Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development

Hernandez-Nunez, I.; Urman, A.; Zhang, X.; Jacobs, W.; Hoffman, C.; Rebba, S.; Harding, E. G.; Li, Q.; Mao, F.; Cani, A. K.; Chen, S.; Dawlaty, M. M.; Rao, R. C.; Ruzycki, P. A.; Edwards, J. R.; Clark, B. S.

doi:10.1101/2025.02.03.636318

bioRxiv · 10.1101/2025.02.03.636318

Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development

Hernandez-Nunez, I.·Urman, A.·Zhang, X.·Jacobs, W.·Hoffman, C.·Rebba, S.·Harding, E. G.·Li, Q.·Mao, F.·Cani, A. K.·Chen, S.·Dawlaty, M. M.·Rao, R. C.·Ruzycki, P. A.·Edwards, J. R.·Clark, B. S.

View original source ↗Export metadata ↓

Abstract

Retinal cell fate specification from multipotent retinal progenitors is governed by dynamic changes in chromatin structure and gene expression. Methylation at cytosines in DNA (5mC) is actively regulated for proper control of gene expression and chromatin architecture. Numerous genes display active DNA demethylation across retinal development; a process that requires oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and is controlled by the ten-eleven translocation methylcytosine dioxygenase (TET) enzymes. Using an allelic series of conditional TET enzyme mutants, we determine that DNA demethylation is required upstream of NRL and NR2E3 expression for the establishment of rod-photoreceptor fate. Using histological, behavioral, transcriptomic, and base-pair resolution DNA methylation analyses, we establish that inhibition of active DNA demethylation results in global changes in gene expression and methylation patterns that prevent photoreceptor precursors from adopting a rod-photoreceptor fate, instead producing a retina in which all photoreceptors specify as cones. Our results establish the TET enzymes and DNA demethylation as critical regulators of retinal development and cell fate specification, elucidating a novel mechanism required for the specification of rod-photoreceptors.

Source connections

Published article ↗

Explore related subjects

developmental biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Hernandez-Nunez, I., Urman, A., Zhang, X., Jacobs, W., Hoffman, C., Rebba, S., Harding, E. G., Li, Q., Mao, F., Cani, A. K., Chen, S., Dawlaty, M. M., Rao, R. C., Ruzycki, P. A., Edwards, J. R., Clark, B. S.. 2025-02-03. Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development. https://doi.org/10.1101/2025.02.03.636318

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Transposable Elements Profiling Reveals DUXA-associated MLT1D Endogenous Retroviral Elements Activation During Bovine Maternal to Zygotic Transition

Transposable elements (TEs) are a major source of genomic diversity in mammals, yet their regulatory roles in the bovine genome remain poorly understood. Through characterizing bovine TE landscape, despite the substantial proportion (25.6%) of ruminant-specific TEs, we observe age- and class-dependent genomic distribution patterns similar to those observed in other mammals. Next, we profile TE and gene expression dynamics in pre-implantation embryos generated in vivo (IVV), by in vitro fertilization (IVT), and through somatic cell nuclear transfer (SCNT). The zygotic genome activation (ZGA) is shifted from the 4-cell stage to the 8-cell stage in IVT and SCNT embryos compared to IVV embryos. SCNT embryos exhibit impaired initiation of early transcription programs at the 4-cell stage and disrupted developmental trajectories, including abnormal activation of pluripotency-associated genes. A subset of retroviral LTR elements are strongly activated at ZGA in IVV and IVT embryos, whereas their activation is markedly muted in SCNT embryos, suggesting that impaired gene and TE reprogramming may contribute to the developmental defects commonly observed in SCNT embryos. By epigenomic profiling, the MLT1D elements from the ERVL-MaLR LTR family lose repressive marks and gain H3K27ac at ZGA, together with DUXA-binding motif enrichment. Knockdown of DUXA in bovine embryos significantly reduced MLT1D expression and ZGA marker genes. We propose that a subset of DUXA-enriched MLT1D functions as enhancers that promote ZGA. Overall, our study provides new insights into the regulatory roles of TEs during bovine embryogenesis and establishes a framework for comparative studies of TE-mediated gene regulation in early mammalian development.

developmental biology↗

bioRxiv2026

Distinct transcriptional responses to mild cold versus warm temperatures in adult Drosophila melanogaster ovaries

Temperature influences fertility across diverse organisms, yet the mechanisms underlying how suboptimal temperatures affect gamete production and quality remain largely unknown. We previously showed that chronic exposure of adult Drosophila melanogaster females to mild cold promotes the maintenance of germline stem cells (GSCs) and high oocyte quality over time despite reducing the rates of oogenesis, while exposure to warm temperature causes death of early germline cysts and vitellogenic follicles and a severe decrease in oocyte quality. To explore potential mechanisms underlying these highly distinct responses, we compared the ovarian transcriptomes of females maintained at these temperatures (18{degrees}C or 29{degrees}C) to that of 25{degrees}C controls. We found that 18{degrees}C upregulates or downregulates ~2.5 times as many genes as 29{degrees}C, indicating that the ovary mounts active physiological responses to mild cold and warm temperatures--as opposed to simply undergoing passive changes driven by thermodynamics. Gene set enrichment analysis revealed modulation of genes involved in neuronal signaling in opposite directions at 18{degrees}C versus 29{degrees}C. Most genes, however, exhibit temperature-specific regulation: 29{degrees}C upregulates synaptic transmission genes and downregulates lipid biosynthesis genes, whereas 18{degrees}C upregulates actin cytoskeleton genes and downregulates cell adhesion and lipid organization genes. Notably, mild cold or warm temperature specifically modulated (either up or down) the expression of distinct sets of transposable elements (TEs), suggesting the existence of temperature-dependent TE regulatory mechanisms and/or downstream effects. Finally, we show that GSCs at 18{degrees}C have increased retrotransposon R2 transcript levels, larger nucleolar size, and elevated levels of the known stemness factor phosphorylated Mad, leading to a working model whereby elevated ribosome biogenesis supports increased stemness signaling to promote GSC maintenance in mild cold. These findings suggest potential mechanisms and open new questions for investigation towards a deeper understanding of how temperature modulates gene expression and impacts germline development and quality--which are essential for the perpetuation of species.

developmental biology↗

bioRxiv2026

Dynamic Changes in Endometrial Folding and Secretory Activity Across the Menstrual Cycle

Embryo implantation remains a major limitation of assisted reproductive technology, with failure occurring in approximately 30% of euploid embryo transfers. Implantation requires a synchronized dialogue between the blastocyst and receptive endometrium during the window of implantation (WOI), yet minimally invasive approaches to characterize the structural and molecular features of receptivity remain limited. We analyzed paired sonohysterogram images and uterine lavage samples collected during the proliferative and mid-secretory phases from subjects with regular ovulatory cycles and proven fertility. Endometrial folds were quantified, and lavage samples were analyzed by Luminex multiplex immunoassay. Folds were present in both phases but were significantly more abundant during the mid-secretory WOI, independent of imaging view and endometrial thickness. Folding correlated strongly with circulating estradiol level during the proliferative phase but not the mid-secretory phase, and folding patterns between phases were not correlated, suggesting distinct regulatory mechanisms. Consistent with these structural patterns, uterine lavage demonstrated phase-specific differences in expression of factors associated with endometrial receptivity and implantation, with glandular epithelium, and myeloid-lineage cells emerging as major contributors. Together, these findings identify coordinated structural and secretory processes during the WOI and support further evaluation of endometrial folding and uterine lavage as complementary, minimally invasive markers of endometrial receptivity.

developmental biology↗