Analysis of Cardiac Development in Ts65Dn Mice

项目概述：Ts65Dn小鼠是唐氏综合征（DS）最具特征的动物模型。这些小鼠携带T65Dn标记染色体，在小鼠16号染色体（MMU16）的一个区域产生三体，其中包括人类21号染色体（HSA21）上约50%的同源基因。40%的DS个体表现出先天性心脏缺陷（CHD），我们假设Ts65Dn小鼠将表现出与心脏发育缺陷相关的表型。我们已经证明，Ts65Dn的节段性三体在出生时的传播率接近预期的50%，但在断奶后的后代中下降到34%，Ts65Dn新生儿在出生48小时内选择性丢失。尸体大体解剖和组织学检查显示Ts65Dn新生儿主动脉弓和房室垫相关的间隔缺损。这些观察结果支持了一个或多个DS - CHD候选基因位于Ts65Dn三体区域的假设。我们将检查远端MMU16中包含的基因和心内膜缓冲中正常表达的蛋白质，以确定对正常心脏分离至关重要的细胞信号传导和分化途径是否受到Ts65Dn基因过表达的调节。我们计划通过以下具体目的来研究Ts65Dn小鼠的心脏发育：1。确定Ts65Dn胚胎心脏异常的范围。使用光镜和扫描电镜对心脏形态发生关键阶段的准体（野生型，wt）和三体兄弟姐妹的胚胎心脏进行大体和组织学检查，以确定Ts65Dn后代心脏缺陷的发生率和表型范围。2. 利用体外EMT/迁移实验分析Ts65Dn转基因心脏上皮细胞向间充质转化（EMT）的过程，这是AV心内膜缓冲形成的关键。3. 研究Ts65Dn和wt心脏组织的生化和分子差异，重点研究Ts65Dn区域内的基因。心脏发育基因标记和候选DS - CHD基因将使用原位杂交和免疫组织学分析，以确定心内膜垫和衍生结构中基因和蛋白质表达的时空模式。原理：小鼠模型通常用于分析人类疾病的潜在遗传原因，以及识别对正常哺乳动物发育至关重要的基因。确定剂量敏感基因及其错误表达导致退行性变性患者先天性心脏缺陷的机制，将有助于了解二倍体基因在正常心脏发育过程中的作用，并可能为退行性变性或整倍体患者先天性心脏缺陷提供改善策略。

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Ts65Dn mice represent the best-characterized animal model for Down syndrome (DS). These mice carry the T65Dn marker chromosome, producing trisomy for a region of mouse chromosome 16 (MMU16) that includes orthologs of about 50% of the genes on human chromosome 21 (HSA21). Forty percent of DS individuals display congenital heart defects (CHD), and we hypothesize that the Ts65Dn mice will display phenotypes related to defective cardiac development. We have demonstrated that the transmission rate of segmental trisomy in Ts65Dn is near the expected 50% at birth, but declines to 34% of offspring by weaning, with selective loss of Ts65Dn neonates within 48 hours of birth. Gross anatomical and histological examination of cadavers indicates aortic arch and AV cushion related septal defects in Ts65Dn neonates. These observations support the hypothesis that one or more candidate genes for DS CHD occur within the trisomic region of Ts65Dn. We will examine genes contained in distal MMU16 and proteins normally expressed in the endocardial cushions to determine if the pathways of cell signaling and differentiation critical to normal cardiac septation are modulated by overexpression of the Ts65Dn genes. We plan to investigate the cardiac development of Ts65Dn mice through the following Specific Aims: 1. Determine the range of cardiac anomalies present in Ts65Dn embryos. Gross plus histological examination using light and scanning electron microscopy will be performed on embryonic hearts of the eupoid (wild type, wt) and trisomic siblings at critical stages of cardiac morphogenesis to determine the incidence and phenotypic range of cardiac defects in Ts65Dn offspring. 2. Analyze the process of epithelial to mesenchymal transformation (EMT), critical to AV endocardial cushion formation, in Ts65Dn transgenic hearts using an in vitro EMT/ migration assay. 3. Investigate the biochemical and molecular differences between Ts65Dn and wt cardiac tissues with emphasis on genes within the Ts65Dn region. Cardiac developmental gene markers and candidate DS CHD genes will be analyzed using in situ hybridization and immunohistology to determine the temporal- spatial patterns of gene and protein expression in endocardial cushions and derivative structures. RATIONALE: Mouse models are commonly used to analyze underlying genetic causes of human disease, as well as to identify the genes that are critical for normal mammalian development. Defining the dosage sensitive genes and the mechanisms by which their misexpression contributes to congenital heart defects in DS will provide insights into the roles of diploid genes during normal cardiac development and may suggest ameliorative strategies for congenital heart defects in DS or euploid individuals.