EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region.
EN and GBX2 play essential roles downstream of FGF8 in patterning the mouse mid/hindbrain region.
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作者：
Aimin Liu;A. Joyner
Fgf8 在胚胎中脑/后脑交界处表达，是诱导中脑和小脑结构形成所必需的且足以诱导中脑和小脑结构的形成。为了了解 FGF8 通过哪些遗传途径发挥作用，我们使用新型小鼠脑外植体培养系统检查了响应 FGF8 的中脑/后脑基因的上位关系。我们发现，在用 FGF8 浸泡的珠子处理的野生型 E9.5 间脑和中脑外植体中，En2 和 Gbx2 是 FGF8 诱导的第一个基因。通过检查En1/2双突变小鼠胚胎中的基因表达，我们发现Fgf8、Wnt1和Pax5不需要En基因来启动表达，但需要En基因来维持它们，并且Pax6表达在缺乏EN功能的情况下扩展到中脑尾部。由于E9.5 En1/2双突变体缺乏中脑/后脑区域，因此用FGF8处理前脑突变体外植体，并且显着地，发现EN转录因子是诱导Pax5所必需的。因此，FGF8调节的Pax5表达依赖于EN蛋白，并且FGF8以外的因子可能参与启动中脑/后脑中的正常Pax5表达。 En 基因在 FGF8 对前脑外植体中 Pax6 的抑制中也发挥着重要但不是绝对的作用。先前的 Gbx2 功能获得研究表明，中脑中 Gbx2 的错误表达可能导致 Otx2 的抑制。然而，在缺乏 Gbx2 的情况下，FGF8 仍然可以抑制中脑外植体中的 Otx2 表达。相比之下，Wnt1 最初在 Gbx2 突变体外植体中被广泛诱导，如在野生型外植体中一样，但随后在通常表达 Gbx2 的 FGF8 附近的细胞中并未受到抑制。因此，GBX2 在抑制 Otx2 时作用于 FGF8 的上游或平行作用，并在抑制 Wnt1 时作用于 FGF8 的下游。这是在小鼠中进行的第一个此类上位研究，结合了功能获得和功能丧失的方法，揭示了中脑/后脑中小鼠基因调控的各个方面，而单独使用任何一种方法都难以解决这些问题。
Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address through what genetic pathways FGF8 acts, we examined the epistatic relationships of mid/hindbrain genes that respond to FGF8, using a novel mouse brain explant culture system. We found that En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, we found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Previous Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone.