Left-Right Axis Determination and Cardiac Development

描述(由申请人提供)：大多数复杂的先天性心脏缺陷发生在同时患有偏侧疾病的个体中，反映出发育中的心脏对左右(LR)模式过程中的障碍极其敏感。这项建议的目的是确定产生LR身体计划的诱导机制，并阐明心肌细胞如何利用这一位置“蓝图”来发展LR不对称。通过对转化生长因子-β超家族受体的研究，我们发现两个激活素样激酶--ALK2和ALK4--对于非洲爪蛙胚胎心脏(和其他器官)的LR发育既是必要的也是充分的。利用非洲爪哇独有的一系列实验操作，我们将测试ALK2和ALK4通过在LR轴确定中发挥关键作用来调节心脏LR发育的假设。使用生化和功能丧失分析，AIM 1将通过定义ALK通路的配体来识别对LR轴形成重要的细胞-细胞信号。使用经典的移植和共培养试验，AIM 2将通过确定ALK是否将位置信号传递到中线，在已知的细胞-细胞相互作用的背景下定位ALK通路。由于参与中线调控和其他LR传递相互作用的许多分子都是未知的，因此AIM 3将通过识别起作用的上游、下游或ALK通路内的基因来填补其中的一些空白。这将通过进行高通量表达克隆筛选来实现，在该筛选中，申请人已经并将继续鉴定新的偏侧基因。利用体内谱系标记，AIM 4将通过确定ALK信号是否最终导致从成对的左和右心野到不同的直心管不同节段的特定不对称细胞贡献，来解决心脏细胞如何利用LR模式信息。总而言之，我们的研究成果将为加快胚胎心脏形成和脊椎动物LR发育这两个交叉领域的进展提供必要的新知识。这项工作的长期意义将是识别可能导致先天性心脏(和其他)LR缺陷的基因和诱导过程，如果受到遗传或环境扰动的影响。

DESCRIPTION (provided by applicant): The majority of complex congenital heart defects occur in individuals who are also afflicted by laterality disease, reflecting the extreme susceptibility of the developing heart to disturbances in left-right (LR) patterning processes. The objective of this proposal is to identify inductive mechanisms that generate the LR body plan and to elucidate how cardiac cells use this positional "blueprint" to develop LR asymmetries. With focus on receptors for the transforming growth factor-Beta superfamily, we have found that two Activin-Like Kinases--ALK2 and ALK4--are both necessary and sufficient for LR development of the heart (and other organs) in embryos of the frog, Xenopus Iaevis. Taking advantage of the range of experimental manipulations that is uniquely possible in Xenopus, we will test the hypothesis that ALK2 and ALK4 modulate cardiac LR development by playing pivotal roles in LR axis determination. Using both biochemical and loss-of-function analyses, Aim 1 will identify cell-cell signals important for LR axis formation by defining ligands for the ALK pathways. Using classic transplantation and co-culture assays, Aim 2 will position ALK pathways in the context of cell-cell interactions already known to be important for LR patterning by determining whether ALKs relay positional signals to the midline. Because many of the molecules involved in midline specification and other LR relay interactions are not known, Aim 3 will fill in some of these gaps by identifying genes that function upstream, downstream, or within ALK pathways. This will be accomplished by performing a high throughput expression cloning screen in which new laterality genes already have been, and will continue to be, identified by the applicant. Using in vivo lineage labeling, Aim 4 will address how cardiac cells utilize LR patterning information by determining whether ALK signaling culminates in specific asymmetric cell contributions from the paired left and right heart fields to different segments of the "straight" heart tube. Together, our results will contribute new knowledge that is necessary to accelerate progress in the intersecting fields of embryonic heart formation and vertebrate LR development. The long-term significance of the work will be to identify genes and inductive processes that may result in congenital cardiac (and other) LR defects if affected by genetic or environmental perturbations.