Different regulation of limb development by p63 transcript variants.
Different regulation of limb development by p63 transcript variants.
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DOI：
10.1371/journal.pone.0174122
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Saito T
中科院分区：
文献类型：
--
作者：
Kawata M;Taniguchi Y;Mori D;Yano F;Ohba S;Chung UI;Shimogori T;Mills AA;Tanaka S;Saito T
顶端外胚层嵴（AER）位于每个肢芽的远端，是通过分泌各种分子控制肢的近端-远端轴的向外生长和图案化的关键信号中心。成纤维细胞生长因子（Fibroblast growth factors，FGF），尤其是Fgf 8和Fgf 4，是AER细胞产生的代表性分子，是维持AER和细胞增殖所必需的，同时Jag 2-Notch通路负调控AER和肢体发育。p63是p53家族的转录因子，在AER中表达，并且是肢体形成所必需的。然而，p63变异体的潜在机制和具体作用尚不清楚。在这里，我们定量了小鼠胚胎10.5天（E）至E12.5天四肢中p63变体的表达，发现Δ Np 63 γ在所有阶段的四肢中都有强表达，而TAp 63 γ表达在后期迅速增加。对来自E11.5的报告小鼠胚胎的肢芽细胞的增殖激活细胞分选分析显示，所有变体在AER细胞中大量表达，并且它们在间充质细胞中的表达非常低。然后，我们通过使小鼠与p63的无效和flox等位基因以及Msx 2-Cre小鼠（Msx 2-Cre;p63Δ/fl）交配来产生AER特异性p63敲除小鼠。Msx 2-Cre;p63Δ/fl新生儿出现肢体畸形，以远侧成分更为明显。在Msx 2-Cre;p63Δ/fl肢芽和由p63敲低诱导的多能干细胞形成的胚状体中，多种AER相关基因的表达降低。启动子分析和染色质免疫沉淀分析表明Fgf 8和Fgf 4是Δ Np 63 γ的转录靶点，Jag 2是TAp 63 γ的转录靶点。此外，TAp 63 γ过表达加重Msx 2-Cre;p63Δ/fl小鼠的表型。这些数据表明Δ Np 63和TAp 63通过转录调节在AER中具有不同作用的不同靶分子来控制肢体发育。我们的研究结果有助于进一步了解肢体发育的分子网络。
The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.