GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 8762983
• 负责人: ALAN PERANTONI
• 金额: $ 114.62万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/8762983
• 关键词: Address; Animal Model; Benign; Birth; Brachyury protein; Calcium; Calcium Signaling; Caricatures; Cell Count; Cell Culture Techniques; Cell Maintenance; Cell Proliferation; Cell Survival; Cells; Chondrocytes; Collaborations; Defect; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Duct (organ) structure; Ectopic Expression; Epithelial; Epithelium; Family; Gene Expression; Genetic Transcription; Genitourinary system; Goals; Growth; Intermediate Mesoderm; Invaded; Investigation; Kidney; Lead; Lesion; Ligands; Link; Maintenance; Malignant Childhood Neoplasm; Mediating; Mesenchymal; Mesenchyme; Mesoderm; Metanephric Diverticulum; Metanephric structure; Molecular Target; Morphogenesis; Movement; Mus; Neoplasms; Nephroblastoma; Nephrogenic Cord; Nephrons; Normal tissue morphology; Nuclear; Organogenesis; Osteoblasts; Osteoclasts; Osteogenesis; Paraxial Mesoderm; Pathogenesis; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Population; Process; Proteins; RORA gene; Regulation; Reporting; Rho-associated kinase; Role; STAT3 gene; Series; Signal Pathway; Signal Transduction; Skeletal system; Skeleton; Small Interfering RNA; Stem cells; Stromal Cells; Structure; Structure of mesonephric duct; Study Section; System; Time; Tissues; Transcriptional Activation; Undifferentiated; Ureter; bone loss; cytokine; drug development; inhibitor/antagonist; interest; kinase inhibitor; knock-down; leukemia inhibitory factor; loss of function; male; malformation; member; mineralization; mouse model; mutant; neoplastic; neoplastic cell; nephrogenesis; novel; progenitor; receptor; reproductive; response; self-renewal; small molecule; stemness; substantia spongiosa; tool; transcription factor; tumor; tumorigenesis

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 SIX2/CITED1-positive blastemal/progenitor population with a restricted capacity for epithelial conversion (MET). It is 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. Inductive signaling in MM progenitors that results in MET can be mediated by a variety of factors, including Wnt4, which is essential for nephron formation. We previously reported that Wnt4 induces MET by a calcium-dependent mechanism and not by canonical Wnt signaling as thought. We also determined that the cytokine leukemia inhibitory factor (LIF) can similarly induce tubule formation (MET) in renal progenitors, as can small molecule GSK3beta inhibitors. The common thread among these inducers is their ability to activate calcium signaling. LIF, for example, induces phosphorylation of PLCgamma, which activates calcium signaling and subsequently NFAT-mediated transcription. In addition to its role in the induction of MET in MM, LIF also appears to function in the maintenance and expansion of the MM progenitor population. LIF induces MET in MM at 30-50 ng/ml through activation of calcium signaling in these progenitors. However, LIF also maintains and expands the progenitor population at levels that fail to induce MET (1 ng/ml) or activate PLCgamma. It is known to function principally through STAT activation, and the lower levels of LIF activate STATs 1, 3 and 5 in renal progenitors without simultaneously activating calcium signaling. At 1 ng/ml, LIF increases proliferation and the number of cells expressing renal stem cell marker and critical maintenance and self-renewal factor Six2. This response is further facilitated with the concurrent addition of a Rho kinase inhibitor (ROCKi). Importantly, LIF/ROCKi-treated cells retain their ability to undergo