The role of the primitive choana in amniote craniofacial morphogenesis

描述(由申请人提供)：我建议研究羊膜后鼻孔的发育，特别强调确定对胚胎口腔的其他关键因素的假定影响，如次级腭部形成。后鼻孔是连接口腔和鼻道的内部鼻孔，对正常的鼻腔呼吸至关重要。在哺乳动物中，后鼻孔被第二上颚覆盖，因此直接开口进入鼻咽。然而，在大多数其他爬行动物羊膜中，后鼻孔直接进入口腔。尽管它们在所有脊椎动物中都存在，但很少有人关注这种结构。这一点尤其值得注意，因为人类后鼻孔发育不正确(例如，将后鼻孔与鼻道分开的口鼻膜滞留)，导致后鼻孔闭锁(CA)，可能导致婴儿死亡。最近的几项研究已经确定了当受到干扰时，可以在小鼠模型中导致CA的遗传途径。此外，我们的初步研究表明，后鼻孔表达关键的发育基因，这些基因与颅骨发育的其他各个方面有关，从牙齿发育的启动到次生腭部的生长和融合。我推测，在非常年轻的胚胎中，后鼻孔距离足够近，足以影响口腔顶部相邻结构的发育。因此，我已经提出了一系列研究，这些研究将有助于描述与有和没有次级上颚的羊膜动物适当的后鼻孔发育相关的组织特异性变化。随后，我试图确定我们观察到的在脊椎动物中特别活跃的分子途径的可能作用。对于这些研究，我将使用两种模式生物--鸡和老鼠，以及一种非模式生物--海龟。这三个羊膜代表了三种不同类型的次级上颚。老鼠有完整的次级上颚，鸡有天然的腭裂，而海龟有原始的上颚。这些比较研究将使我们能够比较后鼻孔周围组织中表达的基因，并识别那些与闭合和开放上颚相关的基因。下一步，我将培养包含后鼻孔的胚胎腭部，并将增加或降低该区域丰富的不同信号分子的水平。我还将植入微小的箔层屏障，以阻止后鼻孔信号到达上颚。这些干预措施对口鼻膜的破裂和腭突的启动的影响将被调查。在鸡身上，将通过在鸡蛋中的胚胎中添加信号或其抑制剂来研究对腭骨的长期影响。这将是关于脊椎动物胚胎发育的第一次研究。将调查面部是否存在新的信号中心，并将发现导致面部畸形(如腭裂)的新的分子原因。

DESCRIPTION (provided by applicant): I propose to study the development of the amniote choana, with specific emphasis on identifying putative influences on other key elements of the embryonic oral cavity such as secondary palate formation. The choanae are the internal nares which connect the oral cavity to the nasal passages and are crucial for proper nasal respiration. In mammals, the choanae are covered by the secondary palate and therefore, open directly into the nasopharynx. In most other reptilian amniotes however, the choanae open directly into the oral cavity. Despite their persistence in all vertebrates, very little focus has been placed on thi structure. This is especially remarkable since incorrect development of the choana (e.g. retention of the oronasal membrane separating the choana from the nasal passage) in humans, which results in choanal atresia (CA), can lead to infant mortality. Several recent studies have identified genetic pathways which, when perturbed, can cause CA in mouse models. Additionally, our preliminary studies have shown that choanae express key developmental genes which are implicated in various other aspects of cranial development ranging from the initiation of tooth development to the growth and fusion of the secondary palate. I hypothesize that in very young embryos the choanae are in close enough proximity to influence development of the adjacent structures in the roof of the oral cavity. Therefore, I have proposed a series of studies which will serve to characterize the tissue specific changes associated with proper choanal development in amniotes with and without a secondary palate. Subsequently, I seek to identify the likely roles of the molecular pathways which we have observed to be specifically active in the choanae. For these studies, I will use two model organisms, chicken and mouse, as well as a non-model organism, the turtle. These three amniotes represent three different types of secondary palates. Mice have a complete secondary palate, chickens have a natural cleft palate and turtles have primitive palate. These comparative studies will allow us to compare genes expressed in tissues surrounding the choanae and identify those that are correlated with a closed versus open palate. Next, I will culture the embryonic palate containing the choanae and will increase or decrease the levels of different signalling molecules that are enriched in this region. I will also implant microscopic foil barriers to block the choana signals from reaching the palate. The effect of these interventions on the breakdown of the oronasal membrane and the initiation of palatal shelves will be investigated. In chicken, the longer term effects on palatine bones will be studied by adding the signals or their inhibitors to the embryos while in the egg. These will be the first studies on the embryonic development of the choanae. The presence of a new signalling center in the face will be investigated, and novel molecular causes for facial abnormalities such as cleft palate will be discovered.