GATA6 is a crucial regulator of Shh in the limb bud.
GATA6 is a crucial regulator of Shh in the limb bud.
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DOI：
10.1371/journal.pgen.1004072
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发表时间：
2014-01
期刊：
影响因子：
4.5
通讯作者：
Lassar AB
中科院分区：
文献类型：
--
作者：
Kozhemyakina E;Ionescu A;Lassar AB
在肢芽中，沿着前后（A-P）轴的模式由Sonic Hedgehog（Shh）控制，Shh是由“极化活动区”分泌的信号分子，位于肢芽后缘的组织者组织。我们已经发现，转录因子GATA 4和GATA 6，这是细胞身份的关键调节因子，在早期肢芽中以前后梯度表达，提高了加塔转录因子可能在该组织的图案化中发挥额外作用的可能性。虽然GATA 4和GATA 6在前肢芽中以A-P梯度表达，但后肢芽主要以A-P梯度表达GATA 6。因此，为了专门研究GATA 6在肢体模式中的作用，我们产生了Prx 1-Cre; GATA 6 fl/fl小鼠，它们有条件地从发育中的肢体芽中删除GATA 6。我们发现，这些动物显示异位表达的Shh和它的转录目标，特别是在前间充质的后肢芽。GATA 6在发育肢体中的缺失导致后肢中轴前多指畸形的形成。相反，GATA 6在整个肢芽中的强制表达抑制Shh的表达，并导致亚形肢。我们已经发现GATA 6可以与编码Shh或Gli 1调控元件的染色质（从肢芽中分离）结合，这些调控元件驱动这些基因在该组织中的表达，并证明GATA 6与FOG辅因子协同作用，以抑制由这些序列驱动的荧光素酶报告基因的表达。最重要的是，我们已经发现，在缺乏GATA 6的肢芽中Shh的条件性丧失阻止了这些复合突变体胚胎中后肢多指畸形的发育，表明肢芽前部区域中的GATA 6表达通过抑制Shh的异位表达来阻断后肢多指畸形。Sonic Hedgehog（Shh）是发育中的肢芽的生长和前后模式的关键调节器，并且产生于肢芽后部的“极化活动区”。在这里，我们表明，GATA 4和GATA 6（成员的加塔家族的转录因子）表达在前间充质的小鼠肢芽和肢芽特异性缺失GATA 6的结果在异位表达的Shh和它的靶基因（如Gli 1）在前肢芽间充质，导致轴前多指（趾）。相反，GATA 6在肢芽中的过度表达会导致Shh及其靶基因的下调，从而导致手指数量减少。我们还表明，GATA 6结合到调节Shh或Gli 1的表达的序列，并且在发育中的肢芽中同时缺失GATA 6和Shh基因可以挽救GATA 6突变体的多趾后肢表型。我们的研究结果表明，GATA 6是必要的抑制异位表达的Shh和刺猬转录靶点在小鼠后肢芽的前部区域，从而表明，加塔转录因子，除了作为调节细胞的身份，是重要的负调节异位Shh表达的肢芽。
In the limb bud, patterning along the anterior-posterior (A-P) axis is controlled by Sonic Hedgehog (Shh), a signaling molecule secreted by the “Zone of Polarizing Activity”, an organizer tissue located in the posterior margin of the limb bud. We have found that the transcription factors GATA4 and GATA6, which are key regulators of cell identity, are expressed in an anterior to posterior gradient in the early limb bud, raising the possibility that GATA transcription factors may play an additional role in patterning this tissue. While both GATA4 and GATA6 are expressed in an A-P gradient in the forelimb buds, the hindlimb buds principally express GATA6 in an A-P gradient. Thus, to specifically examine the role of GATA6 in limb patterning we generated Prx1-Cre; GATA6fl/fl mice, which conditionally delete GATA6 from their developing limb buds. We found that these animals display ectopic expression of both Shh and its transcriptional targets specifically in the anterior mesenchyme of the hindlimb buds. Loss of GATA6 in the developing limbs results in the formation of preaxial polydactyly in the hindlimbs. Conversely, forced expression of GATA6 throughout the limb bud represses expression of Shh and results in hypomorphic limbs. We have found that GATA6 can bind to chromatin (isolated from limb buds) encoding either Shh or Gli1 regulatory elements that drive expression of these genes in this tissue, and demonstrated that GATA6 works synergistically with FOG co-factors to repress expression of luciferase reporters driven by these sequences. Most significantly, we have found that conditional loss of Shh in limb buds lacking GATA6 prevents development of hindlimb polydactyly in these compound mutant embryos, indicating that GATA6 expression in the anterior region of the limb bud blocks hindlimb polydactyly by repressing ectopic expression of Shh. Sonic Hedgehog (Shh) is a crucial regulator of the growth and anterior-posterior patterning of the developing limb bud, and is produced in the “Zone of Polarizing Activity” in the posterior of the limb bud. Here, we demonstrate that GATA4 and GATA6 (members of the GATA family of transcription factors) are expressed in the anterior mesenchyme of mouse limb buds and that limb bud-specific deletion of GATA6 results in ectopic expression of Shh and its target genes (such as Gli1) in the anterior limb bud mesenchyme, resulting in preaxial polydactyly. Conversely, over-expression of GATA6 in limb buds causes down-regulation of Shh and its target genes, resulting in a decreased number of digits. We also show that GATA6 binds to the sequences that regulate expression of either Shh or Gli1, and that simultaneous deletion of both GATA6 and Shh genes in developing limb buds rescues the polydactylous hindlimb phenotype of GATA6 mutants. Our findings indicate that GATA6 is necessary to repress ectopic expression of both Shh and hedgehog transcriptional targets in the anterior region of the mouse hindlimb bud, and thus demonstrate that GATA transcription factors, in addition to being regulators of cell identity, are important negative regulators of ectopic Shh expression in the limb bud.
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