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

Jussen, David; Urbach, Rolf

doi:10.1007/978-1-62703-655-9_2

Cited by 3 publications

(3 citation statements)

References 10 publications

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“…Embryos were dechorionated, fixed, immunostained and flat preparations performed according to published protocols ( Patel, 1994 ; Jussen and Urbach, 2014 ). The antibodies used are detailed in the .…”

Section: Methodsmentioning

confidence: 99%

“…Probes for in situ hybridization were synthesized using the templates shown in . In situ hybridization was performed as described previously ( Jussen and Urbach, 2014 ) and probes processed with NBT/BCIP solution for non-fluorescent staining (Carl Roth) or tyramide signal amplification (TSA Cyanine 3 System; PerkinElmer) for fluorescent stainings. Embryos were then immunolabeled with primary antibody followed by incubation with biotinylated (processed with DAB) or fluorescent dye-coupled secondary antibodies as described in the .…”

Section: Methodsmentioning

confidence: 99%

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

2016

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

2016

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“…Flat preparations of the head/truncal ectoderm of stained embryos and mounting were carried out as described previously [106]. …”

Section: Methodsmentioning

confidence: 99%

Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonicDrosophilabrain

Jussen

Hilchen

Urbach³

2016

Open Biol.

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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.

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Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain

Kraft¹,

Massey²,

Kolb³

et al. 2016

Mechanisms of Development

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The Drosophila mushroom bodies, centers of olfactory learning and memory in the fly 'forebrain', develop from a set of neural stem cells (neuroblasts) that generate a large number of Kenyon cells (KCs) during sustained cell divisions from embryonic to late pupal stage. We show that retinal homeobox (rx), encoding for an evolutionarily conserved transcription factor, is required for proper development of the mushroom bodies. Throughout development rx is expressed in mushroom body neuroblasts (MBNBs), their ganglion mother cells (MB-GMCs) and young KCs. In the absence of rx function, MBNBs form correctly but exhibit a reduction in cell size and mitotic activity, whereas overexpression of rx increases growth of MBNBs. These data suggest that Rx is involved in the control of MBNB growth and proliferation. Rx also promotes cell cycling of MB-GMCs. Moreover, we show that Rx is important for the survival of MBNBs and Kenyon cells which undergo premature cell death in the absence of rx function. Simultaneous blocking of cell death restores the normal set of MBNBs and part of the KCs, demonstrating that both, impaired proliferation and premature cell death (of MBNBs and KCs) account for the observed defects in mushroom body development. We then show that Rx controls proliferation within the MBNB clones independently of Tailless (Tll) and Prospero (Pros), and does not regulate the expression of other key regulators of MB development, Eyeless (Ey) and Dachshund (Dac). Our data support that the role of Rx in forebrain development is conserved between vertebrates and fly.

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

Cited by 3 publications

References 10 publications

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

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

Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonicDrosophilabrain

Retinal homeobox promotes cell growth, proliferation and survival of mushroom body neuroblasts in the Drosophila brain

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