Figure 1. miR-31 indirectly regulates Vegf3 expression domain and skeletogenesis by targeting Eve, Wnts, and Fzds.

(A) A simplified proposed model of miR-31’s indirect regulation of Vegf3 and skeletogenesis through potential targets Eve, Wnt1, Wnt16, and Fzd5/8 during mesenchyme blastula (24 hpf). Genes highlighted in yellow are potential miR-31 targets. Eve is activated by canonical Wnt/β-catenin signaling and is expressed in both the Veg1 endoderm and ectoderm and activates Hox11/13b in the Veg1 endoderm (Cui et al., 2014; Peter and Davidson, 2011; Ransick et al., 2002). Wnt1 and Wnt16 also contribute to the activation of Hox11/13b in the Veg1 cells, and the positive feedback of Hox11/13b, Wnt1, and Wnt16 occurs in Veg1 endodermal cells (Cui et al., 2014). Wnt1 signals through Fzd5/8 receptor to activate the ncWnt/PCP-ROCK/JNK pathway, and Wnt16 signals through Fzd1/2/7 to activate the ncWnt/Ca²⁺-PKC pathway (Martínez-Bartolomé and Range, 2019; Range et al., 2013). Wnt1-Fzd5/8 and Wnt16-Fzd1/2/7 cross-regulate each other (Range et al., 2013). Wnt1-Fzd5/8 and/or Wnt16-Fzd1/2/7 signaling lead to the activation of an unidentified factor which activates transcription factor Pax2/5/8, leading to activation of Vegf3 expression (McIntyre et al., 2013; Rottinger et al., 2008). Eve, Hox11/13b, Fzd5/8 and Fzd1/2/7 are in part regulated through autorepression, while Wnt1 may be regulated through autoactivation (Cui et al., 2014; Cui et al., 2017; Range, 2018; Range et al., 2013). (B) The shorter DVC rods and aberrant PMC patterning defects observed in miR-31 KD gastrulae (Stepicheva and Song, 2015) persist into the larval stage (5dpf). A greater number of miR-31 KD larvae are underdeveloped and/or exhibit body rods that fail to meet at the posterior end of the embryo. Maximum intensity projection of Z-stack confocal images are shown. 2 biological replicates. N is the total number of larvae examined. Scale bar = 50μm.