Growth-Differentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 9556193
• 负责人: ALAN PERANTONI
• 金额: $ 104.26万
• 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/9556193
• 关键词: Ablation; Address; Agonist; Alleles; Anchorage-Independent Growth; Apoptosis; Attenuated; Backcrossings; Behavior; Belief; Benign; Bilateral; Bladder; Bone Development; Bone Diseases; Breeding; Caricatures; Cell Survival; Cells; Competence; Cultured Cells; Cultured Tumor Cells; DNA; DNA Sequence; Data; Development; Differentiation and Growth; Disease; Dominant Genetic Conditions; Down-Regulation; Duct (organ) structure; Epithelial; Epithelium; Family member; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic study; Genitourinary system; Goals; Growth; Growth Factor; Human; Hybrids; Hydronephrosis; Incidence; Individual; Injury; Intermediate Mesoderm; Invaded; Investigation; Kidney; LIF gene; Lead; Ligands; MAPK8 gene; Maintenance; Malignant Childhood Neoplasm; Maps; Mediating; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Metanephric structure; Molecular Target; Morphogenesis; Movement; Multipotent Stem Cells; Mus; Mutant Strains Mice; NCAM1 gene; Natural regeneration; Neoplasms; Nephroblastoma; Nephrogenic Cord; Nephrons; Normal tissue morphology; Nuclear; Organogenesis; Organoids; Osteoblasts; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Population; Process; Proteins; Rat Strains; Rattus; Renal function; Response Elements; Rho-associated kinase; Role; Signal Induction; Signal Pathway; Signal Transduction; Skeleton; Small Interfering RNA; Sorting - Cell Movement; Stem cells; Structure; Structure of mesonephric duct; Study Section; Stuve-Wiedemann syndrome; System; Time; Tissue Model; Tissues; Transcription Coactivator; Tubular formation; Tumor Suppressor Proteins; Tumor Tissue; Tumorigenicity; Undifferentiated; Ureter; Urinary tract; Urine; Work; Xenograft procedure; base; bone; bone loss; campomelic dysplasia; cytokine; demineralization; driving force; experience; in vivo; interest; kidney repair; kinase inhibitor; knock-down; long bone; loss of function; male; malformation; molecular targeted therapies; mouse model; mutant; neoplastic; neoplastic cell; nephrogenesis; novel; personalized therapeutic; progenitor; promoter; rat genome; repaired; reproductive tract; response; screening; small molecule; stem cell population; stemness; substantia spongiosa; tissue repair; tumor; tumorigenesis; tumorigenic; whole genome

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 identified several growth factors and small molecule pathway agonists that preserve and propagate long-term the nephronic progenitor. 