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. Author manuscript; available in PMC: 2020 Feb 1.
Published in final edited form as: Dev Biol. 2018 Dec 13;446(1):102–118. doi: 10.1016/j.ydbio.2018.12.014

Figure 5: ex vivo culture enables analysis of niche compaction.

Figure 5:

A, A’) Gonad expressing six4-Moe-GFP to visualze F-actin enrichment at SGP cell cortices (green), cultured ex vivo for ~5 hours, then post-fixed and stained for the niche (FasIII, red), and nuclei (Hoechst, white). FasIII accumulated properly at niche cell boundaries (not all boundaries are visible in this section). Hub cells and, especially, msSGPs at posterior of gonad accumulate higher levels of F-actin than other SGPs. B, B’) Gonad from an embryo expressing six4-Moe-GFP (green) and HistoneRFP (red), cultured ex vivo for ~5 hours, then post-fixed and stained for ECad (red), and Hoechst (white), also revealing nuclei. E-cad accumulated as expected among niche boundaries (red). Note the metaphase plate of a GSC (asterisk); a division plane perpendicular to the hub-GSC interface, as expected. C, C’) Gonad expressing nos-Moe-GFP to visualize F-actin in germ cells (green), cultured ex vivo for ~5 hours, then post-fixed and stained for STAT92E (white), and nuclei (Hoechst, white), nos-Moe-GFP outlines all germ cells. A GSC (arrow) and a non-GSC (arrowhead, blue dotted line) are marked. Germ cells in contact with the niche in this slice are outlineed with a green dotted line, and represent GSCs. D) Niche cell number (FasIII) per gonad (circles), comparing cases where the niche developed fully in vivo (dissected from late-stage embryos, ~23h AEL, blue closed circles), to gonads cultured ex vivo until the compact architecture was achieved (purple open circles). E) For GSCs (n=46, green closed triangles) and non GSCs (n=35, orange open triangles), the graph plots the deviation of each division from an idealized axis perpendicular to the niche-GSC boundary. F) Average STAT accumualtion (arbirtrary units) in ex vivo culture, comparing the GSC tier to germ cells removed from the hub/GSC boundary (3rd tier germ cells). Connectors report values from the same gonad. In D,E bars represent mean and standard deviation; n.s., not significant; ****, p < 0.0001. For A-C, scale bar is 10 microns.