Nonfluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain.

The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. Here, we investigate the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. We find that localized EGFR signalling in the brain neuroectoderm relies on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, we reveal a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, we show that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, we demonstrate that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.

Gene expression profiles uncover individual identities of gnathal neuroblasts and serial homologies in the embryonic CNS of Drosophila.

The numbers and types of progeny cells generated by neural stem cells in the developing CNS are adapted to its region-specific functional requirements. In Drosophila, segmental units of the CNS develop from well-defined patterns of neuroblasts. Here we constructed comprehensive neuroblast maps for the three gnathal head segments. Based on the spatiotemporal pattern of neuroblast formation and the expression profiles of 46 marker genes (41 transcription factors), each neuroblast can be uniquely identified. Compared with the thoracic ground state, neuroblast numbers are progressively reduced in labial, maxillary and mandibular segments due to smaller sizes of neuroectodermal anlagen and, partially, to suppression of neuroblast formation and induction of programmed cell death by the Hox gene Deformed Neuroblast patterns are further influenced by segmental modifications in dorsoventral and proneural gene expression. With the previously published neuroblast maps and those presented here for the gnathal region, all neuroectodermal neuroblasts building the CNS of the fly (ventral nerve cord and brain, except optic lobes) are now individually identified (in total 2×567 neuroblasts). This allows, for the first time, a comparison of the characteristics of segmental populations of stem cells and to screen for serially homologous neuroblasts throughout the CNS. We show that approximately half of the deutocerebral and all of the tritocerebral (posterior brain) and gnathal neuroblasts, but none of the protocerebral (anterior brain) neuroblasts, display serial homology to neuroblasts in thoracic/abdominal neuromeres. Modifications in the molecular signature of serially homologous neuroblasts are likely to determine the segment-specific characteristics of their lineages.

Non-fluorescent RNA in situ hybridization combined with antibody staining to visualize multiple gene expression patterns in the embryonic brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for whole mount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.