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 principally acts through the JAK/STAT pathway and up regulates the expression of several renal stem cell markers, e.g., Six2 and Pax2. Mechanistically, 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. Our conditions have also proven successful in promoting the growth of mouse and human Six2+ nephronic progenitors. However, this required further culture optimization with a Wnt agonist and Bmp family member in addition to LIF/ROCKi. In this case, Six2+ cells could be maintained for several passages and induced to form all segments of the nephronic epithelia. This culture system of MM provides unique opportunities to comprehensively address key mechanisms involved in renal progenitor maintenance and differentiation and raises the possibility that they may be applied to models of tissue repair/regeneration. We are now collaborating with another lab that studies renal damage in mice in efforts to determine if our cultured progenitors can facilitate kidney repair following injury. We have now also applied progenitor culture conditions to the propagation of human Wilms tumor (WT) cells from several different patients and have determined that these same factors selectively expand the Six2+ progenitor from tumor tissues. These cells also retain the expression of several other "stemness" and WT-associated genes, such as NCAM and PAX2. These cells 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. Notably, WT cells readily form organoids when placed in nonadherent culture dishes. 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. To better understand the role that LIF-induced Stat signaling has in kidney development, we are evaluating Stat3 mutant mice, which develop kidneys that are dramatically reduced in size. 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, we have found that Stat3 is required for maintenance of the trabecular bone, and loss of Stat3 results in shortening and bending of the long bones and downregulation of the osteochondro master regulator Sox9. We further identified two Stat-dependent DNA response elements in the Sox9 promoter. 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 now attempting to clarify the role of Stat3 in the osteoblast lineage as well, since the bones that form in Stat3 mutants are severely demineralized. We have now also found that knockdown of Stats in nephronic progenitors attenuates Sox9 expression in cultured cells and are therefore targeting Stat3 ablation specifically to the Six2+ progenitor in vivo in order to understand its role during development or in response to renal tubular injury. Sox9 has been implicated in the repair of ischemic tubular injury, and, based upon our work, this could be initiated by Stat activation. We continue to investigate two aberrant phenotypes in the urinary tract associated with Wnt5a ablation. The first involves duplex kidney formation, which we have determined is caused by a bilateral duplication of the Wolffian duct, a common malformation in humans. We are also evaluating a second renal phenotype in Wnt5a mutants, 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 Wnt5a mutants, and when only one Shh allele was expressed, we could correct the aberrant phenotype. This work demonstrates for the first time that Wnt5a modulates Shh levels during development and possibly also during tumorigenesis, since Wnt5a can also function as a tumor suppressor. Finally, we have continued our efforts to identify a novel genetic