Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Mustafa K. Khokha

Subject Area(s)

Developmental biology, Medicine

Abstract

Congenital heart disease (CHD) affects 1 in every 130 newborns and is the leading cause of infant mortality (2). Heterotaxy (Htx), a disorder of left-right (LR) development, commonly leads to CHD. Despite aggressive surgical management, patients with Htx have poor survival rates and severe morbidity due to their complex CHD. Many of the genetic causes of Htx remain undefined, however, a recent genetic analysis of Htx patients identified a single mutant allele in the novel candidate gene, TMEM195 (3). TMEM195 is an alkylglyercol monooxygenase that cleaves ether lipids, but neither its molecular target nor its role in development has been described (4).

The aim of this project is to identify the molecular mechanism by which TMEM195 alters LR cardiac development. I examined the role of TMEM195 using morpholino (MO) knockdown and mRNA overexpression in Xenopus tropicalis. At a low dose of MO, the patient's Htx phenotype was recapitulated in Xenopus. At a higher dose of MO, a significant gastrulation defect occurs. Experimental data suggest that the gastrulation defect is secondary to changes in the Wnt signaling pathway. Analysis of TMEM195 knockdown shows a significant decrease in beta-catenin expression and nuclear localization during gastrulation. Nuclear localization of beta-catenin cannot be rescued by stabilizing beta-catenin, but is rescued by adding a nuclear-localization signal (NLS). These results suggest for the first time that TMEM195 plays a role in the nuclear import of beta-catenin.

TMEM195's role in nuclear localization many not only be specific to Wnt, but may also affect TGF-beta signaling. Smad2 is an important transcription factor for TGF-beta signaling and must be phosphorylated prior to entering the nucleus. TMEM195 depletion leads to an increase in phosphorylated but not total Smad2. In contrast, Smad1 is unaffected. Significantly, both Smad2 and beta-catenin lack NLS signals, while Smad1 has one (5). These results suggest a potentially broader role of TMEM195 in nuclear localization of non-NLS tagged proteins.

Defining the role of TMEM195 will permit a better understanding of the relationship between nuclear localization, gastrulation errors and defects in LR axis development. Importantly, it may also inform our understanding of the currently unknown mechanisms regulating nuclear import of several key signaling factors including beta-catenin and Smad2 and thus pave the way for the development of novel clinical treatments. Both Wnt and TGF-beta signaling are important for a myriad of different disease processes including congenital and vascular malformations, stem cells and cancer.

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This is an Open Access Thesis.

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