TGF beta regulation of cilium-dependent signaling

描述（申请人提供）：初级纤毛在细胞信号传导和哺乳动物发育中起重要作用。纤毛缺陷与许多人类发育障碍有关，包括脑积水、慢性肺阻塞、多囊肾病和左右不对称缺陷。初级纤毛对Sonic hedgehog （Shh）信号通路至关重要，并可能在其他信号通路中发挥作用。破坏Shh信号或纤毛功能的突变在小鼠中引起类似的表型，包括肢体缺陷和前脑畸形（HPE），这是一种影响颅面发育的严重遗传疾病。人类编码Shh和Tgif1基因的功能突变缺失与HPE有关。Tgif1是一种转录辅助抑制因子，通过Smad2限制转化生长因子（TGF）信号。然而，目前尚不清楚Tgif1及其相关的Tgif2如何调节胚胎发生，也不清楚Tgif1和TGF信号如何调节纤毛发生和Shh信号。我们提出验证在胚胎发育过程中必须严格限制TGF家族信号的模型，不受限制的TGF家族信号通过Smad2破坏细胞极性和初级纤毛的形成，从而抑制纤毛依赖性信号。通过有条件地靶向小鼠的Tgif1和Tgif2基因，我们创建了TGF信号过剩的小鼠模型，其中细胞极性和纤毛发生被破坏。此外，缺乏所有Tgif功能的小鼠存在胚胎缺陷，包括HPE和左右不对称缺陷，并且多能因子Nanog的表达增加。具体而言，我们将：1)确定tgfs是否通过Smad2抑制TGF/Nodal信号来控制前脑Shh信号，并测试缺乏tgfs的HPE是否由于TGF家族信号过量。2)确定在Tgif功能缺失的情况下，是由于极性缺陷和初级纤毛缺失导致前脑无法对Shh信号做出反应，还是Tgif和TGF信号直接调控Shh基因的表达。3)确定是否必须限制TGF家族信号，以防止其他胚胎组织（包括神经管和淋巴结）的初级纤毛和纤毛依赖信号的中断。最后，我们将测试Nanog是否是tgif抑制的直接靶点，以及Nanog的过度表达是否会导致细胞极性和纤毛发生缺陷。这项工作将确定tgfs和TGF信号如何与Shh通路整合，并确定TGF信号在纤毛发生和纤毛依赖性信号传导中的作用。

DESCRIPTION (provided by applicant): The primary cilium plays important roles in cellular signaling and mammalian development. Cilia defects are associated with a number of human developmental disorders, including hydrocephaly, chronic pulmonary obstruction, polycystic kidney disease and left-right asymmetry defects. Primary cilia are critical for Sonic hedgehog (Shh) signaling, and may play roles in other signaling pathways. Mutations that disrupt Shh signaling or cilia function cause similar phenotypes in mice, including limb defects and holoprosencephaly (HPE), a severe genetic disorder affecting craniofacial development. Loss of function mutations in the human genes encoding Shh and Tgif1 are associated with HPE. Tgif1 is a transcriptional corepressor that limits transforming growth factor (TGF) signaling via Smad2. However, it is not known how Tgif1, and the related Tgif2, regulate embryogenesis, or how Tgifs and TGF signaling regulate ciliogenesis and Shh signaling. We propose to test the model that TGF family signaling must be strictly limited during embryonic development, and that unrestrained TGF family signaling via Smad2 disrupts cell polarity and the formation of primary cilia, thereby inhibiting cilia-dependent signaling. By conditionally targeting both the Tgif1 and Tgif2 genes in mice, we have created a mouse model for excess TGF signaling, in which cell polarity and ciliogenesis are disrupted. Additionally, mice lacking all Tgif function have embryonic defects, including HPE and left-right asymmetry defects, and have increased expression of the pluripotency factor, Nanog. Specifically, we will: 1) Determine whether Tgifs control Shh signaling in the forebrain by restraining TGF/Nodal signaling via Smad2, and test whether HPE in the absence of Tgifs is due to excess TGF family signaling. 2) Determine whether, in the absence of Tgif function, the forebrain is unable to respond to Shh signaling due to polarity defects and the absence of primary cilia, or whether Tgifs and TGF signaling directly regulate Shh gene expression. 3) Determine whether TGF family signaling must be limited to prevent disruption of primary cilia and cilia- dependent signaling in other embryonic tissues, including the neural tube and node. Finally, we will test whether Nanog is a direct target for repression by Tgifs, and whether excess Nanog expression causes the cell polarity and ciliogenesis defects. This work will determine how Tgifs and TGF signaling are integrated with the Shh pathway, and determine the role that TGF signaling plays in ciliogenesis and cilia-dependent signaling.