Convergence of dorsal, dpp, and egfr signaling pathways subdivides the drosophila neuroectoderm into three dorsal-ventral columns

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). Here we investigate the origins of DV patterning within the Drosophila CNS. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm-ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh)-which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. Here we describe the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. We confirm that the vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report we show that vnd is not regulated by Dpp signaling. We show that ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and that abnormally high Dpp can repress ind expression. Finally, we show that the msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. We conclude that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS.     

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila Central Nervous System

The Drosophila embryonic Central Nervous System (CNS) develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.     

Local inhibition of Drosophila homeobox-containing neural dorsoventral patterning genes by Dpp

An important step in Drosophila neurogenesis is to establish the neural dorsoventral (DV) patterning. Here we describe how dpp loss-of- and gain-of-function mutation affects the homeobox-containing neural DV patterning genes expressed in the ventral neuroectoderm. Ventral nervous system defective (vnd), intermediate neuroblast defective (ind), muscle-specific homeobox (msh), and orthodenticle (otd) genes participate in development of the central nervous system and peripheral nervous system, and encode homeodomain proteins. otd and msh genes were ectopically expressed in dpp loss-of-function mutation, but vnd and ind were not affected. However, when dpp was ectopically expressed in the ventral neuroectoderm by rho-GAL4/UAS-dpp system, it caused the repression of vnd, and msh expressions in ventral and dorsal columns of the neuroectoderm, respectively, but not that of ind. The later expression pattern of otd was also restricted by Dpp. The expression pattern of msh, vnd and otd in dpp loss-of-function and gain-of-function mutation indicates that Dpp activity does not reach to the ventral midline and it works locally to establish the dorsal boundary of the ventral neuroectoderm.     

CNS midline cells contribute to maintenance of the initial dorsoventral patterning of the Drosophila ventral neuroectoderm

Dorsoventral patterning of the Drosophila ventral neuroectoderm is established by the expression of three evolutionarily conserved homeodomain genes: ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) in the medial, intermediate, and lateral columns of the ventral neuroectoderm, respectively. It was not clear whether extrinsic factor(s) from the CNS midline cells influence the initial dorsoventral patterning by controlling the expression of the dorsoventral patterning genes. We show here that the CNS midline cells, specified by single-minded (sim), are essential for maintaining expression of the dorsoventral patterning genes. Ectopic expression of sim in the ventral neuroectoderm during the blastoderm stage repressed expression of the three homeodomain genes in the ventral neuroectoderm. This indicates that the identity of the CNS midline cells is established by a series of repressions of the three homeodomain genes in the ventral neuroectoderm. Ectopic expression of sim in the ventral neuroectoderm during initial neurogenesis induced ectopic ind expression in the medial column in addition to that in the intermediate column via EGFR signaling between the ventral neuroectoderm and midline cells. In contrast, it repressed the expression of vnd and msh in the medial and lateral columns, respectively. Our findings demonstrate that the CNS midline cells provide extrinsic positional information via EGFR signaling that maintains the initial subdivision of the ventral neuroectoderm into three dorsoventral columns during initial neurogenesis.     

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.     

Regulated vnd expression is required for both neural and glial specification in Drosophila.

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal-ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes--ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal-ventral location of precursor cells, we reassess the vnd loss- and gain-of-function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts-the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis-specified, and the midline glia are reduced in number. vnd over expression results in the mis-specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain-of-function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis-specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated.     

Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo

The Dorsal gradient produces sequential patterns of gene expression across the dorsoventral axis of early embryos, thereby establishing the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Spatially localized repressors such as Snail and Vnd exclude the expression of neurogenic genes in the mesoderm and ventral neuroectoderm, respectively. However, no repressors have been identified that establish the dorsal limits of neurogenic gene expression. To investigate this issue, we have conducted an analysis of the ind gene, which is selectively expressed in lateral regions of the presumptive nerve cord. A novel silencer element was identified within the ind enhancer that is essential for eliminating expression in the dorsal ectoderm. Evidence is presented that the associated repressor can function over long distances to silence neighboring enhancers. The ind enhancer also contains a variety of known activator and repressor elements. We propose a model whereby Dorsal and EGF signaling, together with the localized Schnurri repressor, define a broad domain of ind expression throughout the entire presumptive neuroectoderm. The ventral limits of gene expression are defined by the Snail and Vnd repressors, while the dorsal border is established by the newly defined silencer element.     

Vnd/nkx, ind/gsh, and msh/msx: conserved regulators of dorsoventral neural patterning?

Expression of vnd in ventral, ind in intermediate, and msh in dorsal columns of fly neurectoderm, and of homologous gene families in corresponding domains of vertebrate neurectoderm, suggests that elements of dorsoventral neural patterning have been evolutionarily conserved. However, upstream signaling pathways regulating this columnar gene expression pattern appear to have diverged significantly throughout evolution. In addition, while recent loss-of-function studies in flies and mice indicate that these three genes may have a conserved role in regional specification, there is no obvious conservation of the particular cell fates deriving from corresponding domains. The three-column expression pattern may thus represent a developmental mechanism that is more resistant to evolutionary changes than genetic events upstream or downstream of it.     

Neuroblast formation and patterning during early brain development in Drosophila

The Drosophila embryo provides a useful model system to study the mechanisms that lead to pattern and cell diversity in the central nervous system (CNS). The Drosophila CNS, which encompasses the brain and the ventral nerve cord, develops from a bilaterally symmetrical neuroectoderm, which gives rise to neural stem cells, called neuroblasts. The structure of the embryonic ventral nerve cord is relatively simple, consisting of a sequence of repeated segmental units (neuromeres), and the mechanisms controlling the formation and specification of the neuroblasts that form these neuromeres are quite well understood. Owing to the much higher complexity and hidden segmental organization of the brain, our understanding of its development is still rudimentary. Recent investigations on the expression and function of proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes have provided new insight into the principles of neuroblast formation and patterning during embryonic development of the fly brain. Comparisons with the same processes in the trunk help us to understand what makes the brain different from the ventral nerve cord. Several parallels in early brain patterning between the fly and the vertebrate systems have become evident.     

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.     

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.     

Comparison of early nerve cord development in insects and vertebrates

It is widely held that the insect and vertebrate CNS evolved independently. This view is now challenged by the concept of dorsoventral axis inversion, which holds that ventral in insects corresponds to dorsal in vertebrates. Here, insect and vertebrate CNS development is compared involving embryological and molecular data. In insects and vertebrates, neurons differentiate towards the body cavity. At early stages of neurogenesis, neural progenitor cells are arranged in three longitudinal columns on either side of the midline, and NK-2/NK-2.2, ind/Gsh and msh/Msx homologs specify the medial, intermediate and lateral columns, respectively. Other pairs of regional specification genes are, however, expressed in transverse stripes in insects, and in longitudinal stripes in the vertebrates. There are differences in the regional distribution of cell types in the developing neuroectoderm. However, within a given neurogenic column in insects and vertebrates some of the emerging cell types are remarkably similar and may thus be phylogenetically old: NK-2/NK-2.2-expressing medial column neuroblasts give rise to interneurons that pioneer the medial longitudinal fascicles, and to motoneurons that exit via lateral nerve roots to then project peripherally. Lateral column neuroblasts produce, among other cell types, nerve root glia and peripheral glia. Midline precursors give rise to glial cells that enwrap outgrowing commissural axons. The midline glia also express netrin homologs to attract commissural axons from a distance.