ETD

• epigenetic
• neurodevelopment
• rescue
• setd5
• zebrafish

Maternal mRNAs deposited in the oocyte provide essential regulatory input during early embryogenesis, particularly before and during the maternal-to-zygotic transition (MZT). The chromatin regulator Setd5 has been linked to neurodevelopmental phenotypes in zebrafish (Danio rerio), suggesting that maternally supplied setd5 mRNA may contribute to early central nervous system (CNS) development. In this thesis, we investigated the maternal effect of setd5 on two key neurodevelopmental readouts: neural progenitor proliferation and CNS apoptosis.
To disentangle maternal and zygotic contributions, embryos were generated through targeted crosses among CRISPR-Cas9-derived setd5 mutant lines (+/+, +/-, and -/-) in order to produce distinct combinations of maternal and zygotic setd5 genotypes, enabling a cross-based rescue via wild-type maternal contribution. Embryos were genotyped by high-resolution melting (HRM) analysis, fixed at early developmental stages, cryosectioned, and analyzed by immunohistochemistry and TUNEL assay. Loss of maternal setd5 led to reduced neural progenitor proliferation and increased apoptosis in the CNS compared to embryos with wild-type maternal contribution, supporting a functional role of the maternal component.
To further test whether the presence of setd5 mRNA during developmental stages preceding MZT is sufficient to rescue proliferation and apoptosis alterations, we established an mRNA injection-based rescue strategy that mimics physiological maternal transcript dynamics. A capped in vitro-transcribed setd5 mRNA carrying the sdr16 3’-UTR, chosen to include regulatory elements linked to maternal transcript clearance mediated by miR-430, was generated by cloning and in vitro transcription. Using co-injected RFP as a procedural reference, qPCR analyses confirmed that the 3’-UTR-containing transcript undergoes decay consistent with maternal mRNA behavior, whereas a transcript lacking this UTR remains comparatively stable. An optimized dose of the UTR-containing setd5 mRNA was then microinjected into one-cell stage setd5 -/- embryos, and CNS proliferation and apoptosis were assessed in correctly injected (RFP-positive) embryos. The mRNA-based approach restored the neurodevelopmental readouts, with a trend toward increased progenitor proliferation in rescued embryos, whereas RFP-only injections did not alter the mutant phenotype, indicating that the observed rescue is attributable to setd5 rather than to the injection procedure.
Overall, these findings demonstrate a biologically relevant maternal effect of setd5 during early zebrafish neurodevelopment and provide an experimental framework to model maternal transcript regulation through controlled, physiologically informed rescue.