GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 9153468
• 负责人: ALAN PERANTONI
• 金额: $ 87.74万
• 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/9153468
• 关键词: 3-Dimensional; Ablation; Address; Attenuated; Back; Benign; Binding; Birth; Bladder; Bone Development; Bone Diseases; Brachyury protein; Caricatures; Cell Maintenance; Cell Survival; Cells; Chondrocytes; Collaborations; Competence; Congenital Abnormality; DNA; Defect; Development; Disease; Down-Regulation; Duct (organ) structure; Embryo; Epithelial; Epithelium; Event; Excision; Family; Gene Expression; Genetic Transcription; Genetic study; Genitourinary system; Goals; Grouping; Hydronephrosis; Image; Intermediate Mesoderm; Invaded; Investigation; Kidney; Lead; Length; Lesion; Ligands; MAPK8 gene; Maintenance; Malignant Childhood Neoplasm; Mediating; Mesenchymal; Mesenchyme; Mesoderm; Mesonephric structure; Metanephric Diverticulum; Metanephric structure; Molecular Target; Morphogenesis; Movement; Multipotent Stem Cells; Mus; Mutation; Neoplasms; Nephroblastoma; Nephrogenic Cord; Nephrons; Normal tissue morphology; Nuclear; Organogenesis; Osteoblasts; Osteoclasts; Osteogenesis; Pathogenesis; Pathway interactions; Pattern; Peritoneum; Phenotype; Population; Primitive Streaks; Process; Promoter Regions; Proteins; Reporter; Response Elements; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Skeletal system; Skeleton; Small Interfering RNA; Source; Stem cells; Structure; Structure of mesonephric duct; Study Section; Stuve-Wiedemann syndrome; System; Time; Tissues; Undifferentiated; Ureter; Urine; Vesico-Ureteral Reflux; bone loss; campomelic dysplasia; congenital anomaly; cytokine; interest; kinase inhibitor; leukemia inhibitory factor; long bone; loss of function; male; malformation; mineralization; mouse model; mutant; neoplastic; neoplastic cell; nephrogenesis; neural plate; novel; oncostatin M; pressure; progenitor; promoter; reproductive; response; stem cell population; 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 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. We have determined that the cytokine leukemia inhibitory factor (LIF) in combination with Rho kinase inhibitor (ROCKi) maintains and selectively expands the Six2+ nephronic stem cell population in culture. Moreover, these propagated stem cells retain their capacity to convert to all segments of the nephron, demonstrating that they are multipotent progenitors. LIF functions principally through activation of STATs 1, 3 and 5 and up regulates the expression of several renal stem cell markers, e.g., Six2 and Pax2. Mechanistically, we have now found that LIF stimulates JNK activation, which induces MM proliferation and enhances cell competence to differentiate. The Rho kinase inhibitor (ROCKi) attenuates the LIF-induced Jnk activation thus inhibiting the differentiation of the progenitor. 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. Since YAP interacts with Tead transcription factors, we also determined that canonical Yap signaling through Tead activation is required for YAP-dependent transcription and MM progenitor cell maintenance. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation. In order to better understand the role that LIF-induced Stat signaling has in kidney development, we are generating mouse line to unravel the redundant functioning of this family of transcription factors. During the course of our mouse genetic studies we discovered a significant role for Stat3 in bone development. We have determined that the conditional loss of Stat3 causes a phenotype typical of two bent bone disorders, campomelic dysplasia and Stuve-Wiedemann syndrome. 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, which appear to tie these two congenital abnormalities together. Specifically, we have found that Stat3 is required for maintenance of the trabecular bone, and the loss of Stat3 results in shortening of the long bones and their improper mineralization. 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. To interrogate the mechanism, we analyzed the Sox9 proximal promoter region and discovered several potential Stat DNA response elements (DRE). Indeed, we found that a Sox9 promoter-driven reporter is activated in cells by oncostatin M in a Stat3-dependent fashion. Further, reporter activation is mediated by the Stat DREs, and Stat3 physically binds the promoter of Sox9. 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 currently looking at the simultaneous ablation of other Stat molecules in the kidney to confirm our in culture findings that Stat activation sustains the Six+ nephronic progenitor. Finally, in collaboration with CDBL PI Terry Yamaguchi, we continue to investigate the role of Wnt5a in metanephric development. 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, we have found that inactivation of Wnt5a in mesoderm using T/Brachyury-Cre results in duplex kidneys and double ureter formation bilaterally, a common malformation in the overall population and part of a major grouping of significant congenital abnormalities called CAKUT (Congenital Anomalies of the Kidney and Urogenital Tract). Interestingly though,Wnt5a expression is already lost in the region where bud outgrowth occurs by the time it is initiated. This suggested that the events/interactions responsible for dysmorphogenesis of the metanephros likely precede its development. Thus, we temporally ablated Wnt5a and found that inactivation at E7.5 but not E8.5 resulted in duplex collecting systems. Since Wnt5a is expressed in the primitive streak and neural plate at these earlier times, it is likely that dysregulation in those tissues is responsible for the phenotype. Consistent with this hypothesis is the fact that the entire length of the nephric duct throughout the mesonephros is aberrantly formed, i.e., it is wider and truncated in the mutant embryo. Furthermore, at its caudal end, it appears as a fused doublet, consistent with double ureter outgrowth. Finally, we have also examined the ablation of Wnt5a using several different tissue-specific Cre lines to isolate the source and timing of Wnt5a in the proper patterning of the kidney. Thus far we have determined that simultaneous inactivation of Wnt5a in the nephric duct and surrounding nephrogenic cord is insufficient to cause the abnormality; whereas, early removal in the primitive streak does result in aberrant development. In addition to duplex kidney and ureter formation, we observed that the Wnt5a mutation also causes another phenotype in the urogenital system - a severe secondary vesicoureteral reflux. In the Wnt5a mouse, the ureter intersects inappropriately with the bladder leading to a failed connectivity and resulting in the improper termination of the ureter. The malformation leads either to the inadvertent release of urine into the peritoneum or to back pressure in the kidney, causing hydronephrosis. Unlike duplex kidney formation, this abnormality is dependent upon proper signaling by Wnt5a later in urogenital development, i.e., E9.5. We are currently developing tools that will allow us to visualize the mutant structures with 3-dimensional imaging.

