Molecular Characterization and Cloning of midrhythm: Identification of cgref1 as a Novel Gene in Vertebrate Left-Right Patterning

Abstract

Vertebrate organ asymmetry is a tightly regulated developmental process essential for proper physiological function. Disruption to left-right (LR) patterning can result in congenital disorders such as heterotaxy and critical congenital heart defects (CCHDs), which carry high morbidity and mortality. However, how early asymmetries are generated and interpreted during embryogenesis to guide organ-specific morphogenesis remains poorly understood. This work investigates the cellular and genetic mechanisms that govern LR axis specification using zebrafish. Through a forward ENU mutagenesis screen, a novel mutant, midrhythm, was identified based on its midline heart laterality in the absence of general morphological abnormalities. Phenotypic analysis revealed that midrhythm disrupts normal asymmetric gene expression, causing reduced dand5, bilateral spaw expression, and increased cilia length in the left-right organizer (LRO). Whole genome sequencing and CRISPR-based validation implicated Cell Growth Regulator with EF-Hand Domain (cgref1) as the causative gene underlying the midrhythm phenotype. Structural predictions and protein interaction modeling provide insight into the possible molecular mechanism of Cgref1 function, pointing to its involvement in regulating components of the Nodal signaling cascade through modulation of flow-induced Ca2+.. These findings contribute to a broader understanding of how early asymmetries are translated into organ patterning. To complement the mechanistic understanding of left-right patterning and its disruption, the final chapter applies a global health policy lens to examine inequalities in the early detection of CCHDs in newborns. Emphasis is placed on pulse oximetry screening (POS) as a proven tool, with a focus on identifying systemic barriers to its implementation and proposing equity-driven solutions to expand access globally.