GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 8552564
• 负责人: ALAN PERANTONI
• 金额: $ 119.49万
• 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/8552564
• 关键词: Affect; Animal Model; Apoptotic; Benign; Binding; Binding Proteins; Biological Assay; Brachyury protein; Calcineurin; Calcium; Calcium Signaling; Caricatures; Cell Count; Cell Culture Techniques; Cell Polarity; Cell Survival; Cells; Cephalic; Collaborations; Computer Simulation; Defect; Development; Disease; Duct (organ) structure; Embryo; Employee Strikes; Epithelial; Epithelium; Family; Family member; Genitourinary system; Goals; Intermediate Mesoderm; Invaded; Kidney; Lead; Ligands; Maintenance; Malignant Childhood Neoplasm; Mediating; Mesenchymal; Mesenchyme; Mesoderm; Metanephric Diverticulum; Metanephric structure; Modeling; Molecular Target; Morphogenesis; Movement; Mus; Neoplasms; Nephroblastoma; Nephrogenic Cord; Nephrons; Normal tissue morphology; Organogenesis; Pathogenesis; Pathway interactions; Pediatric Neoplasm; Phenotype; Phosphorylation; Population; Preclinical Drug Evaluation; Process; Proteins; Regulation; Reporting; Rho-associated kinase; Role; STAT3 gene; STAT5A gene; Signal Transduction; Site; Skeletal system; Stem cells; Structure; Structure of mesonephric duct; Study Section; System; Tamoxifen; Time; Tissues; Transcription Factor AP-2 Alpha; Twin Multiple Birth; Ureter; Xenograft procedure; activating transcription factor; bone; bone loss; cytokine; drug development; inhibitor/antagonist; interest; kinase inhibitor; leukemia inhibitory factor; loss of function; male; mineralization; mouse model; mutant; neoplastic; neoplastic cell; nephrogenesis; novel; overexpression; prevent; progenitor; promoter; reproductive; response; small molecule; substantia spongiosa; tissue culture; tool; tumor; tumorigenesis; tumorigenic

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 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 results in the downstream activation of calcineurin. Calcineurin then dephosphorylates/activates transcription factors from the NFAT family. LIF also up regulates the expression of NFAT family members. Similarly, the GSK3beta inhibitors block the phosphorylation of NFAT, and induce the expression of MET markers. By ChIP assay, we have found that, following Wnt4 treatment of MM cells, NFAT binds directly to the promoter of a critical target of nephron formation, namely, Lim1/Lhx1. These findings confirm the role of calcium signaling and NFAT activation in MET of MM progenitors. This has led us to speculate that disruption of calcium signaling may be involved in Wilms tumor pathogenesis. Studies by M. Bouchard have demonstrated that calcineurin A-binding protein is overexpressed in more than 70% of WTs, consistent with our hypothesis. This protein interferes with the ability of calcineurin to dephosphorylate and thus activate NFAT, presumably also preventing the induction of MET. Thus regulation of signaling through this pathway may provide a useful target for WT therapy. An examination of this hypothesis