Biophysical Regulation of Axial Polarity During Regeneration

描述(由申请人提供)：(1)干细胞和再生研究的临床应用不仅需要了解新生组织的合成，还需要了解调节胚泡形态的图案化机制。这个项目试图阐明由生物物理表观遗传变化控制的调控网络，这些变化定位胚泡相对于现有组织的极性。我们的假设是，H，K-ATPase转运蛋白和缝隙连接通讯(GJC)共同调节着传统信号上游的浮游生物再生过程中的前后极性。目标1是通过抑制H，K-ATPase(已完成)和GJC活性的再生蠕虫的微阵列分析，识别和表征离子转运下游的转录靶标。候选者将使用特定的标准进行选择，通过表达分析进行验证，并使用原位杂交和表型的分子标记分析来确定RNAi敲除后的功能特征。目标2是建立和测试模型，概述离子转运体在再生组织中用来控制轴向一致性的机制。通过识别网络节点，将产生离子转运下游的初步转录网络，使用定量PCR来测试单个候选基因的RNAi敲除如何影响所有其他候选基因的表达。这个网络预测的假说随后将被产生和测试，以阐明在再生极性建立过程中，对相关离子转运体活动的转录和信号响应。长期目标是编制和验证一个整合了生物物理和生化信号的系统级再生网络模型。这有可能被用作形态发生的预测模型，以帮助将基础再生研究转化为实用的生物医学疗法。(2)干细胞和再生研究的前景最终是为了替代受伤、衰老或癌症患者丢失或受损的组织、器官和四肢。虽然新组织的再生是关键，但为了使该组织具有功能，它必须与现有组织相关联地适当地形成图案：本研究旨在通过了解再生的扁虫如何决定在截肢后是否形成新的头部和/或尾巴，来发现新组织和旧组织是如何沟通以调节这种图案的。

DESCRIPTION (provided by applicant): (1) Clinical applications arising from stem cell and regeneration research will require an understanding not only of de novo tissue synthesis but also of the patterning mechanisms regulating blastema morphology. This project seeks to elucidate the regulatory networks controlled by biophysical epigenetic changes which orient blastema polarity relative to existing tissues. Our hypothesis is that ion transport by the H,K ATPase transporter and gap junctional communication (GJC) together regulate anterior-posterior polarity during planarian regeneration upstream of traditional signaling. Aim 1 is to identify and characterize transcriptional targets downstream of ion transport, through microarray analyses of regenerating worms inhibited for H,K ATPase (completed) and GJC activity. Candidates will be selected using specific criteria, validated by expression analyses, and functionally characterized following RNAi knockdown using in situ hybridization and molecular marker analyses of phenotypes. Aim 2 is to build and test models which outline the mechanisms ion transporters use to control axial identity in regenerating tissue. A preliminary transcriptional network downstream of ion transport will be generated by identifying network nodes, using quantitative PCR to test how RNAi knockdown of a single candidate affects the expression of all other candidates. Hypotheses predicted by this network will then be generated and tested, to elucidate the transcriptional and signaling response to the activity of the implicated ion transporters during the establishment of regenerative polarity. The long-term goal is to compile and validate a systems-level regeneration network model integrating biophysical and biochemical signaling. This has the potential for use as a predictive model of morphogenesis to aid in the translation of basic regeneration research into practical biomedical therapies. (2) The promise of stem cell and regeneration research is ultimately to replace lost or damaged tissues, organs and limbs in patients suffering from injury, aging or cancer. Although the regeneration of new tissue is critical, for that tissue to be functional, it must be properly patterned in relationship to the existing tissues: This research aims to discover how new and old tissues communicate to regulate this pattering, by learning how regenerating flatworms determine whether to form a new head and/or tail following amputation.