Role of Ectodermal Signals in Facial Prominence Outgrowth and Development

描述（由申请人提供）：大约75%的出生缺陷涉及头部、面部和口腔组织。虽然颅面裂和其他颅面畸形有明确的环境和遗传原因，但颅面发育机制的信息不足，无法在出生前检测或预防大多数这些缺陷。我们的目标是开发颅面畸形的动物模型，这将导致对相关人类出生缺陷的诊断和治疗机制的见解。颅面骨骼的进化和形成依赖于胚胎发生时神经管边缘产生的一种特殊的细胞群——神经嵴。虽然这些细胞含有重要的内在图案信息，但它们也依赖于面部突出的表面外胚层提供的信号来实现其生长和图案潜力。许多研究，主要是利用鸟类模型系统开始的，已经证明了在外胚层和间质之间运作的几种信号转导通路对面部的形态发生至关重要。事实上，由Fgf、Hedgehog （Hh）、Wnt和BMP家族的分泌因子引发的信号级联反应可以改变小鸡脸的大小和形状。由于小鼠在子宫内发育，这些研究更具挑战性。此外，影响这些信号转导通路关键成分的小鼠基因敲除在胚胎发生早期死亡。因此，为了研究这些信号事件在后期发育过程（如面部形态发生）中的功能，有必要采用条件基因敲除技术。许多Cre重组酶转基因可以靶向发育中的面部外胚层的基因表达，但大多数受其表达的程度和/或时间的限制。最近，我们产生了一种新的Cre重组酶转基因，Crect，它绕过了许多这些问题。在E9.5之前，Crect可以介导整个胚胎外胚层的重组，并且对该组织层及其衍生物具有高度特异性。利用Fgf8、Ctnnb1（¿-catenin）和Shh的条件等位基因与Crect获得的初步数据表明，这些信号分子在外胚层的表达对于面部图案的重要性。这些研究还揭示了这些通路在塑造面部的过程中广泛的相互作用。因此，本应用程序的目标是确定这些蛋白质如何单独发挥作用，并作为外胚层中的网络来调节颅面形成。在Aim I中，我们将使用Crect来研究Wnt/2-catenin信号在外胚层中对面部发育的作用。在Aim II中，我们将对Hh通路进行类似的分析。在Aim III中，我们将测试这两种途径与Fgf8的相互作用。这些分析的结果将揭示这些信号通路如何相互作用来控制小鼠颅面形态发生，并将为面部生长的调节和与人类出生缺陷和面部重建研究相关的模式提供重要的见解。公共卫生相关性：出生缺陷影响约3%的美国出生婴儿，其中约75%涉及头部、面部和口腔组织，严重出生缺陷的存在通常会降低儿童和父母的生活质量。关于颅面发育机制的信息不足，无法在出生前检测或预防大多数这些缺陷。我们正在使用动物模型系统来确定正常和异常颅面发育是如何进行的，并确定调解面部形成的新机制，以便我们可以应用这些知识来理解并最终治疗人类面部出生缺陷的起源。

DESCRIPTION (provided by applicant): About 75% of birth defects involve the head, face, and oral tissues. Although orofacial clefts and other craniofacial malformations have clear environmental and genetic causes, insufficient information exists concerning the mechanisms of craniofacial development to enable the majority of these defects to be detected or prevented pre-natally. Our goal is to develop animal models of craniofacial malformations that will lead to mechanistic insight into the diagnosis and treatment of related human birth defects. The evolution and formation of the craniofacial skeleton relies on a specialized population of cells, the neural crest, arising at the margins of the neural tube during embryogenesis. While these cells contain significant intrinsic patterning information, they also rely on signals supplied by the surface ectoderm of the facial prominences to fulfill their growth and patterning potential. A number of studies, largely initiated using avian model systems, have demonstrated that several signal transduction pathways operating between the ectoderm and mesenchyme are critical for morphogenesis of the face. Indeed, manipulation of signaling cascades initiated by secreted factors belonging to the Fgf, Hedgehog (Hh), Wnt, and BMP families can alter the size and shape of the chick face. These studies have been much more challenging in the mouse due to its in utero development. Moreover, mouse gene knockouts affecting critical components of these signal transduction pathways die early in embryogenesis. Therefore, to study the function of these signaling events in later developmental processes - such as facial morphogenesis - it has been necessary to employ conditional gene knockout technology. A number of Cre recombinase transgenes can target gene expression in the developing facial ectoderm, but most are limited by the extent and/or timing of their expression. Recently, we generated a new Cre recombinase transgene, Crect, which circumvents many of these problems. Crect can mediate recombination in the entire embryonic ectoderm prior to E9.5 and is highly specific for this tissue layer and its derivatives. Preliminary data obtained using conditional alleles of Fgf8, Ctnnb1 (¿-catenin), and Shh with Crect have shown the importance of the expression of these signaling molecules in the ectoderm for facial patterning. These studies have also revealed extensive cross-talk between these pathways in shaping the face. Thus, the goal of this application is to determine how these proteins function individually and as a network in the ectoderm to regulate craniofacial formation. In Aim I we will use Crect to investigate the role of Wnt/2-catenin signaling in the ectoderm for development of the face. In Aim II we will perform a similar analysis on the Hh pathway. In Aim III we will test the interplay of these two pathways with Fgf8. The results of these analyses will reveal how these signaling pathways interact to control mouse craniofacial morphogenesis and will provide significant insight into the regulation of facial growth and patterning pertinent to the study of human birth defects and facial reconstruction. PUBLIC HEALTH RELEVANCE: Birth defects affect ~ 3% of all infants born in the US - with about 75% of these involving the head, face, and oral tissues - and the presence of a major birth defect will frequently reduce the quality of life for both the child and the parents. Insufficient information exists concerning the mechanisms of craniofacial development to enable the majority of these defects to be detected or prevented pre-natally. We are using animal model systems to determine how normal and abnormal craniofacial development proceeds and to identify new mechanisms that mediate face formation so that we may apply this knowledge to understand and ultimately treat the origins of human facial birth defects.