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Transcriptome analysis of Drosophila whole larval lymph glands

ABSTRACT: The balance between hematopoietic progenitors and differentiated hemocytes is finely tuned during development. In the larval hematopoietic organ of Drosophila, called the lymph gland, the receptor tyrosine kinase Pvr signals from differentiated cells to maintain a pool of undifferentiated progenitors. However, little is known about the processes that support Pvr function. The small GTPase Ral is involved in the regulation of several membrane trafficking events. Drosophila has a single Ral protein, Rala, which has been implicated in the development of various tissues. Here, we investigated the involvement of Rala in the larval fly hematopoietic system. We discovered that the loss of Rala activity phenocopies Pvr loss of function by promoting hemocyte progenitor differentiation. Moreover, using epistasis analysis, we found that the guanine exchange factor RalGPS lies upstream of Rala in this event, whereas the exocyst and Rab11 are acting downstream. Strikingly, the loss of Rala activity leads to a considerable accumulation of Pvr at the plasma membrane, hence suggesting a trafficking defect and reduced Pvr function. Consistent with this hypothesis, Rala loss of function phenotype in the lymph gland is fully suppressed by constitutive STAT activity, which normally mediates Pvr function in the lymph gland. Together, our findings unravel a novel RalGPS-Rala-exocyst-Rab11 axis for the maintenance of lymph gland homeostasis through Pvr.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE148035 | GEO | 2020/05/25

REPOSITORIES: GEO

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Project description:Abstract: RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumorigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine. We identify non-canonical roles of RAL GTPases not as RAS effectors, but rather by acting upstream of RAS activation via induction of EGFR internalisation . Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation and damage-induced intestinal regeneration, this function of RAL GTPases drives EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of EGFR-driven tissue homeostasis and malignant transformation. Results: RalA is required within ISCs to induce midgut adult midgut regeneration following damage by oral infection with Erwinia carotovora carotovora 15 (Ecc15) (Johansson et al., 2019). To achieve a global view of intestinal pathways affected by RalA, we performed a transcriptomic analysis by RNAseq of whole midguts from vehicle treated (Mock) or damaged (Ecc15 fed) control animals or following RalA knockdown in intestinal stem and progenitor cells using the escargot-gal4 driver (ISC/EB>) (Micchelli and Perrimon, 2006). Consistent with its effect on ISC proliferation (Johansson et al., 2019), RalA knockdown significantly impaired damage-induced upregulation of cell cycle genes in the midgut. Additionally, levels of multiple transcriptional targets of the EGFR/MAPK pathway (Golembo et al., 1996; Hsu et al., 2001; Jin et al., 2015; Meng and Biteau, 2015), such as argos (aos), rhomboid (rho), Sox21a and string (stg) were increased following Ecc15 infection in control midguts. The upregulation of these target genes was significantly impaired upon RalA knockdown.

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Transcriptome analysis of Drosophila whole larval lymph glands

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