MET in culture, thus producing a powerful tool for studying this process. Cells from these cultures have now been passaged multiple times without a loss of ability to undergo MET. An investigation into the mechanism(s) mediated by LIF/ROCKi in these cells revealed that our conditions facilitate the nuclear localization of Yes-associated protein (YAP), a transcriptional co-activator and component of the Hippo signaling pathway. Furthermore, silencing Yap gene expression by siRNA knockdown in MM cells decreased the expression of progenitor markers and increased levels of MET markers, suggesting that YAP maintains MM cells in an undifferentiated state. Ectopic expression of a series of dominant-negative mutant constructs for YAP confirmed that nuclear YAP is sufficient to sustain MM cell 'stemness' and increase their rate of proliferation. Since YAP interacts with Tead transcription factors, we also knocked down members of the Tead family using siRNA and determined that Tead2 and/or Tead3 are required for YAP-dependent transcription and MM progenitor cell maintenance. Taken together, our results demonstrate that LIF/ROCK signaling controls MM cell maintenance and self-renewal by regulating YAP/Tead2/Tead3-dependent transcription. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation. As part of a collaboration with the Carroll lab/UTexas, we characterized the role of Yap and Fat4 in the regulation of the aggregate Six2-expressing progenitor population during metanephric development. Our studies demonstrated that the Six2+ population is growth limited by the surrounding cortical stroma and that loss of stromal cells causes a dramatic expansion of Six+ stem cells. Furthermore, the studies found that Fat4 in stromal cells is responsible for the suppression of Yap in regulating the Six2+ population. These findings suggest a possible role for dysregulated Fat4/Yap signaling in the pathogenensis of Wilms tumors, since tumors characteristically contain a massively expanded Six2+ population. Immunostaining for Yap in Wilms tumor cells also revealed nuclear localization of the co-activator, which of course is consistent with transcriptional activation. We are currently evaluating the significance of this observation. We have continued our examination of the role of STATs in the developing metanephros, where we have found STATs 1, 3, 5, and 6 to be highly expressed and phosphorylated/activated. Given the responsiveness of MM cells to LIF treatment in inducing progenitor marker expression and MM cell proliferation as described above, we believe that Stat signaling plays a fundamental role in stem cell maintenance in the metanephros and possibly other mesodermal tissues. Using conditional loss-of-function (LOF) mouse models, a preliminary assessment of a LOF mutant for Stat3 has revealed extensive defects in the skeletal system but no obvious alterations in the kidney other than possible size differences. In collaboration with colleagues in CDBL, we have found that Stat3 is required for maintenance of the trabecular bone, and the loss of Stat3 results in the loss of mineralization in this tissue. Signatures consistent with interrupted endochondral bone formation were evident in the expansion of hypertrophic chondrocytes and the observed downregulation of the osteochondro master regulator, Sox9. Further, a rapid depletion of the osteoblast lineage coinciding with elevation of the osteoclast population results in wide-spread osteoporotic lesions soon after birth. These findings demonstrate a critical role for STAT3 in the proper patterning of the mammalian skeleton and implicate Sox9 as a downstream target of STAT3 signaling in this