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Growth-Differentiation Factors in Organogenesis

器官发生中的生长分化因素

基本信息

批准号：
10014274
负责人：
ALAN PERANTONI
金额：
$ 11.01万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014274
关键词：
Ablation Adherent Culture Affinity Anchorage-Independent Growth Apoptosis Apoptotic Behavior Belief Benign Bladder CD3E gene Caricatures Cell Culture Techniques Cell Survival Cells Collaborations Cultured Tumor Cells Development Differentiation and Growth Disease Duct (organ) structure Embryo Epithelial Epithelium Female Gene Expression Profiling Genes Genetic Genitourinary system Goals Histologic Hydronephrosis Individual Inferior Intermediate Mesoderm Invaded Kidney LacZ Genes Lead Ligands LoxP-flanked allele Maintenance Malignant Childhood Neoplasm Manuscripts Mediating Mesenchymal Mesenchyme Metanephric Diverticulum Metanephric structure Minor Modeling Modification Molecular Target Morphogenesis Movement Mus Mutant Strains Mice Neoplasms Nephroblastoma Nephrogenic Cord Nephrons Normal tissue morphology One-Step dentin bonding system Organogenesis Organoids Paraxial Mesoderm Pathogenesis Pathway interactions Patients Phenotype Population Primary Neoplasm Process Publications Renal function Retirement Role Signal Induction Signal Transduction Stem cells Structure Structure of mesonephric duct Study Section System Testing Therapeutic Intervention Time Tissues Tubular formation Ureter Urinary tract Urine Work Xenograft procedure aldehyde dehydrogenase 1 base cell preparation driving force interest loss of function male molecular targeted therapies mutant neoplastic neoplastic cell nephrogenesis novel personalized therapeutic prevent progenitor receptor reproductive tract response screening stemness tumor tumorigenesis tumorigenic urogenital tract

项目摘要

The Renal Differentiation and Neoplasia Section studies inductive signaling in tissue development/morphogenesis and, in parallel, its dysregulation in tumorigenesis with emphasis on the ligands that mediate normal tissue interactions and the pathways and targets that are activated in response to signaling. Our focus has been on development of the urogenital tract, which features reciprocal interactions between two distinct mesodermal progenitors, highly coordinated tissue movements, mesenchymal-epithelial transition (MET), integration of structures from different lineages, reiterative cycles of development, and a tumor that caricatures nephrogenesis. More specifically we are interested in the signaling mechanisms that direct metanephric mesenchyme (MM) to convert to the epithelia of the nephron. Wilms tumor (WT) is characterized by an expanded blastemal/progenitor population with a restricted capacity for epithelial conversion (MET). It has been our long-term goal to identify targets on which WT cells depend for survival or dysregulated signaling that can be reprogrammed to allow tumor cells to differentiate to a more benign phenotype. With my retirement, efforts have been focused on bringing current studies to a reasonable and rapid conclusion leading to publication of the results. Accordingly, we have continued an assessment of cell conditions which allow for the robust expansion of cultured Wilms tumor cells for real-time testing of personalized options for therapeutic intervention. We previously defined conditions for the propagation of nephronic progenitors and applied similar conditions to studies of Wilms tumor. With minor modifications we have successfully grown Wilms tumor cells from primary tumors removed from multiple patients. These cells retain the expression of several "stemness" and WT-associated genes, such as, SIX2, ALDH1, and PAX2. They are stable for several passages and retain a tumorigenic phenotype based upon their ability for anchorage-independent growth. Cultured cells from these tumors have now been xenografted into NSG mice to further assess tumorigenic behavior, and one of the WT cultured cell preparations has formed tumors in mice. These tumors can be continuously passaged and retain their embryonal WT character histologically. All 5 cultured WTs readily form organoids when placed in nonadherent culture wells, but these conditions proved inferior to monolayer cultures in sustaining the expression of stemness markers with passage. Given the belief that these cells provide the driving force for WTs, the massive expansion of this population in culture witnessed in these studies may permit the development of personalized therapeutic screening for individual WT patients. This is a major advance in the field, as heretofore, failed efforts to propagate WT cells in culture have led some to speculate that they cannot be sustained except in xenografts. I am also in the process of revising a manuscript which describes an aberrant phenotype in the urinary tract associated with Wnt5a ablation, i.e., hydronephrosis. In these studies we show that hydronephrosis occurs as the result of a blockage in urine flow at the time renal function becomes active, causing apoptosis of the medullary region in the kidney. The blockage occurs at the interface between the ureter and bladder when cells in the common nephric duct fail to apoptose. Gene expression analysis revealed that Shh was increased in the pericloacal region in Wnt5a mutants, and we could rescue the phenotype with Shh haploinsufficiency. We also determined that constitutive activation of HH signaling yielded hydronephrosis and that Shh, which is produced by the cloacal epithelium, signals directly to the common nephric duct to sustain the tubular structure, preventing its degeneration to allow for ureter insertion into the bladder. This work demonstrates for the first time that Wnt5a suppresses Shh levels during development and possibly also during tumorigenesis, since Wnt5a expression is inversely regulated in tumorigenesis in such tissues as kidney and female reproductive tract. Taking this work one step further, I have ablated Wnt5a expression from all cells that respond to HH signaling during development (Gli1-Cre-ERT2). With the incorporation of a floxed allele for LacZ, I can identify those populations which are responding to Gli activation. It appears from these studies that Wnt5a is a global regulator of HH signaling throughout the embryo. Finally, I am also engaged in ongoing collaborative studies on Fgf8 with Mark Lewandoski's lab. These involve efforts to better understand its function in nephronic progenitor maintenance. This has been pursued using mouse genetics and cell culture models. To this end I have successfully demonstrated the partial rescue of progenitor survival in Fgf8 loss-of-function mutant mouse embryos lacking pro-apoptotic factors bax and bak. In another collaboration, we are evaluating the role of FgfrL1 in Fgf8 signaling in the developing kidney, since this receptor has greatest affinity for Fgf8 yet lacks a cytoplasmic signaling domain. Finally we seek an explanation for the absence of a cortical phenotype in Fgf8 mutant kidneys, when a kidney-specific Cre is used rather than mesodermal localized TCre. This suggests that Fgf8 signaling to the kidney is mediated at least in part by expression from adjacent tissues rather than the metanephros itself - possibly the paraxial mesoderm.

