Defining the master regulator of urethral closure in mouse

© 2017 Dr. Paul Edward GradieHypospadias 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