The Role of AP-2 in Craniofacial Development

描述(申请人提供)：约75%的出生缺陷涉及头部、面部和口腔组织。虽然口面部裂和其他颅面畸形有明显的环境和遗传原因，但关于颅面发育机制的信息不足，使这些缺陷中的大多数能够在出生前被发现或预防。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的机械性洞察。我们将重点放在AP-2转录因子家族上，因为这些基因提供了影响面部的人类出生缺陷的遗传和环境原因之间的联系。在老鼠和人类中，有五个不同的基因编码各种AP-2蛋白。其中三个基因在发育中的小鼠面部表达，Tcfap2a、Tcfap2b和Tcfap2c编码蛋白AP-2a、AP-2？和AP-2？分别进行了分析。人类AP-2a基因在BofS患者中发生突变，BofS患者存在口腔裂隙缺陷。同样，编码AP-2的人类基因突变会导致查尔综合征，其特征是面部、心脏和四肢缺陷。在体外和体内，Tcfap2a和Tcfap2c的表达也对维甲酸的应用做出反应，这些基因构成了受这种强大的致畸剂影响的基因调控网络的重要组成部分。上面讨论的三个小鼠AP-2基因的单基因敲除都会导致胚胎发生期间或出生后不久的死亡。Tcfap2b基因缺失的小鼠不会出现明显的面部缺陷，并会因神经和肾脏问题而死亡。Tcfap2c基因缺失的小鼠在原肠发育过程中死亡，因此很难衡量该基因在脸部形成中的作用。Tcfap2a与头面部发育有最直接的联系。Tcfap2a缺失的胚胎表现出多种缺陷，包括口面部裂开和神经脊源性面部骨骼发育不良，这主要是由于表面外胚层中AP-2a功能的丧失所致。尽管Tcfap2a、Tcfap2b和Tcfap2c在发育过程中具有独特的作用，但最近的数据表明，它们也共享多余的功能。事实上，初步研究表明，这些基因的复合敲除产生的面部表型比单独的基因敲除要严重得多。因此，这项应用的目标是确定这些蛋白质如何共同作用来调节头面部的形成。在目标1中，我们将建立BofS的小鼠模型，我们假设BofS通过显性负向机制抑制AP-2功能。在目标2和目标3中，我们将使用组织特异性Cre-loxP技术来生成和分析面部表达的三个AP-2基因的特定敲除组合。这些分析的结果将揭示AP-2基因在头面部形态发生中的组合作用，并为面部发育的转录控制提供重要的见解。与公共卫生相关：在美国出生的所有婴儿中，约有3%受到出生缺陷的影响，其中约75%涉及头部、面部和口腔组织，而严重出生缺陷的存在往往会降低孩子和父母的生活质量。关于颅面发育的机制还没有足够的信息，使得这些缺陷中的大多数能够在出生前被发现或预防。我们正在使用动物模型系统来确定正常和异常的颅面发育是如何进行的，并确定调节面部形成的新机制，以便我们可以应用这些知识来了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. We focus on the AP-2 family of transcription factors since these genes provide a link between the genetic and environmental causes of human birth defects affecting the face. In mouse and human, five separate genes encode the various AP-2 proteins. Three of these genes are expressed in the developing mouse face, Tcfap2a, Tcfap2b, and Tcfap2c encoding the proteins AP-2a, AP-2¿, and AP-2? respectively. The human gene encoding AP-2a is mutated in Branchio-Oculo-Facial Syndrome (BOFS) in which there are orofacial clefting defects. Similarly, mutations in the human gene encoding AP-2¿ cause Char syndrome, which is characterized by face, heart, and limb defects. Expression of Tcfap2a and Tcfap2c are also responsive to retinoic acid application both in vitro and in vivo and these genes form an important component of the gene regulatory network affected by this powerful teratogen. Single gene knockouts of the three mouse AP-2 genes discussed above all result in death during embryogenesis or soon after birth. Tcfap2b null mice do not display overt facial defects and die due to neurological and kidney problems. Tcfap2c null mice die during gastrulation, and so it has been more difficult to gauge the role of this gene in face formation. Tcfap2a has the most direct link to craniofacial development. The Tcfap2a null embryos display multiple defects including orofacial clefting and hypoplasia of the neural crest derived facial skeleton and this is mainly due to loss of AP-2a function in the surface ectoderm. Although Tcfap2a, Tcfap2b, and Tcfap2c have unique roles in development, recent data show that they also share redundant functions. Indeed, preliminary studies indicate that the facial phenotypes generated by compound knockouts of these genes are far more serious than the individual gene knockouts. Thus, the goal of this application is to determine how these proteins function together to regulate craniofacial formation. In Aim 1, we will generate a mouse model of BOFS, which we hypothesize inhibits AP-2 function via a dominant negative mechanism. In Aims 2 and 3 we will generate and analyze particular knockout combinations of the three AP-2 genes expressed in the face using tissue specific Cre-LoxP technology. The results of these analyses will reveal the combinatorial roles for the AP-2 genes in craniofacial morphogenesis and provide significant insight into the transcriptional control of facial development. 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.