Geminin is required for Hox gene regulation to pattern the developing limb

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and development. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior Shh signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the Hoxd13 target Shh, and the Shh target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of Gli3, a Shh pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb. Six litter and somite-matched pairs of Gmnnf/f; Prx-Cre (mutant) and Gmnn+/f; Prx-Cre (control) E10.5 embryos were used to prepare RNA. Batch correction was used to identify genes that were reproducibly differentially expressed genes (DEGs) across comparisons of paired mutant versus control samples.脊椎动物肢体复杂结构的发育，依赖于多条调控通路与多种蛋白质之间精密协调的相互作用。其中，5' Hox转录因子（5' Hox transcription factor）表达的精准调控，对于肢体芽（limb bud）的模式建成与正常发育至关重要。本研究鉴定出Geminin（Gmnn）为该过程的新型调控因子。 采用Gmnn条件性缺失模型进行研究时，可观察到前肢骨骼结构出现缺失或严重减少，而前肢肢端（autopod）与后肢均未受影响。在该Gmnn缺失模型的胚胎前肢芽中，5' Hox基因的表达范围扩展至更近端与前侧区域。 另一款Gmnn条件性缺失模型则引发了相似但程度更轻的后肢骨骼结构减少，并伴随后肢多指（polydactyly），而前肢未受影响。在此模型的Gmnn缺失胚胎的前侧后肢芽中，可检测到异位形成的后部Shh信号中心。该信号中心异位表达Hoxd13、Hoxd13的靶基因Shh，以及Shh的靶基因Ptch1；同时，此类突变体的后肢芽中，作为Shh通路拮抗因子的Gli3切割型阻遏形式的表达水平显著降低。 综上，本研究阐明了Gmnn通过调控Hox基因表达以塑造脊椎动物肢体模式的全新功能。本研究选取6对同窝且体节匹配的Gmnnf/f; Prx-Cre（突变型）与Gmnn+/f; Prx-Cre（对照型）E10.5期胚胎，制备RNA样本。通过批次校正（batch correction）分析，我们在配对的突变型与对照样本的比对中，筛选得到可重复的差异表达基因（differentially expressed genes, DEGs）。