Table3_Pax3/7 regulates neural tube closure and patterning in a non-vertebrate chordate.DOCX

Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis-regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates.

Pax3/7因子在脊椎动物背侧神经系统的发育中扮演着多重角色。从在神经板边缘指定神经嵴，到调节神经管闭合及形成神经管的模式，其作用不可或缺。然而，在非脊椎动物圆口类中，这些角色中哪些得以保留尚不明确。在本研究中，我们探讨了模型海鞘Ciona中Pax3/7的表达和功能。Pax3/7在神经管形成期间表达于神经板边缘细胞，并在神经管闭合后不久表达于中枢神经系统祖细胞。我们发现，不同的顺式调控元件分别控制了这两个不同谱系中的表达。通过CRISPR/Cas9介导的基因编辑技术，我们在F0胚胎的这两个独立区域特异性地敲除了Pax3/7。在神经板边缘敲除Pax3/7导致神经管闭合缺陷，表明Pax3/7在此圆口类特有过程中的古老作用。此外，敲除Pax3/7导致神经受损运动神经节神经元特异性受损，进一步证实了该基因在神经管模式形成中的保守作用。综合这些结果，表明Pax3/7在神经管发育中的关键功能在进化上源远流长，至少可追溯到脊椎动物和海鞘的共同祖先。