Tissue heterogeneity permits diverse biological outputs in response to systemic signals but requires context-dependent spatiotemporal regulation of a limited number of signaling circuits. In addition to their stereotypical roles of transport and cargo sorting, endocytic networks provide rapid, adaptable, and often reversible means of signaling. Aberrant function of the Endosomal Sorting Complex Required for Transport (ESCRT) components results in ubiquitinated cargo accumulation, uncontrolled signaling and neoplastic transformation. However, context-specific effects of ESCRT on developmental decisions are not resolved. By a comprehensive spatiotemporal profiling of ESCRT in Drosophila hematopoiesis in vivo, here we show that pleiotropic ESCRT components have distinct effects on blood progenitor maintenance, lineage choice and response to immune challenge. Of all 13 core ESCRT components tested, only Vps28 and Vp36 were required in all progenitors, whereas others maintained spatiotemporally defined progenitor subsets. ESCRT depletion also sensitized posterior progenitors that normally resist differentiation, to respond to immunogenic cues. Depletion of the critical Notch signaling regulator Vps25 did not promote progenitor differentiation at steady state but made younger progenitors highly sensitive to wasp infestation, resulting in robust lamellocyte differentiation. We identify key heterotypic roles for ESCRT in controlling Notch activation and thereby progenitor proliferation and differentiation. Further, we show that ESCRT ability to regulate Notch activation depends on progenitor age and position along the anterior-posterior axis. The phenotypic range and disparity in signaling upon depletion of components provides insight into how ESCRT may tailor developmental diversity. These mechanisms for subtle control of cell phenotype may be applicable in multiple contexts.
Link to full article: https://www.biorxiv.org/content/10.1101/2021.11.29.470366v1
ESCRT components regulate Notch activation and NICD trafficking in the lymph gland.
(A) Whole-mount larval lymph gland showing NRE-GFP+ve (Notch reporter) cells (green) and dome+ve 624 progenitors (red) in primary lobes upon progenitor-specific knockdown of 8 representative ESCRT 625 components indicated [Hrs, Stam (ESCRT-0); Vps28, Tsg101 (ESCRT-I); Vps25, Vps22 (ESCRT-II); Vps32, 626 Vps24 (ESCRT-III)]. Scale bar: 100 µm. Bar diagrams show quantification of the number of NRE-GFP 627 positive cells in primary, secondary and tertiary lobes upon knockdown of the same 8 ESCRT 628 components. (B) Whole-mount larval lymph gland showing NICD expression (shown in red in the upper 629 panel and in gray scale in the lower panel) in primary lobes upon progenitor-specific knockdown of 630 the same 8 aforementioned ESCRT components. Progenitors are marked by dome>2xEGFP (green). 631 Scale bar: 100 µm. (B’) Magnified view showing lymph gland hemocytes with (arrow) or without 632 (arrowhead) NICD accumulation. Scale bar: 10 µm. Bar diagrams show quantification of the number 633 of NICD accumulating cells in primary, secondary and tertiary lobes. n indicates the number of 634 individual lobes analysed and N indicates the number of larvae analysed. Error bars represent SEM. 635 One-way ANOVA was performed to determine the statistical significance. *P<0.05, **P<0.01, 636 ***P<0.001. See also Fig S4.
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