肾分化和肿瘤部分研究组织发育/形态发生中的诱导信号传导，同时研究肿瘤发生中的失调，重点是介导正常组织相互作用的配体以及响应信号传导而激活的途径和靶点。我们的重点一直是泌尿生殖道的发展，其特点是两个不同的中胚层祖细胞之间的相互作用，高度协调的组织运动，间充质上皮转化（MET），整合不同谱系的结构，发育的循环周期，以及一个肿瘤，讽刺肾发生。更具体地说，我们对指导后肾间充质（MM）转化为肾单位上皮的信号机制感兴趣。Wilms肿瘤（WT）的特征在于具有有限的上皮转化（MET）能力的扩增的胚基/祖细胞群体。我们的长期目标是确定WT细胞生存或失调信号所依赖的靶点，这些靶点可以被重新编程以允许肿瘤细胞分化为更良性的表型。随着我的退休，我的工作重点是使目前的研究得出合理和迅速的结论，从而发表结果。因此，我们继续评估细胞条件，其允许培养的Wilms肿瘤细胞的稳健扩增，用于治疗干预的个性化选择的实时测试。我们以前定义的条件下，为繁殖的肾祖细胞和应用类似的条件下，研究肾母细胞瘤。通过微小的修改，我们已经成功地从多个患者的原发性肿瘤中培养了Wilms肿瘤细胞。这些细胞保留了几个“干性”和WT相关基因的表达，如SIX 2，ALDH 1和PAX 2。它们在几代中是稳定的，并且基于其锚定非依赖性生长的能力保留致瘤表型。现在已将来自这些肿瘤的培养细胞异种移植到NSG小鼠中以进一步评估致瘤行为，并且其中一种WT培养细胞制剂已在小鼠中形成肿瘤。这些肿瘤可以连续传代并在组织学上保留其胚胎WT特征。当置于非贴壁培养威尔斯孔中时，所有5种培养的WT都容易形成类器官，但这些条件证明在维持传代的干性标志物表达方面劣于单层培养。鉴于这些细胞为WT提供驱动力的信念，这些研究中见证的培养物中该群体的大规模扩增可能允许开发针对个体WT患者的个性化治疗筛选。这是该领域的一个重大进展，因为迄今为止，在培养中繁殖WT细胞的失败努力已经导致一些人推测它们除了在异种移植物中之外不能持续。我也在修订一份手稿，该手稿描述了与Wnt 5a消融相关的尿路异常表型，即，肾积水在这些研究中，我们发现肾积水是由于肾功能活跃时尿流受阻，导致肾脏髓质区细胞凋亡而发生的。当肾总管中的细胞不能消化时，在输尿管和膀胱之间的界面处发生阻塞。基因表达分析表明，Shh在Wnt 5a突变体的盲肠周围区域增加，我们可以挽救Shh单倍不足的表型。我们还确定HH信号传导的组成性激活产生肾积水，并且由泄殖腔上皮产生的Shh直接向肾总管发出信号以维持管状结构，防止其变性以允许输尿管插入膀胱。这项工作首次证明了Wnt 5a在发育过程中以及可能在肿瘤发生过程中抑制Shh水平，因为Wnt 5a表达在肾脏和女性生殖道等组织的肿瘤发生中被反向调节。将这项工作更进一步，我已经消除了发育过程中对HH信号传导有反应的所有细胞的Wnt 5a表达（Gli 1-Cre-ERT 2）。通过将LacZ的floxed等位基因并入，我可以识别那些对Gli激活有反应的人群。从这些研究中可以看出，Wnt 5a是整个胚胎中HH信号传导的全局调节剂。最后，我还与Mark Lewandoski的实验室正在进行Fgf 8的合作研究。这些努力涉及更好地了解其在肾单位祖细胞维持中的功能。这已经使用小鼠遗传学和细胞培养模型进行了研究。为此，我已经成功地证明了部分拯救祖细胞的生存FGF 8功能丧失突变小鼠胚胎缺乏促凋亡因子bax和巴克。在另一项合作中，我们正在评估FgfrL 1在发育中的肾脏中Fgf 8信号传导中的作用，因为这种受体对Fgf 8具有最大的亲和力，但缺乏胞质信号传导结构域。最后，我们寻求一个解释FGF 8突变肾脏皮质表型的情况下，当使用肾脏特异性的Cre，而不是中胚层本地化的TCre。这表明Fgf 8信号传导至肾脏至少部分地由邻近组织的表达介导，而不是后肾本身-可能是近轴中胚层。