline Protein Regulates Axon Guidance by Blocking the Reiteration of Neuroblast Rows within the Drosophila Ventral Nerve Cord

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.     

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.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Expression of gooseberry-proximal in the Drosophila developing nervous system responds to cues provided by segment polarity genes

Summary Segment polarity genes define the cell states that are required for proper organization of each metameric unit of the Drosophila embryo. Among these, the gooseberry locus has been shown to be composed of two closely related genes which are expressed in an overlapping single-segment periodicity. We have used specific antibodies raised against the protein product of the gooseberry proximal (gsb-p) gene to determine the spatial distribution of this antigen in wild type embryos, and to monitor the effects of segment polarity mutants on the pattern of the gsb-p protein distribution. We find that the gsb-p protein accumulates beneath each posterior axonal commissure in the progeny of neuroblasts deriving from the epidermal compartments of wingless (wg) and engrailed (en) expression. The results of this analysis support the idea that gsb-p has a specific role in the control of cell fates during neurogenesis, and indicate that en and wg provide critical positional cues to define the domain in which gsbp will be activated. Furthermore, these data suggest that, in order to be expressed in the embryonic CNS, gsb-p may preliminarily require activity of the gooseberry-distal gene in the epidermis. Offprint requests to: S. Côté     

Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka

Background: The orientation of the mitotic spindle influences the asymmetric distribution of cytoplasmic determinants and the positioning of the sibling cell, and therefore has important influences on cell-fate determination and patterning of the embryo. Both the establishment of an axis of polarity and the adjustment of this axis with respect to the coordinates of the embryo have to be controlled. None of the genes identified so far that are involved in these processes seems to have been conserved between flies and nematodes.Results: Here, we show that the bazooka gene encodes a protein with three putative protein-interaction motifs known as PDZ domains and is the first Drosophila representative of the par gene family of Caenorhabditis elegans, members of which are required for establishment of anterior–posterior polarity of the nematode embryo. The bazooka RNA and protein were found to be restricted to the apical cortical cytoplasm of epithelial cells and neuroblasts. Embryos that were mutant for bazooka frequently failed to coordinate the axis of cell polarity with that of the embryo. This was manifested as defective spindle orientation and mispositioning of the daughter cell after division.Conclusions: The Drosophila gene bazooka is likely to be part of a regulatory mechanism required to coordinate the axis of polarity of a cell with that of the embryo. The PDZ domains of Bazooka provide several protein–protein interfaces, which possibly participate in the assembly of a multiprotein complex at the apical pole.     

