Mechanisms of Eph/Ephrin signaling in craniofacial morphogenesis and craniofrontonasal syndrome

项目摘要 先天性畸形影响1%的活产婴儿，其中三分之一显示颅面畸形。 我们对许多这些疾病的遗传原因的理解已经取得了很大的进步，但是 对潜在的发育、细胞和分子机制缺乏了解， 阻碍了个性化医疗方法来治疗这些疾病。颅额鼻综合征（CFNS）是一种 由影响颅面和胸部骨骼的EFNB 1基因突变引起的X连锁疾病，以及 神经发育EFNB 1功能的完全丧失会导致距离过远和腭裂，但 仅在一些细胞中的EFNB 1镶嵌性丢失，具有更严重的后果，也导致面中部 发育不全、冠状颅缝早闭、肢体畸形和胼胝体发育不全。小鼠Efnb 1 突变体有效地模拟CFNS，表现出大多数相同的表型。Efnb 1编码Ephrin-B1 a EphB是一种跨膜信号分子，与EphB受体酪氨酸激酶结合以调节细胞位置。在 Efnb 1 +/-杂合子女性，X染色体失活导致Ephrin-B1功能嵌合， 随后从Ephrin-B1突变细胞中分选出Ephrin-B1表达细胞。这种自我组织 容量是许多不同组织中Eph/Ephrin信号传导的一般特征。在颅面发育中，我们 假设Ephrin-B1在颅面间充质的组织中发挥关键作用，但如何发挥作用 这种情况的发生，以及它如何影响颅面形状，仍然是未知的。因此，我们的目标是了解 Ephrin-B1在发育、细胞和分子水平上调节正常颅面形态发生。 首先，为了了解Ephrin-B1如何调节特定颅面胚胎原基形状的变化， 我们将利用微计算机断层扫描进行基于地标的形态测量分析， 在特定结构中缺乏Ephrin-B1的小鼠胚胎。使用类似的方法，我们将评估相对 三种受体EphB 1、EphB 2和EphB 3参与颅面形态发生。二是 利用新的工具来研究正常颅面间充质的分布、形状、密度和极性， 和Efnb 1突变体胚胎。最后，我们将利用CRISPR基因组编辑技术来执行一个 候选基因的功能筛选以鉴定Ephrin-B1/EphB下游的信号传导途径 发信号。这项工作将提供新的见解的机制，其中Ephrin-B1通常控制 间充质细胞的行为在颅面形态发生，并确定其损失如何导致病理性 颅面形状的改变。因此，这些研究将为我们长期的研究提供基础知识。 长期目标是开发用于治疗颅面畸形和 提高对颅面形态发生基本过程的理解。潜在的细胞 并且分子机制也可能与Eph/Ephrin信号传导 对发育和疾病都很重要。

Project summary Congenital anomalies affect 1% of live births, with one-third of these displaying craniofacial abnormalities. Great advances have been made in our understanding of the genetic causes of many of these conditions, but lack of understanding of the underlying developmental, cellular, and molecular mechanisms continues to stymie personalized medicine approaches to treat these conditions. Craniofrontonasal syndrome (CFNS) is an X-linked condition caused by mutations in the EFNB1 gene that affects craniofacial and thoracic skeleton, and neurological development. Complete loss of EFNB1 function results in hypertelorism and cleft palate, but mosaic loss of EFNB1 in only some cells, has more severe consequences, resulting also in midfacial hypoplasia, coronal craniosynostosis, limb abnormalities, and agenesis of the corpus callosum. Mouse Efnb1 mutants effectively model CFNS, exhibiting most of the same phenotypes. Efnb1 encodes Ephrin-B1 a transmembrane signaling molecule that binds to EphB receptor tyrosine kinases to regulate cell position. In Efnb1+/- heterozygous females, X chromosome inactivation leads to mosaicism for Ephrin-B1 function and subsequent sorting-out of Ephrin-B1-expressing cells from Ephrin-B1 mutant cells. This self-organizing capacity is a general feature of Eph/Ephrin signaling in many different tissues. In craniofacial development, we hypothesize that Ephrin-B1 plays a critical role in the organization of the craniofacial mesenchyme, though how this occurs, and how it affects craniofacial shape, remains unknown. It is therefore our goal to understand how Ephrin-B1 regulates normal craniofacial morphogenesis at the developmental, cellular and molecular scales. First, to understand how Ephrin-B1 regulates changes in shape of specific craniofacial embryonic primordia, we will utilize micro-computed tomography scanning to perform landmark-based morphometric analysis of mouse embryos lacking Ephrin-B1 in specific structures. Using a similar approach, we will evaluate the relative involvement of three receptors EphB1, EphB2 and EphB3 in craniofacial morphogenesis. Second, we will utilize new tools to study the distribution, shape, density, and polarity of the craniofacial mesenchyme in normal and Efnb1 mutant embryos. Finally, we will harness CRISPR genome editing technology to perform a functional screen of candidate genes to identify the signaling pathways downstream of Ephrin-B1/EphB signaling. This work will provide new insights into the mechanisms by which Ephrin-B1 normally controls mesenchymal cell behaviors in craniofacial morphogenesis, and determine how its loss results in pathological changes in craniofacial shape. These studies will therefore provide foundational knowledge toward our long- term goal of developing improved therapies for the treatment of individuals with craniofacial anomalies and an improved understanding of the fundamental processes of craniofacial morphogenesis. The underlying cellular and molecular mechanisms may also be relevant to any of the numerous contexts where Eph/Ephrin signaling is important in development and disease.