Date of Award

12-31-2018

Document Type

Campus Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology/Molecular, Cellular, and Organismal Biology

First Advisor

Kellee R. Siegfried

Second Advisor

Linda Huang

Third Advisor

Richard Kesseli

Abstract

Disorders of sexual development (DSD) are congenital conditions affecting chromosomal, gonadal, or anatomical sex. Incidence of DSD is ~1 in 500, with pathologies ranging from ambiguous sex at birth to adult infertility. Zebrafish have emerged as an exceptional model to study gonad development and fertility, as their genome contains orthologs to most genes controlling sex in mammals. However, it is unknown how sex is specified in zebrafish, which lack sex chromosomes. The quantity of embryonic germ cells and signaling from these cells to the somatic gonad are factors known to influence ovary versus testis fate in vertebrates. To understand the genetic regulation underlying germ cell development and gonadal sex differentiation in zebrafish, forward and reverse genetic approaches were used to investigate two candidate genes. The first, dmrt1 (doublesex and mab-3 related transcription factor 1), is a key regulator of sex across metazoans. We isolatedv mutations disrupting dmrt1 and found the majority of mutants develop as fertile females. Few mutants became males, all of which lost germ cells and displayed testicular dysgenesis. We found that Dmrt1 is necessary for transcriptional regulation of amh (anti-Müllerian hormone) and foxl2 (forkhead box L2), which are important for male or female sexual development, respectively. The second candidate, cdk21 (cyclin-dependent kinase 21), was identified in a mutagenesis screen for gonadogenesis regulators. Mouse models of CDKs (which regulate cell cycle progression) exhibit hypoplasia and depletion of progenitor lineages, including germ cell defects, suggesting a conserved role of CDKs in germ cell differentiation. We demonstrate that mutations disrupting the kinase domain of the Cdk21 protein are linked to testis hypoplasia, gradual loss of germ cells, and 100% male sex development. Histological and biochemical evidence of cell cycle delay in cdk21 mutant testes suggests a role in maintenance of mitotic spermatogonia, and progression of spermatocytes through meiosis. Furthermore, cdk21 mutants exhibit reduced phosphorylation of the tumor suppressor Retinoblastoma protein 1 (Rb1), indicating this kinase regulates cell cycle progression similarly to the closely related Cdk4 and Cdk6 proteins. We conclude dmrt1 and cdk21 are required for spermatogenesis: dmrt1 promotes male differentiation by transcriptionally regulating sex-specific genes, whereas cdk21 regulates mitotic and meiotic cell divisions in testicular germ cells.

Comments

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