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。母细胞瘤（Wilms tumor， WT）的特点是胚母细胞/祖细胞群体扩大，上皮细胞转化（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。我们已经确定细胞因子白血病抑制因子（LIF）与Rho激酶抑制剂（ROCKi）联合维持并选择性地扩大培养中的Six2+肾干细胞群。此外，这些增殖的干细胞保留了转化为肾元所有部分的能力，表明它们是多能祖细胞。LIF主要通过激活stat1、3和5发挥作用，并上调几种肾干细胞标记物的表达，如Six2和Pax2。在机制上，我们现在已经发现LIF刺激JNK激活，从而诱导MM增殖并增强细胞分化能力。Rho激酶抑制剂（ROCKi）减弱了liff诱导的Jnk激活，从而抑制了祖细胞的分化。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。由于YAP与Tead转录因子相互作用，我们还确定通过Tead激活的典型YAP信号是YAP依赖性转录和MM祖细胞维持所必需的。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。为了更好地理解liff诱导的Stat信号在肾脏发育中的作用，我们正在生成小鼠系来揭示这一转录因子家族的冗余功能。在我们的小鼠遗传研究过程中，我们发现Stat3在骨骼发育中的重要作用。我们已经确定Stat3的条件缺失导致了两种典型的弯曲骨疾病，弯曲性异常增生和Stuve-Wiedemann综合征。使用条件功能丧失（LOF）小鼠模型，对Stat3的LOF突变体的初步评估揭示了骨骼系统中的广泛缺陷，这些缺陷似乎将这两种先天性异常联系在一起。具体来说，我们发现Stat3是维持小梁骨所必需的，而Stat3的缺失会导致长骨缩短及其矿化不当。与软骨内骨形成中断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。为了探究其机制，我们分析了Sox9近端启动子区域，并发现了几个潜在的Stat DNA应答元件（DRE）。事实上，我们发现一个Sox9启动子驱动的报告子在细胞中以stat3依赖的方式被oncostatin M激活。此外，报告基因激活由statdres介导，Stat3物理结合Sox9的启动子。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是Stat3信号传导的下游靶点。我们目前正在研究同时消融肾脏中其他Stat分子，以证实我们的培养结果，Stat激活维持6 +肾祖细胞。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。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。然而，我们发现使用T/Brachyury-Cre对中胚层的Wnt5a进行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，也是称为ckut（肾脏和泌尿生殖道先天性异常）的重大先天性异常的主要组的一部分。有趣的是，Wnt5a的表达在芽萌发时就已经在芽生长的区域丢失了。这表明导致后肾畸形形成的事件/相互作用可能先于后肾发育。因此，我们暂时消融了Wnt5a，发现在E7.5而不是E8.5时失活会导致双收集系统。由于Wnt5a在这些早期的原始条纹和神经板中表达，这些组织中的失调可能是导致表型的原因。与这一假设相一致的事实是，贯穿中肾的整个肾管长度是异常形成的，即在突变胚胎中，它更宽且截短。此外，在其尾端，它表现为融合的双重，与双输尿管的生长一致。最后，我们还使用几种不同的组织特异性Cre系研究了Wnt5a的消融，以分离Wnt5a在肾脏适当模式中的来源和时间。到目前为止，我们已经确定，肾管和周围肾原索中Wnt5a的同时失活不足以引起异常；然而，原始条纹的早期移除会导致异常发育。除了双肾和输尿管形成外，我们观察到Wnt5a突变还会导致泌尿生殖系统的另一种表型——严重的继发性膀胱输尿管反流。在Wnt5a小鼠中，输尿管与膀胱不适当相交，导致连通性失败，导致输尿管不正确终止。这种畸形会导致尿液不经意地释放到腹膜中，或者导致肾脏的背压，从而导致肾积水。与双肾形成不同，这种异常依赖于Wnt5a在泌尿生殖发育后期的适当信号传导，即E9.5。我们目前正在开发工具，使我们能够用三维成像来可视化突变结构。