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. 2016 Oct 15;143(20):3852–3862. doi: 10.1242/dev.136267

Fig. 8.

Fig. 8.

Ebf is an independent regulator of siphon muscle fate in the B7.5 lineage. (A-D) Close-up of ASM (marked with open arrowheads), and first heart precursors (FHP, marked with white arrows) and second heart precursors (SHP, marked with white arrowheads), derived from the B7.5 lineage, revealing Mrf mRNA (blue) and Bhlh-tun-1 mRNA (green), with the B7.5 lineage marked by Mesp>H2B:mCherry. (A,B) Larvae under CRISPR control condition either expressing Tbx1/10>LacZ (A) or overexpressing Ebf with Tbx1/10>Ebf (B). In B, note that expression of Mrf and Bhlh-tun-1 has expanded to the SHP, due to earlier Tbx1>Ebf expression in the secondary TVCs. (C,D) Larvae with Tbx1/10 mutated specifically in the B7.5 lineage, and expressing Tbx1/10>LacZ (C) or Tbx1/10>Ebf (D). Note that in C there is a complete loss of Mrf or Bhlh-tun-1 expression, whereas in D, Mrf and Bhlh-tun1 show wild-type expression patterns in the ASM, and are also expressed in the SHP. Scale bars: 25 μm. (E) Bar chart showing percentage of larvae expressing Mrf or Bhlh-tun1 in ASMP or SHP under indicated conditions. Each bar represents the pooled scores from two biological replicates, with error bars showing estimated standard error and sample sizes indicated. ***P<0.00001, by χ2 analysis with continuity correction. (F) Schematic diagram comparing core regulatory interactions upstream of morphogenesis and Mrf-driven differentiation in ASM and OSM. In black, documented shared expression of Hand-r, Ebf and Tbx1/10 in cells that give rise to, among other tissues, siphon muscles, where Hand-r might be involved in activation of the core common regulators Tbx1/10 and Ebf, as indicated by green dashed arrows. Distinct regulatory relationships in place in ASM versus OSM are indicated by orange and brown arrows, respectively. The shared direct input from Ebf to Mrf in both ASM and OSM is indicated by the solid green arrow. The dashed green arrows from Ebf and Tbx1/10 to morphogenesis are inferred from our results that mutagenesis of Mrf affects differentiation, but not morphogenesis of both ASM and OSM (see Fig. 4).