Abstract Body (Do not enter title and authors here): Introduction
DEAD-box RNA helicase 21 (DDX21) plays critical roles in fundamental biological processes, including ribosome biogenesis, gene transcription, cell cycle regulation, and cell differentiation. However, its role in cardiovascular development remains poorly understood.
Hypotheses
We hypothesize that DDX21 is essential for proper development of the cardiovascular system.
Methods
Embryonic cardiomyocytes(CMs) and Vascular smooth muscle cells (VSMCs)-specific Ddx21 knockout mice were generated via crossbreeding Sm22-Cre mice with Ddx21^flox/flox mice. Embryos at various developmental stages (E10.5, E15.5, E18.5 and E19.5), as well as neonatal P0 animals were harvested for morphological assessment. Hearts and aortas were harvested at E18.5 and P0, then processed for paraffin-sectioning, followed by Hematoxylin and Eosin staining and immunofluorescence analysis. VSMCs were treated with siRNA targeting DDX21, followed by analysis of immunofluorescence staining and RNA-sequencing.
Results
Ddx21 conditional knockout (cKO) embryos did not exhibit overt morphological abnormalities until E18.5 and later stages. The viability of cKO embryos declined markedly between embryonic day 18.5 (E18.5) and birth (P0). Although a limited number of neonatal Ddx21 cKO animals were obtained, none survived beyond postnatal day 0.5 (P0.5). At E18.5, cardiac development was impaired, with a particularly underdeveloped right ventricular chamber. Thoracic aortas from P0 cKO animals exhibited reduced diameter and wall thickness compared to controls. Loss of DDX21 in CMs and VSMCs impaired their proliferative capacity, as indicated by a reduction in Ki67+ or EdU+ cells. DDX21 ablation led to increased tri-methylation of lysine 27 on histone H3 (H3K27me3), a repressive epigenetic mark. RNA-sequencing of VSMCs treated with siRNA-DDX21 led to a significant downregulation of 487 genes and an upregulation of 181 genes, indicating a global suppression of gene expression following DDX21 knockdown. Pathway analysis of the downregulated genes revealed significant enrichment in cell cycle regulation.
Conclusion
DDX21 is critical for cardiovascular development by promoting the proliferation of CMs and VSMCs. Its loss leads to elevated H3K27me3 levels and widespread gene repression, particularly affecting cell cycle-related pathways. These findings highlight the essential role of DDX21 in regulating epigenetic and transcriptional programs that support cell proliferation.