Zoology - Theses
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ItemDefining the master regulator of urethral closure in mouse( 2017)Hypospadias is the ectopic placement of the urethral opening on the underside of the penis and is one of the most common developmental abnormalities in humans, occurring in approximately 1 in every 125 live male births. In addition, we have observed a doubling in the incidence of hypospadias over the past several decades suggesting an environmental component likely in the form of estrogen mimicking chemicals generally referred to as environmental endocrine disruptors (EEDs). Current models fail to explain these observations. The goal of this thesis is to produce a theory that describes the development and genetic regulation of urethral closure, and use it to explain the aetiology, spectrum, and rise in incidence of hypospadias observed in humans. The work presented in this thesis was performed using a novel mouse model (OVE442) with isolated hypospadias. This model was used to define the role of the urorectal septum (URS) during urethral closure. The process of urethral closure is generally thought to occur by tissue fusion. However, we provide immunohistological evidence that suggests the urethra is internalized by growth of the URS, which contributes tissue to the ventral aspect of the penis during embryonic development. The OVE442 model was next used to define a key regulator of the URS during urethral closure. Initial characterization of a genomic mutation in OVE442 model led us to discover a long non-coding RNA, designated Leat1, which was deleted near EfnB2. Loss of signalling through the EPHRINB2 protein was previously shown to cause severe hypospadias in mouse, however little is known about EfnB2 gene regulation during urethral closure. Leat1 was characterized, functionally examined, and shown to regulate EfnB2 expression through direct interaction with the EPHRINB2 protein. We further showed that Leat1 expression is differentially regulated in males and females, and that it is supressed by estrogen. These results showed that EfnB2 drives growth of the URS during urethral closure and provided the first experimental evidence revealing the genetic mechanism that causes male and female urethral anatomy to diverge. These observations were used together with our anatomical descriptions to produce a developmental theory that explains urethral formation in mouse. We extended our understanding further by using comparative time series RNA-Seq to describe global transcription and ChIP-Seq to identify genes actively regulated by estrogen and androgen during urethral. From these data, we identify potential urethral closure genes downstream of Leat1 and EfnB2 including genes that are likely responsive to sex hormones. This work has provided fundamental insights on the anatomy and genetic regulation of urethral closure. I have shown that male urethral closure is driven by growth of the URS and that this growth is regulated by the long non-coding RNA Leat1 in mouse. Furthermore, I have produced a list of potential EED targets that may lead to better understanding the causes of hypospadias. Through this work I have produced a theory that explains the spectrum of urethral malformations observed in human, defects associated with hypospadias such as chordee, and the genetic mechanism that is likely disrupted by EEDs. These findings fundamentally change the way we consider urethral development and may help to find ways to reduce the incidence or prevent hypospadias in humans.
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ItemDefining the role of endogenous estrogen in penile urethral development( 2017)Hypospadias, the ectopic placement of the urethral meatus along the ventral aspect of the penis, is one of the most common birth defects in boys. The prevalence of hypospadias has increased in recent decades, a phenomenon that has been attributed to growing exposure to endocrine disrupting chemicals(EDCs)in the environment that disrupt androgen and estrogen signaling. Mice have been used to model hypospadias using knockout and transgenic lines and hormonal manipulations. While mice present a tractable model to study penile development, several structures differ between mice and humans and there is a lack of consensus in the literature on their annotation and developmental origins. Furthermore, extensive postnatal development occurs in mice, complicating comparisons to human penis development. Using, section histology, gross morphology, and magnetic resonance imaging, this thesis first provides an extensive examination of the adult mouse penis and addresses differences in the mouse and human penis to facilitate comparisons between human and murine hypospadias phenotypes. Secondly, this thesis presents a hypospadias phenotype in mice with reduced estrogen signaling. Mice lacking estrogen receptor α(αERKO)have a mild hypospadias phenotype, which corresponds to the most common phenotype seen in humans. The third chapter of this thesis describes this phenotype through section histology and micro computed tomography and outlines the delamination events that occur postnatally to develop the mature mouse penis. This study offers the first direct evidence of a role for endogenous estrogen in distal penile urethral development. To identify how estrogen may be acting alongside androgen to drive penis development, this thesis next identifies direct targets of estrogen and androgen in the developing penis of human and mice using chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq). Select target genes identified from ChIP-Seq were validated as being hormonally responsive using mouse genital tubercle cultures and quantitative polymerase chain reaction (qPCR). The result is a list of potential targets of androgen and estrogen in the development of the penile urethra. This data represents a key step in determining which genes are potential targets of endocrine disrupting chemicals and which may be affected by EDCs to cause hypospadias. Taken together, this thesis proposes a precise balance between estrogens and androgens is critical in driving normal urethral closure. Determining which EDCs can induce hypospadias will require examination of not only anti-androgenic and estrogen-mimicking chemicals, but those that reduce estrogen signaling as well. This thesis provides a comprehensive guide to postnatal mouse penis anatomy as well as a list of estrogen targets in the developing penis, which together represent a useful resource for the field of hypospadias.