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scRNA-Seq uncovers cell type-specific impairments in developing Tet loss-of-function retinae

Heilman, Shea (2024) scRNA-Seq uncovers cell type-specific impairments in developing Tet loss-of-function retinae. Doctoral Dissertation, University of Pittsburgh.

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Abstract

DNA methylation is an epigenetic modification that typically occurs on cytosines, and impacts countless biological processes. Balanced methylation signatures are required for normal physiology and are regulated by proteins that either add, modify, or remove methylated bases. The major methylation-modifying factors include Tet family methylcytosine dioxygenases (Tets). Tets recognize and oxidize 5-methyl-cytosine (5mC) to 5-hydroxymethylcytosine (5hmC); 5hmC functions either as an intermediate in demethylation or as a stable regulatory mark1–3. In DNA, 5hmC deposition is known to regulate gene expression, and in several contexts, promotes open chromatin states and gene upregulation4–6 . Functionally, Tets are required for the development of many distinct tissues and cell types that include, but are not limited to, B-cells7,8, hematopoietic stem cells9, the heart10, and the retina11. While many studies have correlated broad patterns of 5hmC deposition with gene expression in specific developmental contexts—particularly in enhancers during organogenesis5,6, and in gene bodies during cellular maturation12–18—other accounts revealed that Tet regulation at similar stages also occurred via other mechanisms and in other gene regions19,20. To add to this complexity, Tet expression and 5hmC deposition are distributed unequally across different tissue types, and between cell types that compose a given tissue 13, and balanced Tet activity also supports the maintenance of cell non-autonomous signals that promote differentiation-supportive microenvironments11. The cell population specificity of Tet regulation and the various mechanisms co-opted by Tets to exert control suggest that Tets and 5hmC could differentially regulate separate cell populations that compose a cellularly complex tissue. Previous work assessing retinogenesis in a Tet-null environment suggested broad differentiation impairment across many retinal cell populations, but did not identify the full scope of cell type-specific impairment. In this study, we utilized single-cell approaches to compare the cellular composition and cell type- specific phenotypes in control and Tet-null (tet2-/-;tet3-/-) retinae, and outlined the methods used to collect our scRNAseq samples. Our data revealed molecular signs of impaired terminal differentiation across cones, and disproportionate expansions of differentiation-impaired subpopulations of horizontal cells and bipolar cells. We also found unexpected changes in the cell type composition of tet2-/-;tet3-/- retinae; in particular, we saw robust expansions of ON Bipolar cells and reductions in cones. Of the tet2-/-;tet3-/- cones that did differentiate, there were disproportionate expansions of UV and blue cones at the expense of red and green cones. Together, these data suggest that Tet proteins play a major role in both cell type specification and terminal differentiation.

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Item Type: University of Pittsburgh ETD
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Heilman, Sheasheaheilman@gmail.comshh1360000-0003-4337-5446
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee MemberAldiri, Issamaldirii@pitt.edualdirii@pitt.edu0000-0002-0813-2943
Thesis AdvisorGross, Jeffreyjmgross@austin.utexas.edugrossjm@pitt.edu0000-0002-9422-6312
Committee MemberKostka, Denniskostka@pitt.edukostka@pitt.edu0000-0002-1460-5487
Committee MemberRoman, Bethromanb@pitt.eduromanb@pitt.edu0000-0002-1250-1705
Committee ChairShin, Donghundonghuns@pitt.edudonghuns@pitt.edu0000-0002-7975-9014
Date: 12 December 2024
Defense Date: 19 November 2024
Approval Date: 13 February 2025
Submission Date: 11 December 2024
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 179
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Genetics and Developmental Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Methylation, Hydroxymethylation, Retinal Biology, Retinal Development
Date Deposited: 13 Feb 2025 14:48
Last Modified: 10 Apr 2025 15:12
URI: http://d-scholarship.pitt.edu/id/eprint/47237

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