locus associated with Wilms tumor/nephroblastoma. The Noble rat strain is exquisitely sensitive to transplacental induction of nephroblastoma at high incidence; whereas the Fisher rat is completely insensitive to nephroblastoma induction. Cross breeding between these two rat strains suggests inheritance occurs as an incomplete dominant trait. To map the responsible locus, we have backcrossed F1 hybrids to the Fisher strain and then whole-genome sequenced the DNAs from all rats that developed nephroblastoma (25) as well as several controls. George Nelson is responsible for coordinating and assessing all sequencing data. Since the rat genome is not well annotated, we are experiencing technical issues with alignments, but are now sorting through those problems.

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点。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。我们鉴定了几种生长因子和小分子途径激动剂，可以长期保存和繁殖肾病祖细胞。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) 组合可维持并选择性扩增培养物中的 Six2+ 肾病干细胞群。此外，这些增殖的干细胞保留了转化为肾单位所有部分的能力，表明它们是多能祖细胞。 LIF 主要通过 JAK/STAT 通路发挥作用，上调多种肾干细胞标志物的表达，例如 Six2 和 Pax2。从机制上讲，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 LIF 诱导的 JNK 激活，从而抑制祖细胞的分化。对这些细胞中 LIF/ROCKi 介导的机制的研究表明，我们的条件促进 Yes 相关蛋白 (YAP) 的核定位，YAP 是一种转录辅激活因子，也是 Hippo 信号通路的组成部分。此外，通过 MM 细胞中的 siRNA 敲低来沉默 Yap 基因表达会降低祖细胞标记的表达并增加 MET 标记的水平，表明 YAP 使 MM 细胞维持在未分化状态。我们的条件也被证明可以成功促进小鼠和人类 Six2+ 肾单位祖细胞的生长。然而，这需要除 LIF/ROCKi 之外还使用 Wnt 激动剂和 Bmp 家族成员进行进一步的培养优化。在这种情况下，Six2+细胞可以维持数代并被诱导形成肾单位上皮的所有部分。这种 MM 培养系统提供了独特的机会来全面解决涉及肾祖细胞维持和分化的关键机制，并提高了它们应用于组织修复/再生模型的可能性。我们现在正在与另一个研究小鼠肾损伤的实验室合作，以确定我们培养的祖细胞是否可以促进损伤后的肾脏修复。我们现在还将祖细胞培养条件应用于来自几位不同患者的人类肾母细胞瘤（WT）细胞的繁殖，并确定这些相同的因子选择性地从肿瘤组织中扩增 Six2+ 祖细胞。这些细胞还保留了其他几种“干性”和 WT 相关基因的表达，例如 NCAM 和 PAX2。这些细胞可稳定传代数次，并根据其不依赖贴壁的生长能力保留致瘤表型。来自这些肿瘤的培养细胞现已被异种移植到 NSG 小鼠中，以进一步评估致瘤行为。值得注意的是，当放置在非贴壁培养皿中时，WT 细胞很容易形成类器官。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张可能允许为个体 WT 患者开发个性化治疗筛查。这是该领域的一项重大进展，因为迄今为止，在培养物中繁殖 WT 细胞的失败努力导致一些人推测它们只能在异种移植物中才能维持。为了更好地了解 LIF 诱导的 Stat 信号在肾脏发育中的作用，我们正在评估 Stat3 突变小鼠，这些小鼠发育出的肾脏尺寸急剧减小。在我们的小鼠遗传学研究过程中，我们发现 Stat3 在骨骼发育中发挥着重要作用。我们已经确定 Stat3 的条件性缺失会导致两种弯曲骨疾病的典型表型，即弯曲骨发育不良和 Stuve-Wiedemann 综合征。使用条件性功能丧失（LOF）小鼠模型，我们发现 Stat3 是维持小梁骨所必需的，并且 Stat3 的缺失会导致长骨缩短和弯曲以及骨软骨主调节因子 Sox9 的下调。我们进一步鉴定了 Sox9 启动子中的两个 Stat 依赖性 DNA 反应元件。这些发现表明 Stat3 在哺乳动物骨骼的正确模式中发挥着关键作用，并表明 Sox9 是该过程中 Stat3 信号传导的下游靶标。我们现在正试图阐明 Stat3 在成骨细胞谱系中的作用，因为 Stat3 突变体中形成的骨骼严重脱矿质。我们现在还发现，肾病祖细胞中 Stats 的敲低会减弱培养细胞中 Sox9 的表达，因此将 Stat3 消融专门针对体内 Six2+ 祖细胞，以了解其在发育过程中或对肾小管损伤的反应中的作用。 Sox9 与缺血性肾小管损伤的修复有关，根据我们的工作，这可能是由 Stat 激活启动的。我们继续研究与 Wnt5a 消融相关的泌尿道中的两种异常表型。第一个涉及双肾形成，我们已确定这是由沃尔夫管双侧重复引起的，这是人类常见的畸形。我们还在评估 Wnt5a 突变体的第二种肾脏表型，即肾积水。在这些研究中，我们发现肾积水的发生是由于肾功能活跃时尿流受阻，导致肾脏髓质区域细胞凋亡。当肾总管中的细胞未能凋亡时，输尿管和膀胱之间的界面就会发生阻塞。基因表达分析显示，Wnt5a突变体中Shh增加，当仅表达一个Shh等位基因时，我们可以纠正异常表型。这项工作首次证明 Wnt5a 在发育过程中调节 Shh 水平，也可能在肿瘤发生过程中调节 Shh 水平，因为 Wnt5a 还可以起到肿瘤抑制因子的作用。最后，我们继续努力确定与肾母细胞瘤/肾母细胞瘤相关的新遗传位点。 Noble 大鼠品系对高发病率的经胎盘诱导肾母细胞瘤极其敏感；而Fisher大鼠对肾母细胞瘤诱导完全不敏感。这两种大鼠品系之间的杂交表明遗传是作为不完整的显性性状发生的。为了绘制负责基因座的图谱，我们将 F1 杂交体与 Fisher 品系回交，然后对所有患肾母细胞瘤 (25) 以及一些对照大鼠的 DNA 进行全基因组测序。 George Nelson 负责协调和评估所有测序数据。由于大鼠基因组没有得到很好的注释，我们在比对方面遇到了技术问题，但现在正在解决这些问题。