End stage liver disease is a major cause of morbidity and mortality worldwide and in the USA, where it is the 12th leading cause of death. Hence, innovative research to promote liver repair is critical. Critically, we and others identified the Wnt/b-catenin signaling pathway as key for liver regeneration and repair. We also showed this pathway?s key roles in hepatic metabolic zonation (MZ) and liver regeneration (LR) after partial hepatectomy (PH). The impetus behind requesting a diversity supplement to our current R01 is two-fold. An exceptionally talented and driven graduate student from an underrepresented minority with an interest in Wnt signaling and organ regeneration is highly interested in pursuing her graduate training in our lab. And, her work and interests are a logical extension of the existing R01?s specific aims. Thus, broadening the scope of the current studies through innovating imaging and spatial transcriptomics with focus on Wnt-Frizzled signaling would add further impact and innovation and promote diversity in the lab. Specifically, we are extending aims 2 and 3 of the original grant by proposing two aims.
Specific Aim 1 for the Diversity Supplement will create a temporospatial atlas of Fzd receptor mRNA expression, first in the normal liver as a baseline, and then in liver undergoing regeneration after PH. We will employ multiplex RNAscope to directly and quantitatively visualize zone-specific expression of all 10 Fzd receptors. These studies will shed light on aspects of basic liver zonation, which is crucial for creating a foundation to further investigate liver biology, both structurally and functionally. It would also allow for a differential comparison of zonation and Fzd expression between normal and regenerating liver post- PH, which will help reveal molecular mechanisms responsible for LR as well as re-establishing zones after PH.
This aim i s an extension of the current Aim 2 of the existing proposal which focuses on examining an in-depth role of Fzd7 in zonation and after PH. Based on analysis performed on hepatocyte-specific Fzd7-knockout mice, we found no defect in MZ or LR after PH. This led us to propose spatial transcriptomics through RNA scope for all Fzds at baseline in liver and then after PH.
Specific Aim 2 of the supplement will elucidate the early changes in the expression of Wnt2 and Wnt9b during the process of LR. Using RNAscope, we will study changes in temporospatial patterns of Wnt2 and Wnt9b expression at early time points after PH in normal mice and in specific KO models, including endothelial cell-specific Wnt2 and Wnt9b single and double KOs. This is an extension of Aim 3 which examines hypoxia and shear stress as upstream effectors of Wnt signaling in liver. However, visualizing cell-specific changes in gene expression of Wnt2/9b by RNAscope as a function of time, will provide us a more solid rationale for studying dynamics of Wnt2/9b control of LR. In conclusion, by creating an temporospatial atlas of Fzd receptor expression in the normal and regenerating liver and investigating the spatiotemporal Wnt2/9b expression in LR, we will be one step closer to manipulating the regenerative process as a therapeutic measure for various kinds of disease and injury like fatty liver disease, cirrhosis, and liver failure.
Chronic liver disease and its sequela, end stage liver disease, are the 12th commonest cause of morbidity in the USA, yet liver transplantation is not an option for all due to ineligibility for many as well as shortage of available livers. For novel hepatic regenerative medicine approaches to become a reality, it is of utmost importance to elucidate the complex cell-molecule circuitry for relevant pathways that drive hepatocyte differentiation and repair after injury. The current proposal examines a major regulator of these processes, the Wnt/b-catenin pathway, importantly expanding on our currently funded research and, in so doing, promotes diversity by encouraging the career development of a highly promising member of an underrepresented group.
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