Polarization of Drosophila Neuroblasts During Asymmetric Division

During Drosophila development, neuroblasts divide to generate progeny with two different fates. One daughter cell self-renews to maintain the neuroblast pool, whereas the other differentiates to populate the central nervous system. The difference in fate arises from the asymmetric distribution of proteins that specify either self-renewal or differentiation, which is brought about by their polarization into separate apical and basal cortical domains during mitosis. Neuroblast symmetry breaking is regulated by numerous proteins, many of which have only recently been discovered. The atypical protein kinase C (aPKC) is a broad regulator of polarity that localizes to the neuroblast apical cortical region and directs the polarization of the basal domain. Recent work suggests that polarity can be explained in large part by the mechanisms that restrict aPKC activity to the apical domain and those that couple asymmetric aPKC activity to the polarization of downstream factors. Polarized aPKC activity is created by a network of regulatory molecules, including Bazooka/Par-3, Cdc42, and the tumor suppressor Lgl, which represses basal recruitment. Direct phosphorylation by aPKC leads to cortical release of basal domain factors, preventing them from occupying the apical domain. In this framework, neuroblast polarity arises from a complex system that orchestrates robust aPKC polarity, which in turn polarizes substrates by coupling phosphorylation to cortical release.Cells use polarity for remarkably diverse functions. In this article, I discuss a polarity that is harnessed to generate daughter cells with different fates. Using polarity to divide asymmetrically addresses several challenges that complex organisms face. The diversification of cell types and tissues that occurs during the development of complex organisms is one such challenge. Drosophila neuroblasts, the subject of this article, undergo repeated symmetry breaking asymmetric cell divisions (ACDs) to populate the central nervous system. In a similar manner in adult organisms, ACDs are important for adult homeostasis, replenishing cells that are turned over during the course of normal physiology (Betschinger and Knoblich 2004).A fundamental aspect of ACD is the production of daughter cells containing distinct fate determinants. To segregate fate determinants, the cell becomes polarized to form mutually exclusive cortical domains, each with a set of fate determinants appropriate for one of the two daughter cells. The cleavage furrow forms at the interface of the two domains, partitioning the fate determinants into the two daughter cells where they function to either self-renew (to keep the progenitor population) or to differentiate (e.g., by changing the pattern of gene expression). One of the unique features of the symmetry breaking that occurs during ACD, at least as implemented by the neuroblast, is that it is remarkably dynamic, developing early in mitosis and depolarizing following the completion of cytokinesis.Since the discovery of the first polarized components, neuroblasts have been an excellent model system for investigating the mechanisms of cell polarization and have been extensively analyzed. Although aspects of neuroblast polarity remain unclear, a core framework for how polarity is created and maintained is emerging. In this article, I focus on neuroblast polarity as centered around the activity of atypical protein kinase C, which has emerged as a key regulator of the process. In this framework, neuroblast polarity can be explained by events that polarize aPKC and those that couple aPKC activity to the polarization of fate determinants.     

ming is expressed in neuroblast sublineages and regulates gene expression in the Drosophila central nervous system.

Cell diversity in the Drosophila central nervous system (CNS) is primarily generated by the invariant lineage of neural precursors called neuroblasts. We used an enhancer trap screen to identify the ming gene, which is transiently expressed in a subset of neuroblasts at reproducible points in their cell lineage (i.e. in neuroblast 'sublineages'), suggesting that neuroblast identity can be altered during its cell lineage. ming encodes a predicted zinc finger protein and loss of ming function results in precise alterations in CNS gene expression, defects in axonogenesis and embryonic lethality. We propose that ming controls cell fate within neuroblast cell lineages.     

Zebrafish segmentation and pair-rule patterning

Segmentation in the vertebrate embryo is evident within the paraxial mesoderm in the form of somites, which are repeated structures that give rise to the vertebrae and muscle of the trunk and tail. In the zebrafish, our genetic screen identified two groups of mutants that affect somite formation and pattern. Mutations of one class, the fss-type mutants, disrupt the formation of the anterior-posterior somite boundaries during somitogenesis. However, segmentation within the paraxial mesoderm is not completely eliminated in these mutants. Irregular somite boundaries form later during embryogenesis and, strikingly, the vertebrae are not fused. Here, we show that formation of the irregular somite boundaries in these mutants is dependent upon the activity of a second group of genes, the you-type genes, which include sonic you, the zebrafish homologue of the Drosophila segment polarity gene, sonic hedgehog. Further to characterize the defects caused by the fss-type mutations, we examined their effects on the expression of her1, a zebrafish homologue of the Drosophila pair-rule gene hairy. In wild-type embryos, her1 is expressed in a dynamic, repeating pattern, remarkably similar to that of its Drosophila and Tribolium counterparts, suggesting that a pair-rule mechanism also functions in the segmentation of the vertebrate paraxial mesoderm. We have found that the fss-type mutants have abnormal pair-rule patterning. Although a her1 mutant could not be identified, analysis of a double mutant that abolishes most her1 expression suggests that a her1 mutant may not display a pair-rule phenotype analogous to the hairy phenotype observed in Drosophila. Cumulatively, our data indicate that zebrafish homologues of both the Drosophila segment polarity genes and pair-rule genes are involved in segmenting the paraxial mesoderm. However, both the relationship between these two groups of genes within the genetic heirarchy governing segmentation and the precise roles that they play during segmentation likely differ significantly between the two organisms. Dev. Genet. 23:65–76, 1998. © 1998 Wiley-Liss, Inc.     

Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC). There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.