is ongoing. In addition to its role in the induction of MET in MM, LIF also appears to have a 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 (1-5 ng/ml). LIF functions principally through STAT activation, and the lower levels of LIF activate both STAT3 and STAT5 in renal progenitors. At 1 ng/ml, LIF increases the number of cells expressing renal stem cell marker Six2 and this is further increased with the concurrent addition of a Rho kinase inhibitor. The combination in fact causes a massive expansion of Six2-expressing cells in tissue culture. Importantly, these 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 differentiate in culture. Similar culture conditions have now also been applied to dissociated tissue fragments from WTs and support the propagation of Six2-expressing tumor cells, which have been shown to populate the blastemal portions of these tumors. We are currently characterizing the lines generated from these primary cultures. If tumorigenic in xenografts, these lines could provide an important new model for drug screening. We have also 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. 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 loss of mineralization in this tissue. We are currently generating conditional double mutants for Stats 1 and 3 and Stats 1 and 5 in order to circumvent the complications of redundancy and further assess the role of these Stats in renal development. Since mesodermal populations are significantly affected in the bone, we are also evaluating the MM progenitors in the metanephros for apoptotic cells. Finally, in collaboration with CDBL PI Terry Yamaguchi, we are investigating 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 bifid ureter formation bilaterally, a condition that afflicts some 1% of 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. However, Wnt5a is normally not expressed around the site of UB outgrowth at E10.5, suggesting that the cause of duplex kidney precedes this development in the IM. In fact, Wnt5a is expressed in a gradient at both the cranial and caudal ends of the IM at E9.5. To examine the effect of its expression on duplex kidney formation, we ablated Wnt5a in the posterior of the embryo using several tissue-specific Cre lines. T-Cre, Pax3-Cre, and Hoxb6-Cre eliminate Wnt5a expression in the posterior IM, whereas the combination of AP2-Cre and Rarb2-Cre or Dll1-Cre, which are active within the kidney itself, do not. T-Cre, Pax3-Cre and Hoxb6-Cre mediated Wnt5a mutants have a truncated and widened IM and double ureters, while AP2-Cre and Rarb2-Cre or Dll1-Cre-mediated Wnt5a mutants have a normal IM and single ureters. Moreover, tamoxifen induction of Hoxb6-Cre-ERT2 at E6.5 yielded Wnt5a-deficient embryos with an aberrant IM and double ureters; whereas tamoxifen induction at E8.5 resulted in mutants with normal IM extension and single ureters. Further studies using progenitor-specific markers reveal that at E9.5, Wnt5a mutant embryos with an IM disturbance have a striking misorientation of ND cells, typical of disrupted planar cell polarity signaling. Moreover, the ND bifurcates at its caudal terminus, thus forming twin epithelial UB outgrowths in the surrounding MM. These results suggest that Wnt5a regulates IM extension and that its loss in the IM causes double ureter and duplex kidney formation. We are currently collaborating with a computer modeling group to develop hypotheses that might explain this phenotype.

