Differential effects of EGF receptor signalling on neuroblast lineages along the dorsoventral axis of the Drosophila CNS

The Drosophila ventral nerve cord derives from a stereotype population of about 30 neural stem cells, the neuroblasts, per hemineuromere. Previous experiments provided indications for inductive signals at ventral sites of the neuroectoderm that confer neuroblast identities. Using cell lineage analysis, molecular markers and cell transplantation, we show here that EGF receptor signalling plays an instructive role in CNS patterning and exerts differential effects on dorsoventral subpopulations of neuroblasts. The Drosophila EGF receptor (DER) is capable of cell autonomously specifiying medial and intermediate neuroblast cell fates. DER signalling appears to be most critical for proper development of intermediate neuroblasts and less important for medial neuroblasts. It is not required for lateral neuroblast lineages or for cells to adopt CNS midline cell fate. Thus, dorsoventral patterning of the CNS involves both DER-dependent and -independent regulatory pathways. Furthermore, we discuss the possibility that different phases of DER activation exist during neuroectodermal patterning with an early phase independent of midline-derived signals.     

Dorsoventral patterning in the Drosophila central nervous system: the vnd homeobox gene specifies ventral column identity

The Drosophila CNS develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. In this and the accompanying paper, we investigate the role of two homeobox genes, vnd and ind, in establishing ventral and intermediate cell fates within the Drosophila CNS. During early neurogenesis, Vnd protein is restricted to ventral column neuroectoderm and neuroblasts; later it is detected in a complex pattern of neurons. We use molecular markers that distinguish ventral, intermediate, and dorsal column neuroectoderm and neuroblasts, and a cell lineage marker for selected neuroblasts, to show that loss of vnd transforms ventral into intermediate column identity and that specific ventral neuroblasts fail to form. Conversely, ectopic vnd produces an intermediate to ventral column transformation. Thus, vnd is necessary and sufficient to induce ventral fates and repress intermediate fates within the Drosophila CNS. Vertebrate homologs of vnd (Nkx2.1 and 2.2) are similarly expressed in the ventral CNS, raising the possibility that DV patterning within the CNS is evolutionarily conserved.     

msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm

Many of the mechanisms that govern the patterning of the Drosophila neuroectoderm and mesoderm are still unknown. Here we report the sequence, expression, and regulation of the homeobox gene msh, which is likely to play an important role in the early patterning events of these two tissue primordia. msh expression is first observed in late blastoderm embryos and occurs in longitudinal bands of cells that are fated to become lateral neuroectoderm. This expression is under the control of dorsoventral axis-determination genes and depends on dpp-mediated repression in the dorsal half of the embryo and on fib-(EGF-) mediated repression ventrally. The bands of msh expression define the cells that will form the lateral columns of proneural gene expression and give rise to the lateral row of SI neuroblasts. This suggests that msh may be one of the upstream regulators of the achaete-scute (AS-C) genes and may play a role that is analogous to that of the homeobox gene vnd/NK2 in the medial sector of the neuroectoderm. During neuroblast segregation, msh expression is maintained in a subset of neuroblasts, indicating that msh, like vnd/NK2, could function in both dorsoventral patterning of the neuroectoderm and neuroblast specification. The later phase of msh expression that occurs after the first wave of neuroblast segregation in defined ectodermal and mesodermal clusters of cells points to similar roles of msh in patterning and cell fate specification of the peripheral nervous system, dorsal musculature, and the fat body. A comparison of the expression patterns of the vertebrate homologs of msh, vnd/NK2, and AS-C genes reveals striking similarities in dorsoventral patterning of the Drosophila and vertebrate neuroectoderm and indicates that genetic circuitries in neural patterning are evolutionarily conserved.     

