BACKGROUND Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. METHODS We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. RESULTS We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. CONCLUSIONS We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain - suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically needed to promote p1 fates.

中文翻译：

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斑马鱼prdm12b独立于nkx6.1抑制起作用，以促进eng1b在神经管p1域中的表达。

背景技术成人神经系统的功能取决于胚胎发生过程中神经回路的建立。在脊椎动物中，组成运动回路的神经元沿着神经管的背腹轴在不同的区域形成。每个域的特征是转录因子（TF）的独特组合，可促进特定的命运，同时抑制相邻域的命运。eng1b的表达和p1域中V1中间神经元的生成需要prdm12 TF，但其功能的细节仍不清楚。方法我们使用CRISPR / Cas9生成了prdm12的第一个种系突变体，并利用该资源，结合经典的荧光素酶报告基因分析和免疫共沉淀实验，研究了prdm12b在斑马鱼中的功能。我们还生成了bhlhe22和nkx6的种系突变体。1来研究这些TF如何与prdm12b相互作用以控制p1的形成。结果我们发现prdm12b突变体在p1域中缺乏eng1b表达，并且还具有异常的触摸诱发的逃避反应。使用萤光素酶报告基因测定，我们证明Prdm12b充当转录阻遏物。我们还显示Bhlhe22 TF通过Prdm12b锌指结构域结合形成复合物。但是，bhlhe22突变体在p1域中显示正常的eng1b表达。虽然提出了prdm12通过抑制nkx6.1 TF的表达来促进p1命运，但我们并未观察到prdm12b功能丧失时nkx6.1域的扩展，也不会在prdm12b和nkx6同时丧失时恢复eng1b表达。 1。结论我们得出结论，prdm12b生殖系突变产生的表型与吗啉代介导的prdm12功能丧失的表型没有区别。就prdm12b功能而言，我们的结果表明Prdm12b充当转录阻遏物，并与EHMT2 / G9a和Bhlhe22相互作用。但是，体内eng1b表达不需要bhlhe22功能，这可能表明其他bhlh基因可以在胚胎发生过程中进行补偿。最后，我们没有找到证据证明nkx6.1和prdm12b在p1域的形成中起阻遏对的作用-这表明prdm12b不仅需要抑制非p1命运，而且特别需要促进p1命运。我们的结果表明Prdm12b充当转录阻遏物，并与EHMT2 / G9a和Bhlhe22相互作用。但是，体内eng1b表达不需要bhlhe22功能，这可能表明其他bhlh基因可以在胚胎发生过程中进行补偿。最后，我们没有找到证据证明nkx6.1和prdm12b在p1域的形成中起阻遏对的作用-这表明prdm12b不仅需要抑制非p1命运，而且特别需要促进p1命运。我们的结果表明Prdm12b充当转录阻遏物，并与EHMT2 / G9a和Bhlhe22相互作用。但是，体内eng1b表达不需要bhlhe22功能，这可能表明其他bhlh基因可以在胚胎发生过程中进行补偿。最后，我们没有找到证据证明nkx6.1和prdm12b在p1域的形成中起阻遏对的作用-这表明prdm12b不仅需要抑制非p1命运，而且特别需要促进p1命运。