Hoxa2 genes provide critical patterning signals during development, and their regulation and function have been extensively studied. We report a previously uncharacterized significant sequence divergence of a highly conserved hindbrain hoxa2b enhancer element in the family syngnathidae (pipefishes, seahorses, pipehorses, seadragons). We compared the hox cis-regulatory element variation in the Gulf pipefish and two species of seahorse against eight other species of fish, as well as human and mouse. We annotated the hoxa2b enhancer element binding sites across three species of seahorse, four species of pipefish, and one species of ghost pipefish. Finally, we performed in situ hybridization analysis of hoxa2b expression in Gulf pipefish embryos. We found that all syngnathid fish examined share a modified rhombomere 4 hoxa2b enhancer element, despite the fact that this element has been found to be highly conserved across all vertebrates examined previously. Binding element sequence motifs and spacing between binding elements have been modified for the hoxa2b enhancer in several species of pipefish and seahorse, and that the loss of the Prep/Meis binding site and further space shortening happened after ghost pipefish split from the rest of the syngnathid clade. We showed that expression of this gene in rhombomere 4 is lower relative to the surrounding rhombomeres in developing Gulf pipefish embryos, reflecting previously published functional tests for this enhancer. Our findings highlight the benefits of studying highly derived, diverse taxa for understanding of gene regulatory evolution and support the hypothesis that natural mutations can occur in deeply conserved pathways in ways potentially related to phenotypic diversity.

Hoxa2基因在发育过程中提供关键的模式信号，其调控和功能已被广泛研究。我们报告了海龙科（海龙、海马、海龙）中高度保守的后脑hoxa2b增强子元件的先前未表征的显着序列差异。我们将墨西哥湾尖嘴鱼和两种海马的hox 顺式调节元件变异与其他八种鱼类以及人类和小鼠进行了比较。我们注释了三种海马、四种海龙和一种鬼海龙的hoxa2b增强子元件结合位点。最后，我们对海湾海龙胚胎中hoxa2b的表达进行了原位杂交分析。我们发现所有检查过的合颌鱼都共享一个经过修饰的菱形 4 hoxa2b增强子元件，尽管该元件已被发现在之前检查过的所有脊椎动物中高度保守。几种海龙和海马中的hoxa2b增强子的结合元件序列基序和结合元件之间的间距已被修改，并且在鬼海龙与海马的其余部分分离后，Prep/Meis 结合位点的丢失和进一步的空间缩短发生进化枝。我们发现，在发育中的海湾海龙胚胎中，该基因在菱形节 4 中的表达相对于周围菱形节较低，这反映了先前发表的该增强子的功能测试。我们的研究结果强调了研究高度衍生的多样化分类群对于理解基因调控进化的好处，并支持这样的假设：自然突变可能以与表型多样性潜在相关的方式发生在深度保守的途径中。