Interaction between Drosophila EGF receptor and vnd determines three dorsoventral domains of the neuroectoderm

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side of the neuroectoderm. Here we show that, in the intermediate column, the receptor tyrosine kinase DER represses expression of proneural genes, achaete and scute, and is required for the formation of neuroblasts. Most of the early function of DER is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that in DER mutants. MAP-kinase activation was also observed in the medial column where esg and proneural gene expression is unaffected by DER. We found that the homeobox gene vnd is required for the expression of esg and scute in the medial column, and show that vnd acts through the negative regulatory region of the esg enhancer that mediates the DER signal, suggesting the role of vnd is to counteract DER-dependent repression. Thus nested expression of vnd and the DER activator rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains.     

Formation and specification of ventral neuroblasts is controlled by vnd in Drosophila neurogenesis

During Drosophila neural development, neuroblasts delaminate from the neuroectoderm of each hemisegment in a stereotypic orthogonal array of five rows and three columns (ventral, intermediate, and dorsal). Prevailing evidence indicates that the individual neuroblast fate is determined by the domain-specific expression of genes along the dorsoventral and anteroposterior axis. Here, we analyze the role of Vnd, a NK-2 homeodomain protein, expressed initially in the ventral neuroectoderm adjacent to the ventral midline, in the dorsoventral patterning of the neuroectoderm and the neuroblasts. We show that in vnd null mutants most ventral neuroblasts do not form and the few that form do not develop ventral fates, but instead develop intermediate-like fates. Furthermore, we demonstrate that Vnd influences the gene expression patterns in the ventral proneural clusters and neuroectoderm, and that its action in neuroblast formation includes, but is not exclusive to the activation of proneural AS-C genes. Through the use of GAL4/UAS gene-expression system we show that ectopic Vnd expression can promote ventral-like fates in intermediate and dorsal neuroblasts and can suppress certain normal characteristics of the intermediate and dorsal neuroectoderm. Our results are discussed in the context of the current evidence in dorsoventral patterning in the Drosophila neuroectoderm.     

Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the msh homeobox gene. Here we describe a new Drosophila homeobox gene, intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. Vertebrate genes closely related to vnd (Nkx2.1 and Nkx2.2), ind (Gsh1 and Gsh2), and msh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved.     

Argos transcription is induced by the Drosophila EGF receptor pathway to form an inhibitory feedback loop

Argos is a secreted molecule with an atypical EGF motif. It was recently shown to function as an inhibitor of the signaling triggered by the Drosophila EGF receptor (DER). In this work, we determine the contribution of Argos to the establishment of cell fates in the embryonic ventral ectoderm. Graded activation of DER is essential for patterning the ventral ectoderm. argos mutant embryos show expansion of ventral cell fates suggesting hyperactivation of the DER pathway. In the embryonic ventral ectoderm, argos is expressed in the ventralmost row of cells. We show that argos expression in the ventral ectoderm is induced by the DER pathway: argos is not expressed in DER mutant embryos, while it is ectopically expressed in the entire ventral ectoderm following ubiquitous activation of the DER pathway. argos expression appears to be triggered directly by the DER pathway, since induction can also be observed in cell culture, following activation of DER by its ligand, Spitz. Argos therefore functions in a sequential manner, to restrict the duration and level of DER signaling. This type of inhibitory feedback loop may represent a general paradigm for signaling pathways inducing diverse cell fates within a population of non-committed cells.     

orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis

We describe mutations in the orb gene, identified previously as an ovarian-specific member of a large family of RNA-binding proteins. Strong orb alleles arrest oogenesis prior to egg chamber formation, an early step of oogenesis, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of oskar and gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway. Orb protein, which is localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. These data will be discussed in the context of a model proposing that Orb is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and that this function contributes to both D/V and A/P axis specification during oogenesis.     

