The homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported. Using transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes. These data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates.

中文翻译：

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Amphioxus Pdx和Cdx的突变证明了ParaHox基因在肠道，肛门和尾巴模式中的保守作用。

同源盒基因Pdx和Cdx遍布整个动物界和ParaHox小基因簇的一部分。基因表达模式暗示了Pdx和Cdx在塑造双边动物通肠的过程中起着古老的作用，尽管功能数据对于少数血统来说是可用的。为了检查Pdx和Cdx基因功能的进化保守性，我们集中于两栖类，小型海洋动物，它们在碳酸盐进化中占有举足轻重的地位，并且首次报道了ParaHox基因的聚类。使用转录激活因子样效应子核酸酶（TALENs），我们设计了两栖类佛罗里达分支杆菌的Pdx和Cdx基因的移码突变，并建立了突变株系。纯合的Pdx突变体在两栖动物内胚层中有缺陷，表现为表达内源性GFP的中肠区域的缺失。纯合的Cdx突变体无法打开肛门，该突变体在后身伸展和表皮尾鳍发育方面也有缺陷。用视黄酸（RA）信号反向激动剂进行的治疗可部分挽救轴向和尾鳍表型，表明它们是由RA信号增强引起的。基因表达分析和荧光素酶测定表明，通过Cdx对Cyp26基因的直接转录调控，野生型动物的后RA水平保持较低。转录组分析揭示了突变体中广泛的基因表达变化，其中Pdx和Cdx对肠道富集基因的影响不成比例，而Cdx对Hox基因的共线性影响。这些数据表明，文昌鱼Pdx和Cdx在确定肠内胚层中和后细胞的命运方面具有重要作用，可能追溯到Bilateria起源的角色。这一结论与这两个ParaHox基因在具有双侧肛门直肠的双侧肠道的起源中发挥作用是一致的，形态学的创新促成了寒武纪的生态变化。此外，我们发现文昌鱼Cdx通过脊椎动物的保守分子机制来促进体轴延伸。Cdx的轴向延伸作用至少可以追溯到Chordata的起源，并且可能促进了肛门后尾巴的进化和脊索中的主动运动。我们发现，文昌鱼Cdx通过与脊椎动物守恒的分子机制促进了身体轴的延伸。Cdx的轴向延伸作用至少可以追溯到Chordata的起源，并且可能已经促进了肛门后尾巴的进化和脊索中的主动运动。我们发现，文昌鱼Cdx通过与脊椎动物守恒的分子机制促进了身体轴的延伸。Cdx的轴向延伸作用至少可以追溯到Chordata的起源，并且可能已经促进了肛门后尾巴的进化和脊索中的主动运动。