To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.

中文翻译：

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Hoxa13调节与软骨发育有关的常见Hox靶基因的表达，以协调足突的扩张。

为了阐明Hox基因在四肢软骨发育中的作用，我们通过ChIP-Seq鉴定了HOXA11和HOXA13的靶基因。ChIP DNA片段包含进化保守的序列和多个高度保守的HOX结合位点。HOXA11 ChIP片段的很大一部分与HOXA13 ChIP片段重叠，表明这两个因子共享相同的靶标。删除邻近Bmp2或Tshz2的目标区域会降低它们在autopod中的表达，表明它们充当肢体芽特异性增强子。我们通过基因芯片分析鉴定了Hox下游基因表现出Hoxa13基因敲除（KO）和Hoxd11-13缺失双突变体（Hox13 dKO）autopod中的表达变化。与ChIP片段相邻的Hox下游基因被定义为Hox的直接靶标。我们分析了在自足发育和Hox13dKO肢芽过程中编码两种不同类别的转录因子的Hox靶基因的空间表达模式。（a）编码软骨分化阻遏物的Bcl11a在E11.5自足足中表达，并在Hox13dKO中显着减少。（b）由于Hox13在E11.5的抑制或相对较弱的转录活性，转录因子Aff3，Bnc2，Nfib和Runx1t1在斑足类软骨中表达，但不在足足中表达。有趣的是，这些基因的表达后来在E12.5的自足软骨中观察到。这些结果表明，Hox13通过多条途径暂时中止了足趾软骨的软骨分化，直到建立产生五位手指所需的桨状结构为止。