Role of integrin alpha 5 in the formation of the pharyngeal arch arteries

项目总结 咽弓动脉(PAAS)的异常形成导致严重和致命的先天性 心脏病。PaaS通过血管生成形成，其中内皮细胞(EC)神经丛侵入 咽弓，然后结合成专利的PaaS。我们的实验室已经表明，大多数制造 向上的PaaS来源于一群被称为第二心区(SHF)的中胚层前体细胞。 此外，我们的实验室已经证明整合素ɑ5(itgɑ5)在ISL1谱系中的表达，包括 第二心区、咽内胚层和外胚层通过 未知的机制。整合素是细胞表面的受体，它组织细胞外基质并传递细胞外基质信号以诱导 细胞反应，包括细胞增殖、存活、迁移和分化。初步数据显示 ITGɑ5调节产生PAAS的SHF来源的内皮细胞的数量。我们假设整合素ɑ5 Isl1系的表达通过中介调节SHF来源的咽弓内皮细胞的数量 SHF-祖细胞存活和/或增殖、迁移和/或分化。在具体的目标一中，我们将 使用ITGɑ5f/-；Isl1Cre突变体和ITGɑ5f/+；Isl1Cre对照胚胎来研究这一假设，这些胚胎携带 Rosa26nTnG记者。记者用核GFP的表达来标记来源于Isl1系的细胞。 利用这种条件基因敲除小鼠模型和免疫组织化学方法，我们将研究其增殖情况。 以及产生的Isl1来源的细胞的凋亡率、迁移模式和分化能力 给PaaS的ECS。我的初步数据还显示，SHF来源的ECs在 在我们的突变体体内发育咽部区域。在脊椎动物的发育过程中，细胞一般不会交叉 中线确保了正确的左右图案。ECM组织和程序化细胞死亡反应 中线形成了一个屏障，阻止细胞在中线上迁移。因为我们知道ITGɑ5调节 细胞外基质组织我们假设ItgSHF5在Is11系中的表达调节ɑ来源的EC模式 通过调节细胞外基质组织和中线的程序性细胞死亡反应。我们会 利用我们的条件性小鼠模型和免疫组织化学，在《特定目标2》中研究这一假说。 我们将比较对照和突变胚胎中线的细胞外基质组织和细胞凋亡率。 此外，我们还将研究Isl1来源的细胞在胚胎发育早期中线的分布。 因为如果Isl1来源的细胞能够越过我们突变体的中线，那么导致 PAAS可能被引导到错误的一侧，导致异常的PAA形成。该计划的完成 这项建议中概述的实验将提供对参与发展的机制的见解 PaaS，因此将增加我们对相关先天性心脏病是如何发生的理解，创造新的 为临床检测、预防和治疗这些先天性心脏病提供了可能性。

PROJECT SUMMARY The aberrant formation of the Pharyngeal Arch Arteries (PAAs) results in severe and lethal forms of congenital heart disease. The PAAs form through vasculogenesis, where an endothelial cell (EC) plexus invades the pharyngeal arches before coalescing into patent PAAs. Our lab has shown that a majority of the ECs that make up the PAAs derives from a population of mesodermal progenitor cells known as the Second Heart Field (SHF). Additionally, our lab has shown that Integrin ɑ5 (Itgɑ5) expression within the Isl1-lineages, encompassing the second heart field and pharyngeal endoderm and ectoderm, regulates the formation of the PAAs through unknown mechanisms. Integrins are cell surface receptors that organize the ECM and relay ECM signals to elicit cellular responses, including cell proliferation, survival, migration, and differentiation. Preliminary data suggests Itgɑ5 regulates the number of SHF-derived ECs that give rise to the PAAs. We hypothesize that integrin ɑ5 expression in the Isl1-lineages regulates the number of SHF-derived ECs in the pharyngeal arches by mediating SHF-progenitor cell survival and/or proliferation, migration, and/or differentiation. In Specific Aim I, we will investigate this hypothesis using Itgɑ5f/- ; Isl1Cre mutant and Itgɑ5f/+ ; Isl1Cre control embryos, which carry the Rosa26nTnG reporter. This reporter marks cells derived from the Isl1-lineages with the expression of nuclear GFP. Utilizing this conditional knockout mouse model and immunohistochemistry, we will investigate the proliferation and apoptosis frequencies, migration patterns, and differentiation capacity, of the Isl1-derived cells that give rise to the ECs of the PAAs. My preliminary data additionally shows SHF-derived ECs crossing the midline in the developing pharyngeal region within our mutants. During vertebrate development, cells in general do not cross the midline ensuring proper left-right patterning. The ECM organization and programmed cell death response at the midline create a barrier that stops cells from migrating across the midline. Because we know Itgɑ5 regulates ECM organization we hypothesize that Itgɑ5 expression in the Isl1-lineages regulates SHF-derived EC patterning through mediating the ECM organization and the programmed cell death response at the midline. We will investigate this hypothesis in Specific Aim II, utilizing our conditional mouse model and immunohistochemistry. We will compare the ECM organization and apoptosis frequency at the midline in control and mutant embryos. Additionally, we will investigate the distribution of Isl1-derived cells at the midline during early embryogenesis because if Isl1-derived cells are able to cross the midline in our mutants, the Isl1-derived cells that give rise to the PAAs could be directed to the wrong side, resulting in aberrant PAA formation. The completion of the experiments outlined in this proposal will provide insights into mechanisms involved in the development of the PAAs and will therefore increase our understanding of how related congenital heart diseases arise, creating new possibilities for the clinical detection, prevention, and treatments of these congenital heart diseases.