肾脏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，同时研究其在肿瘤发生中的失调，重点关注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标。我们的重点是泌尿生殖道的发育，其特征是两个不同的中胚层祖细胞之间的相互相互作用、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讽刺肾发生的肿瘤。更具体地说，我们对引导后肾间充质（MM）转化为肾单位上皮的信号传导机制感兴趣。肾母细胞瘤 (WT) 的特点是胚细胞/祖细胞群增多，但上皮转化 (MET) 能力有限。我们的长期目标是确定 WT 细胞生存或失调信号传导所依赖的靶标，这些靶标可以重新编程以使肿瘤细胞分化为更良性的表型。 MM 祖细胞中导致 MET 的诱导信号传导可以由多种因素介导，包括 Wnt4，它对于肾单位的形成至关重要。 我们之前报道过 Wnt4 通过钙依赖性机制诱导 MET，而不是像想象的那样通过经典 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中的肾小管形成 (MET)，小分子 GSK3β 抑制剂也可以。 这些诱导剂的共同点是它们激活钙信号传导的能力。 例如，LIF 会诱导 PLCgamma 磷酸化，从而导致下游钙调磷酸酶激活。 然后钙调神经磷酸酶去磷酸化/激活 NFAT 家族的转录因子。 LIF 还上调 NFAT 家族成员的表达。 同样，GSK3beta 抑制剂阻断 NFAT 的磷酸化，并诱导 MET 标记物的表达。 通过 ChIP 检测，我们发现，在 Wnt4 处理 MM 细胞后，NFAT 直接与肾单位形成关键靶点（即 Lim1/Lhx1）的启动子结合。 这些发现证实了钙信号传导和 NFAT 激活在 MM 祖细胞 MET 中的作用。这使我们推测钙信号传导的破坏可能与肾母细胞瘤的发病机制有关。 M. Bouchard 的研究表明，钙调神经磷酸酶 A 结合蛋白在超过 70% 的 WT 中过度表达，这与我们的假设一致。 这种蛋白质干扰钙调神经磷酸酶去磷酸化的能力，从而激活 NFAT，大概也阻止了 MET 的诱导。因此，通过该途径调节信号传导可能为 WT 治疗提供有用的靶点。对这一假设的检验正在进行中。 除了在 MM 中诱导 MET 中发挥作用外，LIF 似乎还具有维持和扩大 MM 祖细胞群的功能。 LIF 通过激活这些祖细胞中的钙信号传导，以 30-50 ng/ml 的浓度在 MM 中诱导 MET。 然而，LIF 也会维持和扩大祖细胞群至无法诱导 MET 的水平 (1-5 ng/ml)。 LIF 主要通过 STAT 激活发挥作用，较低水平的 LIF 会激活肾祖细胞中的 STAT3 和 STAT5。 1 ng/ml 的 LIF 会增加表达肾干细胞标记 Six2 的细胞数量，并且随着同时添加 Rho 激酶抑制剂，这种情况会进一步增加。 事实上，这种组合会导致组织培养中表达 Six2 的细胞大量扩增。 重要的是，这些细胞保留了在培养物中进行 MET 的能力，从而为研究这一过程提供了强大的工具。来自这些培养物的细胞现已传代多次，且不丧失在培养物中分化的能力。 类似的培养条件现在也已应用于从 WT 中分离的组织碎片，并支持表达 Six2 的肿瘤细胞的增殖，这些细胞已被证明可以填充这些肿瘤的胚基部分。 我们目前正在表征这些原代文化产生的品系。 如果异种移植物具有致瘤性，这些细胞系可以为药物筛选提供重要的新模型。 我们还继续研究 STAT 在发育中的后肾中的作用，我们发现 STAT 1、3、5 和 6 高度表达并被磷酸化/激活。 使用条件性功能丧失 (LOF) 小鼠模型，对 Stat3 的 LOF 突变体进行的初步评估显示，骨骼系统存在广泛缺陷，但除了可能的大小差异之外，肾脏没有明显的改变。 通过与 CDBL 的同事合作，我们发现 Stat3 是维持骨小梁所必需的，而 Stat3 的缺失会导致该组织矿化的丧失。 我们目前正在为统计 1 和 3 以及统计 1 和 5 生成条件双突变体，以避免冗余的并发症并进一步评估这些统计在肾脏发育中的作用。由于骨骼中的中胚层群体受到显着影响，我们还在评估后肾中 MM 祖细胞的凋亡细胞。 最后，我们与 CDBL PI Terry Yamaguchi 合作，正在研究 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会导致双侧双肾和双歧输尿管形成，这种情况困扰着总人口的约 1%。通常情况下，形成集合管和输尿管的输尿管芽从小鼠 E10.5 处的中间中胚层 (IM) 中的沃尔夫管 (WD) 中作为单个分支延伸。然而，Wnt5a 通常在 E10.5 的 UB 生长部位周围不表达，这表明双肾的原因早于 IM 中的这种发育。事实上，Wnt5a 在 E9.5 的 IM 头端和尾端均以梯度表示。为了检查其表达对双肾形成的影响，我们使用几个组织特异性 Cre 系消除了胚胎后部的 Wnt5a。 T-Cre、Pax3-Cre 和 Hoxb6-Cre 消除了后 IM 中的 Wnt5a 表达，而在肾脏本身内活跃的 AP2-Cre 和 Rarb2-Cre 或 Dll1-Cre 的组合则不会。 T-Cre、Pax3-Cre 和 Hoxb6-Cre 介导的 Wnt5a 突变体具有截短且加宽的 IM 和双输尿管，而 AP2-Cre 和 Rarb2-Cre 或 Dll1-Cre 介导的 Wnt5a 突变体具有正常的 IM 和单输尿管。此外，他莫昔芬在 E6.5 诱导 Hoxb6-Cre-ERT2 产生了 Wnt5a 缺陷胚胎，具有异常 IM 和双输尿管；而 E8.5 处的他莫昔芬诱导产生具有正常 IM 延伸和单个输尿管的突变体。使用祖细胞特异性标记的进一步研究表明，在 E9.5，IM 干扰的 Wnt5a 突变胚胎具有 ND 细胞的显着错误方向，这是平面细胞极性信号传导破坏的典型特征。此外，ND 在其尾端分叉，从而在周围 MM 中形成双上皮 UB 生长物。这些结果表明 Wnt5a 调节 IM 延伸，并且其在 IM 中的丢失导致双输尿管和双肾形成。 我们目前正在与计算机建模小组合作，提出可能解释这种表型的假设。