Supplementary MaterialsFigure 1source data 1: Hair cell progenitors are replenished via proliferation of other support cells elife-43736-fig1-data1. distinct support cell populations are correspondingly rotated (Figure 2figure supplement 1). We also generated GFP lines for each insertion site. We did not observe GFP labeling in hair cells in stable lines (Figure 2figure supplement 2). Open in a separate window Figure 2. Genetic labeling of distinct support cell populations.(A, C, E) Maximum projections of neuromasts from locus using CRISPR (Tg[expression in DV cells, as defined by the transgene. At three dpf, Klf2 soon after the initiation of transgene expression, we see considerable overlap between NTR-GFP and nlsEos. All NTR-GFP?+cells were also positive for nlsEos, while an additional subset of cells expressed nlsEos alone. When we compared expression at five dpf, the size of the double-positive (NTR-GFP+; nlsEos+) population did not change, whereas the number of cells expressing nlsEos alone increased significantly, occupying a more central location (Figure 5ACB, arrowheads; Figure 5C; NTR-GFP/nlsEos: 9.04??2.39 [3 dpf] vs. 8.47??2.27 [5 dpf]; nlsEos only: 6.10??2.27 [3 dpf] vs. 10.86??2.72 (5 dpf); p 0.9999 [NTR-GFP/nlsEos], p 0.0001 [nlsEos only]). These observations are consistent with the idea that both transgenes initiate expression at the same time, but that nlsEos protein is retained longer than NTR-GFP protein as cells mature and as a result, NTR-GFP is expressed in a subset of DV cells. We next tested to the efficacy of DV cell ablation at 3 and 5 dpf. Treatment of these fish with 10 mM Mtz for 8 hr was sufficient to ablate the majority of NTR-GFP cells. Treating fish with Mtz for 8 hr at five dpf (Mtz5) slightly but significantly decreased the number of support cells solely expressing nlsEos by about 13%. Treating fish with Mtz for 8 hr at three dpf, followed by a second 8 hr Mtz treatment at five dpf (Mtz3/5) decreased the number of solely nlsEos-positive cells even further, by about 40% (Figure 5DCG; Mock: 11.18??2.04; Mtz5: 9.72??2.03; Mtz3/5: 6.76??2.12; p=0.0288 [Mock vs. Mtz5], p 0.0001 [Mock vs. Mtz3/5, Mtz5 vs. Mtz3/5]). Open in a separate window Figure 5. Differences in overlap between function, yet these double positive larvae have the same number of hair cells during development (five dpf) and after hair cell regeneration as their non-transgenic and heterozygotic siblings (Figure 6figure supplement 2). This would suggest that function is dispensable for hair cell development order ACP-196 and regeneration, in spite of the contribution DV cells make to both processes. However, we did not formally test whether function was actually disrupted by transgene insertion, so it is possible that these double-positive larvae are not indicative of true loss-of-function or that there are mechanisms to compensate for the loss of have similar patterns to those of the transgenic insertions reported here. We stress that the purpose of this study is not to correlate progenitor function to specific gene function, but to examine the functional differences between populations of order ACP-196 support cells marked by transgene insertion. While our study may not definitively link the action of underlying loci with progenitor identity, our experiments demonstrate that these genetically labeled support cells have distinct progenitor functions, and can serve as important tools in future studies determining the precise mechanisms underlying regeneration in the lateral line. The role of Planar Cell Polarity and progenitor order ACP-196 localization Neuromasts located on the trunk develop at different times from different migrating primordia. Within a given neuromast, hair cells are arranged such that their apical order ACP-196 stereocilia respond to directional deflection in one of two directions along the body axis. Hair cells derived from the first primordium (primI) respond along the anteroposterior axis, and hair cells derived from the second primordium (primII) respond along the dorsoventral axis (Lpez-Schier et al., 2004; Lpez-Schier and Hudspeth, 2006). Spatial restriction of support cell proliferation is orthogonal to hair cell planar polarity, with proliferation occurring dorsoventrally in primI-derived neuromasts and anteroposteriorly in primII-derived neuromasts (Romero-Carvajal et al., 2015). This 90-degree switch between prim1- and primII-derived neuromasts is reflected in the distribution of labeled cell populations as well: (Romero-Carvajal et al., 2015). It is possible that Notch signaling may repress Wnt signaling in AP cells, further contributing to their low regenerative capacity. More.