You Have to CUT&WALK Before You Can CUT&RUN: Regulation of Gene Expression by the Vitamin D Receptor (VDR) in the Annual Killifish, <i>Austrofundulus limnaeus</i>

The annual killifish, Austrofundulus limnaeus, found in ephemeral ponds in South America, possesses a remarkable ability to survive extended periods without water and oxygen. This ability is associated with developmental dormancy known as diapause. Numerous genetic and environmental factors influence the entry into diapause and tolerance to environmental stress. Among these is vitamin D3 signaling mediated by the vitamin D receptor (VDR), which plays a crucial role in regulating entry into diapause and the continuous development of an embryo in a temperature-dependent manner. Vitamin D3 signaling influences a variety of physiological processes in vertebrates. While extensively characterized in mammalian systems, the role of VDR in non-mammalian vertebrates, particularly in the context of development, remains poorly understood. In this study, I explore the role of VDR signaling in regulating the development of A. limnaeus embryos. I employ a new technique, called a CUT&RUN assay, that uses a custom anti-VDR A. limnaeus antibody to identify genomic binding sites occupied by the VDR. Here, I report a new dissociation protocol to generate single-cell suspensions from early-stage embryos, enabling the first implementation of CUT&RUN in this species. Sequencing data processed through motif enrichment analysis and Gene Ontology (GO) analysis generated a de novo VDR motif sequence as well as VDR-associated genomic sites and biological processes potentially regulated by the VDR. These data provide preliminary evidence for VDR-mediated transcriptional regulation in early embryonic stages of A. limnaeus, while also laying methodological groundwork for future chromatin profiling research in a non-mammalian model.