Investigations into the roles of Shroom3 in development and adult physiology

Carleton, Jennifer

Investigations into the roles of Shroom3 in development and adult physiology

Files

Carleton_Jennifer_PhD_2026_thesis.pdf (5.41 MB)

Date

2026-03-25

Authors

Carleton, Jennifer

Abstract

The actin binding protein Shroom3 is best known for its role in mammalian neural tube closure through control of apical constriction and apicobasal elongation of epithelial cells. More roles for Shroom3 have recently been uncovered, both developmentally and postnatally, where its loss causes congenital heart and kidney defects and is genetically linked to chronic kidney disease in patients. However, questions still remain about the range of Shroom3 expression and the differences in its spatial and temporal functions. I hypothesize that Shroom3 has a wider postnatal expression range than previously understood, and that in organs with both developmental and postnatal expression, it is necessary for normal morphogenesis and physiology. Using a LacZ reporter system, postnatal Shroom3 expression was characterized in the brain, whisker pad, heart, lungs, intestines, kidney, ovary, uterus, testes, and bladder. Fluorescent staining with a custom Shroom3 antibody supported these results with additional expression in the retina. Many of these tissues do not demonstrate Shroom3’s characteristic apical constriction, suggesting that it has differing functions in other tissues. The heart and kidney were selected for further research due to having both developmental and postnatal expression and known phenotypes upon Shroom3 loss. Our lab previously found that global loss of Shroom3 during development resulted in congenital heart defects. Building on this, a novel floxed Shroom3 allele was used to create a myocardial specific knockout during development. Interestingly, no morphological defects were observed, suggesting that myocardial loss was not responsible for the known defects. The same floxed allele was used to induce Shroom3 loss in adult mice. After six months of monitoring, no morphological alterations in the kidneys were observed, and no changes in total urine soluble protein was seen. This suggests that kidney phenotypes from Shroom3 loss originate in development. Overall, this thesis presents evidence that Shroom3 expression in the adult mouse has a wider range than previously understood, evidence that Shroom3 acts in a non-cell autonomous manner and emphasizes the developmental impact of Shroom3 on postnatal physiological function. This raises that Shroom3 likely functions in different roles developmentally and postnatally, even in the same tissue.

Summary for Lay Audience

Cytoskeletal proteins provide scaffolding within cells, giving them structural integrity and allowing for movement and communication. The smallest of these is actin, which controls cell movement and shape. Shroom3 is a protein that binds actin and controls its location in the cell. This interaction was first discovered in the developing brain and spinal cord, where Shroom3 loss caused neural tube defects in mice. Shroom3 has since been identified in other organs during development and into adult life. This poses a question about the extent of Shroom3 expression in adults and what its functional differences are at different periods of life. I hypothesize that Shroom3 has a wider postnatal expression range than previously understood, and that in organs with both developmental and postnatal expression, it is necessary for normal morphogenesis and physiology. To identify the range of adult expression, we used chemical and antibody stains for Shroom3 in major adult mouse organs. Expression was seen in the brain, heart, kidney, lungs, retina, intestines, bladder, and the male and female reproductive organs. The heart and kidney were selected for further study as these both have developmental and adult Shroom3 expression and have known congenital defects associated with its loss. Shroom3 function was blocked in a tissue and time specific manner. Neonates with loss specifically in the developing heart showed no congenital heart defects and were able to live into adulthood with no long-term health consequences. This poses that other cell populations with Shroom3 expression are key contributors to proper heart development. The same knockout system was used in adult mice. After 6 months of Shroom3 loss, no changes in overall health or kidney structure were seen, and no detectable protein was found in the urine. This suggests that Shroom3 in proper kidney development is more influential on long term kidney function. Overall, this thesis argues for further research in adult Shroom3 expression, with a focus on the differences between developmental and adult function.