process. We are continuiing our examination of Stat redundancy in the metanephros through the acquisition of a conditional Stat3/5 mouse line. This line will also allow us to generate a Stat1/3/5 triple mutant and assess the role of their combined effects on renal development. Finally, in collaboration with CDBL PI Terry Yamaguchi, we continue to investigate the role of Wnt5a in metanephric development. We have found that its specific inactivation in mesoderm using T/Brachyury-Cre results in duplex kidneys and double ureter formation bilaterally, a common malformation in the overall population. Normally the ureteric bud, which forms the collecting ducts and ureter, extends as a single outgrowth from the Wolffian duct (WD) in the intermediate mesoderm (IM) at E10.5 in the mouse. Since Wnt5a is expressed in both the IM and the adjacent paraxial mesoderm (PM), which directs axis extension, its loss from either or both tissues may cause the WD malformation. To address this directly, we have acquired IM and PM specific Cre lines to establish each tissue's contribution to formation of the WD. We are also investigating the mechanism by which Wnt5a signals to regulate WD formation. To this end, we have demonstrated that the Wnt receptor ROR2 is linked genetically to the Wnt5a duplex ureter phenotype.

肾分化和肿瘤组研究组织发育/形态发生中的诱导信号，同时研究其在肿瘤发生中的失调，重点研究介导正常组织相互作用的配体以及响应信号激活的途径和靶标。我们的重点是泌尿生殖道的发育，其特点是两种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），不同谱系结构的整合，反复的发育周期，以及一种讽刺肾发生的肿瘤。更具体地说，我们感兴趣的是指示后肾间质（MM）转化为肾元上皮的信号机制。Wilms肿瘤（WT）的特点是SIX2/ cited1阳性胚/祖细胞群体扩大，上皮转化（MET）能力有限。我们的长期目标是确定WT细胞赖以生存或信号失调的靶标，这些靶标可以被重新编程，使肿瘤细胞分化为更良性的表型。导致MET的MM祖细胞诱导信号可以由多种因素介导，包括对肾元形成至关重要的Wnt4。我们之前报道过Wnt4通过钙依赖机制诱导MET，而不是像我们认为的那样通过典型的Wnt信号传导。我们还确定细胞因子白血病抑制因子（LIF）可以类似地诱导肾祖细胞的小管形成（MET），小分子gsk3 β抑制剂也可以。这些诱导剂的共同点是它们激活钙信号的能力。例如，LIF诱导plcγ的磷酸化，激活钙信号和随后的nfat介导的转录。除了在MM中诱导MET的作用外，LIF似乎还在MM祖细胞群体的维持和扩大中起作用。LIF通过激活这些祖细胞中的钙信号，以30-50 ng/ml诱导MM中的MET。然而，在不能诱导MET （1 ng/ml）或激活PLCgamma的水平上，LIF也能维持和扩大祖细胞群。已知LIF主要通过STAT激活起作用，较低水平的LIF激活肾祖细胞中的stat1、3和5，而不同时激活钙信号。当浓度为1 ng/ml时，LIF可增加增殖和表达肾干细胞标志物及关键维持和自我更新因子Six2的细胞数量。同时加入Rho激酶抑制剂（ROCKi）进一步促进了这种反应。重要的是，LIF/ rocki处理的细胞在培养中保留了MET的能力，从而为研究这一过程提供了有力的工具。来自这些培养的细胞现在已经传代多次，而没有失去经历MET的能力。对这些细胞中由LIF/ROCKi介导的机制的研究表明，我们的条件促进了yes相关蛋白（YAP）的核定位，YAP是一种转录共激活因子和Hippo信号通路的组成部分。此外，通过敲低siRNA沉默MM细胞中Yap基因的表达，降低了祖标记的表达，增加了MET标记的水平，表明Yap维持MM细胞处于未分化状态。一系列YAP显性阴性突变体的异位表达证实了核YAP足以维持MM细胞的“干性”并提高其增殖速度。由于YAP与Tead转录因子相互作用，我们也使用siRNA敲除了Tead家族的成员，并确定Tead2和/或Tead3是YAP依赖性转录和MM祖细胞维持所必需的。综上所述，我们的研究结果表明，LIF/ROCK信号通过调节YAP/Tead2/ tead3依赖性转录来控制MM细胞的维持和自我更新。这种MM培养系统提供了独特的机会，以全面解决涉及肾祖细胞维持和分化的关键机制。作为与Carroll实验室/ texas合作的一部分，我们表征了Yap和Fat4在后细胞发育过程中对总six2表达祖细胞群体的调节中的作用。我们的研究表明，Six2+细胞群的生长受到周围皮质基质的限制，基质细胞的缺失会导致Six+干细胞的急剧扩增。此外，研究发现基质细胞中的Fat4在调节Six2+群体中抑制Yap。这些发现提示Fat4/Yap信号失调可能在Wilms肿瘤的发病过程中起作用，因为肿瘤的特点是含有大量扩增的Six2+群体。在Wilms肿瘤细胞中对Yap的免疫染色也显示了共激活子的核定位，这当然与转录激活一致。我们目前正在评估这一观察结果的意义。我们继续研究STATs在发育中的后肾中的作用，发现STATs 1、3、5和6是高表达和磷酸化/激活的。鉴于MM细胞对LIF治疗的反应性，诱导祖细胞标志物表达和MM细胞增殖，我们认为Stat信号在后肾和其他中胚层组织的干细胞维持中起着重要作用。使用条件功能丧失（LOF）小鼠模型，对Stat3的LOF突变体的初步评估显示，骨骼系统存在广泛的缺陷，但除了可能的大小差异外，肾脏没有明显的改变。在与CDBL同事的合作中，我们发现Stat3是维持小梁骨所必需的，Stat3的丢失会导致该组织矿化的丢失。与软骨内骨形成中断相一致的特征在肥大软骨细胞的扩张和观察到的骨软骨总调节因子Sox9的下调中很明显。此外，成骨细胞谱系的快速消耗与破骨细胞群的升高相一致，导致出生后不久广泛的骨质疏松病变。这些发现表明STAT3在哺乳动物骨骼的正确模式中起着关键作用，并暗示Sox9在这一过程中是STAT3信号传导的下游目标。我们通过获得条件Stat3/5小鼠系，继续研究后肾中的Stat冗余。这条细胞系也将允许我们产生Stat1/3/5三重突变体，并评估它们在肾脏发育中的联合作用。最后，在与CDBL PI Terry Yamaguchi的合作下，我们继续研究Wnt5a在后肾发育中的作用。我们发现，使用T/Brachyury-Cre在中胚层中特异性失活可导致双肾和双输尿管形成，这是总体人群中常见的畸形。通常情况下，在小鼠E10.5岁时，输尿管芽从Wolffian管（WD）中作为一个单独的外生物从中胚层（IM）中延伸出来，形成集合管和输尿管。由于Wnt5a在IM和邻近的指导轴向延伸的近轴中胚层（PM）中均表达，因此其在任一组织或两组织中的缺失都可能导致WD畸形。为了直接解决这个问题，我们已经获得了IM和PM特定的Cre线，以确定每个组织对WD形成的贡献。我们也在研究Wnt5a信号调控WD形成的机制。为此，我们证明了Wnt受体ROR2与Wnt5a双输尿管表型有遗传联系。