Effect of Elf3 Knockout on Zebrafish Early Development

Abstract

Elf3 (E74 like ETS transcription factor 3) is a member of the ETS (erythroblast 26 transformation-specific) family of transcription factors that regulate gene expression through a conserved by DNA-binding domain. Disruptions in elf3 in zebrafish have been linked to abnormalities such as craniofacial cartilage malformation and impaired notochord development, particularly in the context of prenatal alcohol exposure, recapitulating effects of fetal alcohol spectrum disorder (FASD). Previous morpholino oligonucleotide (MO) studies showed that elf3 is required for epidermal, mesenchymal, and neural tissue development. This study hypothesizes that knockout of elf3 in zebrafish embryos disrupts embryogenesis through dysregulation of extracellular matrix (ECM) remodeling proteins. To test this, a zebrafish elf3 mutant line was generated using CRISPR-Cas9 mutagenesis. Heterozygous adult zebrafish displayed skeletal abnormalities indicating that even partial loss of elf3 function produces noticeable structural abnormalities. Homozygous elf3-/- embryos displayed morphogenesis defects and cell death beginning around 10 hours post fertilization (hpf), most prominently within the head anlagen or anterior neural plate, resulting in embryonic lethality. Molecular analysis revealed significant dysregulation of ECM-related transcripts in elf3-/- embryos. elf3+/- embryos also showed phenotypic consequences, including delayed growth during early development and the emergence of a chordoma-like phenotype in later stages. Chordomas are rare tumors characterized by the invasion of notochordal tissue into surrounding structures. Tissue architecture and cellular changes associated with the chordoma-like phenotype were examined to assess tissue organization and adhesion properties. Similar craniofacial and notochordal defects have been reported previously elf3 morpholino knockdown experiments, supporting the validity of the mutant phenotypes. Together, these results suggest that reduced elf3 expression alters the regulation of ECM proteins during epithelial to mesenchymal (EMT) transition, including matrix metalloproteases (Mmps) and Snail2, which are critical for morphogenesis and maintenance of tissue structure. These findings highlight elf3 as a critical regulator of zebrafish embryogenesis, neural crest cell migration, and tissue morphogenesis, with implications for congenital disorders such as FASD and notochordrelated diseases, including cancer. This elf3 mutant line thus provides a valuable model to further investigate molecular pathways regulated by elf3 and its relevance to vertebrate development and disease.