The role of signalling pathways in urogenital ridge differentiation

Abstract

A key question in trying to understand embryonic development is what are the signals required to drive the differentiation of a progenitor cell population into distinct cell types? Upon receiving differentiation signals, cells undergo a myriad of responses such as mitosis, changing morphology, migration, changing adhesive properties, integrating with other cells and changing potentiality. The intermediate mesoderm is the progenitor tissue that gives rise to the majority of cell types in the urogenital system. While many factors that determine the specification of the intermediate mesoderm are known, the signalling pathways that determine the partitioning and differentiation of the intermediate mesoderm into the gonad, reproductive tract and kidney are unclear. Defining the fundamental signals and morphogenic processes that regulate the differentiation of the urogenital system will assist in our understanding of the basis of Disorders of Sex Development and Congenital Anomalies of the Kidney and Urinary Tract. In this project, the function of several signalling pathways that determine the patterning of the urogenital ridge in mouse were investigated.First, the function of the ROBO2-SLIT2 pathway in patterning the kidney domain in the early urogenital ridge was examined. Loss of Robo2 in mouse and human is known to result in the formation of supernumerary kidneys driven by an expansion of Gdnf expression. Examination of the early urogenital ridge revealed that the number of cells in the nephrogenic cord was increased in Robo2-null mice, resulting in ectopic caudal mesonephric tubules and an expanded metanephric mesenchyme. In addition, the metanephric mesenchyme fails to separate from the Wolffian duct during kidney development, suggesting that the morphogenesis of the metanephric mesenchyme away from the Wolffian duct may result from active cell migration.Second, the anterior-posterior extension of the intermediate mesoderm and the upper and lower urinary tract differentiation was investigated in Wnt5a-null mice. It was observed that Wnt5a-null mice develop a horseshoe kidney. Underlying horseshoe kidney formation, Wolffian duct migration and insertion into the cloaca are disrupted in Wnt5a-null mice. These defects were reminiscent of mice with perturbed retinoic acid signalling, which led to the finding that the Wolffian duct is exposed to abnormally high levels of retinoic acid in Wnt5a-null mice. Therefore, the likely underlying cause of Wolffian duct migration defects in Wnt5a-null mice is exposure to increased retinoic acid concentrations, secondary to the failure of global axis extension.Third, the anterior-posterior extension of the gonad domain in intermediate mesoderm patterning was investigated by studying mouse mutants with a defect in retrograde trafficking of primary cilia. Mouse mutants of the primary cilium component Ift144, develop XY and XX gonads that are larger than wild-type. Investigation of the early patterning of the urogenital ridge found the anterior-posterior domain of the gonad was extended with a concomitant extension of the embryo trunk axis. Extension of the anterior-posterior gonad axis resulted in an increase in testis cord number, suggesting that the gonad domain is partitioned along the available space rather than partitioned a finite number of times. In addition, somitogenesis in the trunk of the embryo was disrupted, with somitogenesis in the tail proceeding correctly, suggesting that perturbed somite segmentation may be the cause of the increased trunk extension. Thus, analysis of Ift144 mouse mutants has suggested that the length of the trunk axis of the embryo is the main determinant of the gonad anterior-posterior domain and helped to clarify the paradigm of testis cord formation.Fourth, the expression, regulation and function of the microRNA miR-202 was examined in testis differentiation. miR-202-5p/3p was detected as being expressed specifically in the Sertoli cells, during XY gonad differentiation. Over expression of pri-miR-202 in XX gonads did not perturb XX gonad differentiation, suggesting that pri-miR-202 does not directly antagonize the XX differentiation pathway. Furthermore, investigation of the pri-miR-202 promoter regions identified that pri-miR-202 is a direct target gene of the key male determining factor SOX9, suggesting that miR-202-5p/3p may function to enforce XY testis differentiation.In summary, work presented in this thesis has elucidated signalling pathways and morphogenic events that are required for differentiation of the intermediate mesoderm into the urogenital system. This work has identified key determinants of anterior-posterior axis of the kidney and gonad domains, and examined the consequences of expansion or reduction of these fields. In addition, a non-coding RNA was identified in testis development downstream of the male determining gene SOX9, which may function in the testis differentiation pathway. Importantly, investigation of the genetic networks and basic biology of urogenital ridge formation informs our understanding of the basis of human developmental disorders such as Disorders of Sex Development and Congenital Anomalies of the Kidney and Urinary Tract.