The differentiation between neuroglia and connective tissue sheath in insect ganglia revisited: the neural lamella and perineurial sheath cells are absent in a mesodermless mutant of Drosophila

Two morphogenetic mutations, twist and Delta, that affect the embryonic development of Drosophila in known ways were used to examine the derivation and function of the outer layers of the central nervous system (CNS). Both the extracellular neural lamella, which ensheaths the CNS, and its source, the underlying perineurial sheath cell layer, fail to develop in Drosophila embryos that are homozygous for a loss of function mutation in the twist gene, and which thus lack mesodermal derivatives. The cell layer immediately below the perineurial sheath cells, here termed barrier glial cells, constitute the ion permeability barrier in wild-type embryos. They are present in twist mutant embryos, appear to be normal at the ultrastructural level, and function as a blood-brain ion barrier. The apparent derivation of perineurial sheath cells from mesodermal precursors distinguishes them from neurons, glia and other nonneural components of the CNS, such as tracheae, all of which are of ectodermal origin. We confirm Scharrer's interpretation of the relationship between the perineurium and underlying neuroglia. In embryos homozygous for the neurogenic mutant Delta, an embryonic lethal in which excess ventral blastoderm gives rise to neuroblasts, the CNS forms as an amorphous cell mass, with discontinuous perineurial sheath and barrier glial cell layers. We propose that the cell mass is permeable to lanthanum ions and fails to form a blood-brain barrier because volume growth prevents the formation of continuous surface cell layers.     

Induction of a secondary body axis in Xenopus by antibodies to beta-catenin

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.     

Cellular pathways acting along the germband and in the amnioserosa may participate in germband retraction of the Drosophila melanogaster embryo

Germband retraction in Drosophila melanogaster, like most embryonic morphogenetic events in this organism and in higher eukaryotes, is not well understood. We have taken several approaches to study the relationships between previously identified mutations (u-shaped, serpent, hindsight and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, DER/faint little ball. Our observations from genetic, immunohistochemical, and embryo culture experiments suggest that the former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germband retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by DER/faint little ball, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of DER in the germband may be mediated by the cytoskeleton.     

The role of the msh homeobox gene during Drosophila neurogenesis: implication for the dorsoventral specification of the neuroectoderm

Development of the Drosophila central nervous system begins with the delamination of neural and glial precursors, called neuroblasts, from the neuroectoderm. An early and important step in the generation of neural diversity is the specification of individual neuroblasts according to their position. In this study, we describe the genetic analysis of the msh gene which is likely to play a role in this process. The msh/Msx genes are one of the most highly conserved families of homeobox genes. During vertebrate spinal cord development, Msx genes (Msx1-3) are regionally expressed in the dorsal portion of the developing neuroectoderm. Similarly in Drosophila, msh is expressed in two longitudinal bands that correspond to the dorsal half of the neuroectoderm, and subsequently in many dorsal neuroblasts and their progeny. We showed that Drosophila msh loss-of-function mutations led to cell fate alterations of neuroblasts formed in the dorsal aspect of the neuroectoderm, including a possible dorsal-to-ventral fate switch. Conversely, ectopic expression of msh in the entire neuroectoderm severely disrupted the proper development of the midline and ventral neuroblasts. The results provide the first in vivo evidence for the role of the msh/Msx genes in neural development, and support the notion that they may perform phylogenetically conserved functions in the dorsoventral patterning of the neuroectoderm.     

Integration of the head and trunk segmentation systems controls cephalic furrow formation in Drosophila

Genetic and molecular analyses of patterning of the Drosophila embryo have shown that the process of segmentation of the head is fundamentally different from the process of segmentation of the trunk. The cephalic furrow (CF), one of the first morphological manifestations of the patterning process, forms at the juxtaposition of these two patterning systems. We report here that the initial step in CF formation is a change in shape and apical positioning of a single row of cells. The anteroposterior position of these initiator cells may be defined by the overlapping expression of the head gap gene buttonhead (btd) and the primary pair-rule gene even-skipped (eve). Re-examination of the btd and eve phenotypes in live embryos indicated that both genes are required for CF formation. Further, Eve expression in initiator cells was found to be dependent upon btd activity. The control of eve expression by btd in these cells is the first indication of a new level of integrated regulation that interfaces the head and trunk segmentation systems. In conjunction with previous data on the btd and eve embryonic phenotypes, our results suggest that interaction between these two genes both controls initiation of a specific morphogenetic movement that separates two morphogenetic fields and contributes to patterning the hinge region that demarcates the procephalon from the segmented germ band.