A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development.
A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development.
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DOI：
10.1371/journal.pgen.1002757
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发表时间：
2012-07
期刊：
影响因子：
4.5
通讯作者：
Arnheiter H
中科院分区：
文献类型：
--
作者：
Bharti K;Gasper M;Ou J;Brucato M;Clore-Gronenborn K;Pickel J;Arnheiter H
视神经上皮细胞分离为视网膜和视网膜色素上皮细胞（RPE）是脊椎动物早期眼发育的关键事件。在这里，我们在小鼠中发现，以其视网膜促进活性而闻名的转录因子PAX 6在早期色素上皮发育中也起着至关重要的作用。然而，这种作用仅在色素细胞转录因子MITF突变遗传致敏的背景中才能观察到。事实上，Pax 6基因剂量的减少加剧了在Mitf无效等位基因纯合的胚胎中观察到的RPE到视网膜的转分化，并且它在Mitf无效等位基因杂合或靶向突变产生的RPE特异性亚型Mitf等位基因纯合的胚胎中诱导这种转分化。相反，Pax 6基因剂量的增加干扰转分化，即使在纯合子Mitf无效胚胎。基因表达分析显示，PAX 6与MITF或其partial TFEC一起抑制Fgf 15和Dkk 3的表达。外植体培养实验表明，FGF和DKK 3的组合通过抑制经典WNT信号传导和刺激包括Six 6和Vsx 2在内的视网膜形成基因的表达来促进视网膜形成。我们的研究结果表明，与Mitf/Tfec Pax 6作为一个抗视网膜生成因子，而与视网膜生成基因，它作为一个促视网膜生成因子。结果表明，仔细操纵Pax 6调节电路可能有助于从胚胎或诱导多能干细胞生成视网膜和色素上皮细胞。视网膜色素上皮细胞或视网膜色素上皮细胞在眼睛的后面是至关重要的视网膜的正常功能，其异常可导致视网膜疾病，如成人发病性黄斑变性。深入了解这些疾病的发病机制和潜在的治疗方法，可能来自于使用体外诱导多能干细胞产生的RPE细胞。为了在体外获得真实的RPE细胞，我们需要彻底了解它们在体内发育的正常过程。在这里，我们发现有效的视网膜诱导转录因子PAX 6在小鼠RPE中起着关键的抗视网膜生成作用。但是PAX 6如何在视网膜中促视网膜生成，而在RPE中抗视网膜生成？为了解决这个问题，我们使用了基因表达研究，并将其与染色质免疫沉淀试验相结合，该试验分析了转录因子与染色质在体内的相互作用。我们的研究结果表明，在RPE中，PAX 6与两种相关RPE转录因子MITF和TFEC中的一种（或两种）合作，以抑制细胞外信号，所述细胞外信号在正常视网膜中诱导促进视网膜形成的信号级联。因此，这项研究提供了对RPE发育的机制性见解，这对于从诱导多能干细胞有效生成视网膜和RPE可能变得重要。
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE–specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells. The retinal pigment epithelium or RPE in the back of the eye is critical for the normal function of the retina, and its abnormalities can lead to retinal disorders such as adult-onset macular degeneration. Insights into the pathogenesis of such disorders, and potential therapies, may come from using RPE cells generated in vitro from induced pluripotent stem cells. To obtain authentic RPE cells in vitro, we need to thoroughly understand the normal process of their development in vivo. Here we find that the potent retina-inducing transcription factor PAX6 plays a critical anti-retinogenic role in the RPE of mice. But how can PAX6 be pro-retinogenic in the retina and anti-retinogenic in the RPE? To address this question, we used gene expression studies and combined them with chromatin immunoprecipitation assays, which analyze the interaction of transcription factors with chromatin in vivo. Our findings show that, in the RPE, PAX6 cooperates with either one (or both) of two related RPE transcription factors, MITF and TFEC, to suppress extracellular signals that in the normal retina induce a signaling cascade promoting retina formation. Hence, this study provides mechanistic insights into RPE development that may become important for the efficient generation of retina and RPE from induced pluripotent stem cells.
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