microRNAs (miRNAs) play a critical role in a variety of biological processes, including embryogenesis and the physiological functions of cells. Evolutionarily conserved microRNA-31 (miR-31) has been found to be involved in cancer, bone formation, and lymphatic development. We previously discovered that, in the sea urchin, miR-31 knockdown (KD) embryos have shortened dorsoventral connecting rods, mispatterned skeletogenic primary mesenchyme cells (PMCs) and shifted and expanded Vegf3 expression domain. Vegf3 itself does not contain miR-31 binding sites; however, we identified its upstream regulators Eve and Wnt1 to be directly suppressed by miR-31. Removal of miR-31’s suppression of Eve and Wnt1 resulted in skeletal and PMC patterning defects, similar to miR-31 KD phenotypes. Additionally, removal of miR-31’s suppression of Eve and Wnt1 results in an expansion and anterior shift in expression of Veg1 ectodermal genes, including Vegf3 in the blastulae. This indicates that miR-31 indirectly regulates Vegf3 expression through directly suppressing Eve and Wnt1. Furthermore, removing miR-31 suppression of Eve is sufficient to cause skeletogenic defects, revealing a novel regulatory role of Eve in skeletogenesis and PMC patterning. Overall, this study provides a proposed molecular mechanism of miR-31’s regulation of skeletogenesis and PMC patterning through its cross-regulation of a Wnt signaling ligand and a transcription factor of the endodermal and ectodermal gene regulatory network.

microRNAs (miRNAs) 在包括胚胎发生和细胞生理功能在内的多种生物学过程中发挥着关键作用。已发现进化上保守的 microRNA-31 (miR-31) 与癌症、骨形成和淋巴发育有关。我们之前发现，在海胆中，敲低 miR-31 (KD) 的胚胎缩短了背腹连接杆，使成骨原代间充质细胞 (PMC) 发生了错误排列，并转移和扩展了Vegf3表达域。Vegf3本身不含 miR-31 结合位点；然而，我们发现其上游调节因子 Eve 和 Wnt1 被 miR-31 直接抑制。消除 miR-31 对Eve和Wnt1的抑制导致骨骼和 PMC 图案缺陷，类似于 miR-31 KD 表型。此外，消除 miR-31 对Eve和Wnt1的抑制会导致 Veg1 外胚层基因（包括囊胚中的Vegf3）表达的扩增和前移。这表明 miR-31通过直接抑制Eve和Wnt1间接调节Vegf3的表达。此外，消除Eve的miR-31 抑制足以导致骨骼缺陷，揭示Eve的新调节作用在骨骼形成和 PMC 模式中。总体而言，这项研究提供了 miR-31 通过其对 Wnt 信号配体和内胚层和外胚层基因调控网络的转录因子的交叉调控来调控骨骼发生和 PMC 模式的分子机制。