Developmental reprogramming and transorganogenesis

总结 拟议研究的主要目标是阐明细胞和分子机制， 控制发育的可塑性，并研究如何有丝分裂后分化的细胞在一个完整的动物， 在转分化（Td）过程中重编程并重塑为新的细胞类型。描述良好的 C.内胚层发育途径elegans将被应用于分子解剖Td， “transorganogenesis”（一个器官转化为另一个器官）。该途径中的一个组分，C。elegans ELT-7加塔转录因子能够转化两种器官的分化的有丝分裂后细胞， 咽部和子宫，转化为具有正常肠道基因表达模式和超微结构特征的细胞 细胞在这个非凡的过程中，转录组发生了高度动态的变化， （滥交、衰减、消退、反弹、持续和重塑）可以在基于Td的治疗期间解决。 基因表达和改变细胞和器官形态。这些事件定义了Td感受态（Td+）细胞 这些细胞转化为肠样细胞和Td-抗性（Td-）细胞，仅允许ELT-7短暂激活其靶点。 在Td期间表达上调的基因中，有一些与蛋白质相关的基因， 周转，自噬和细胞内病原体反应（IPR），表明这些途径发挥作用， 在Td.在ELT-7诱导的发育停滞的基础上，开发了两种遗传选择， 大量的Td缺陷突变体。有了这些初步的发现，我们将探讨机制， 通过三个具体目标。在SA 1中，我们将研究细胞重塑的动力学，测试细胞内蛋白质的表达。 假设蛋白质降解和自噬的周转过程参与了Td的关键阶段， 重塑，评估IPR在Td中的作用，并研究细胞周期退出在Td过程中的作用。在 SA 2，我们将评估不同分化细胞类型的单个Td+和Td-细胞之间的差异是否可以 将归因于其独特的转录组动力学，并将测试染色质变化的假设， 通过ATAC-seq评估的Td-细胞的结构，最初和短暂地通过类似的模式进行， Td+细胞的那些，弹性回复到原始状态。在SA 3中，我们将表征来自两个 在ELT-7介导的发育停滞和Td中有缺陷的遗传选择，并将使用它们来 研究TD六个阶段的相互依赖性。我们将找出大数字背后的因果基因 通过高通量方法，基于合并样本中变异体的统计分析， 全基因组序列，包括编码蛋白质和非编码RNA的序列。这些研究可能会推进 我们对消化道癌前化生机制的理解。他们还将 提供了对出生缺陷中器官畸形机制的深入了解，并可能导致方法， 用于再生医学中器官生成的重编程分化。

SUMMARY The major objectives of the proposed research are to illuminate the cellular and molecular mechanisms that control developmental plasticity and to investigate how post-mitotic differentiated cells in an intact animal can be reprogrammed and remodeled into new cell types in the process of transdifferentiation (Td). The well-described pathway for endoderm development in C. elegans will be applied to molecularly dissecting Td and “transorganogenesis” (conversion of one organ into another). One component in this pathway, the C. elegans ELT-7 GATA transcription factor, is capable of converting differentiated, post-mitotic cells of two organs, the pharynx and uterus, into cells with gene expression patterns and ultrastructural characteristics of normal gut cells. Highly dynamic changes in the transcriptome occur during this remarkable process, and six stages (promiscuity, attenuation, extinction, rebound, persistence, and remodeling) can be resolved during Td based on gene expression and altered cellular and organ morphology. These events define Td-competent (Td+) cells that convert to gut-like cells and Td-resistant (Td-) cells that only transiently permit ELT-7 to activate its target. Among the genes whose expression undergoes upregulation during Td are sets of genes associated with protein turnover, autophagy, and the intracellular pathogen response (IPR), suggesting that these pathways play a role in Td. On the basis of ELT-7-induced developmental arrest, two genetic selections were developed that identify large numbers of mutants defective in Td. With these preliminary findings in hand, we will probe the mechanisms of Td through three Specific Aims. In SA1, we will investigate the dynamics of cellular remodeling, test the hypothesis that turnover processes of protein degradation and autophagy participate in key stages of Td and remodeling, assess the role of the IPR in Td, and investigate the action of cell-cycle exit in the Td process. In SA2, we will evaluate whether differences between single Td+ and Td- cells of varied differentiated cell types can be ascribed to their unique transcriptome dynamics and will test the hypothesis that changes in chromatin architecture of Td- cells, assessed by ATAC-seq, initially and transiently proceed through similar patterns to those of Td+ cells, with elastic reversion to the original state. In SA3, we will characterize mutants from two genetic selections that are defective in ELT-7-mediated developmental arrest and Td and will use them to investigate interdependence of the six stages of Td. We will identify the causal gene(s) underlying large numbers of mutants defective in Td by a high-throughput method based on statistical analysis of variants in pooled genome-wide sequences, including those coding for proteins and non-coding RNAs. These studies may advance our understanding of mechanisms involved in pre-cancerous metaplasias of the digestive tract. They will also provide insights into the mechanisms of organ malformation in birth defects and could lead to methods for reprogramming differentiation for organ generation in regenerative medicine.