The Role of CBX2 in Zebrafish Development: Crosstalk Between Polycomb Repression, WNT/β-Catenin, and Retinoic Acid Signaling

Polycomb group (PcG) proteins are highly conserved chromatin modifiers that maintain transcriptional repression during development through the epigenetic regulation of key developmental genes. Among these, Chromobox homolog 2 (CBX2) is a core component of the canonical Polycomb Repressive Complex 1 (PRC1), which catalyzes histone H2A monoubiquitination (H2AK119ub) and mediates chromatin compaction to preserve transcriptional silencing. Although CBX2 has been extensively studied in metazoan, its functional role during vertebrate embryogenesis remains poorly characterized. In this study, we investigated the developmental and molecular functions of cbx2 in zebrafish (Danio rerio), which shares key aspects of epigenetic regulation with humans, using a combination of morpholino-mediated knockdown, pharmacological modulation, rescue assays, and CRISPR/Cas9-based mutagenesis. Knockdown of cbx2 resulted in pleiotropic developmental abnormalities, including microphthalmia, cardiac edema, craniofacial cartilage malformations, and pectoral fin truncations, accompanied by impaired locomotor activity and increased apoptosis in the head region. These phenotypes were partially rescued by co-injection of cbx2 mRNA, confirming the specificity of the knockdown phenotype. Morpholino-mediated depletion ofcbx2 led to upregulation of ALDH1A2 (an RA-synthesizing enzyme), resulting in excessive retinoic acid (RA) signaling. Elevated RA activity was confirmed by increased expression of downstream RA-responsive genes (cyp26a1, dhrs3a, hoxb1a, hoxb5b and meis3a) and by phenotypic rescue upon treatment with the RA synthesis inhibitor DEAB. Mechanistically, cbx2 depletion caused a marked reduction in PRC1-mediated H2AK119ub levels, consistent with its role as a key chromatin repressor. ChIP-qPCR analysis revealed that CBX2 maintains PRC1 occupancy and repressive histone marks at RA-responsive promoters, thereby preventing aberrant activation of the RA pathway. Excessive RA signaling in cbx2-deficient embryos was associated with enhanced apoptosis, mitochondrial depolarization, and activation of the intrinsic apoptotic pathway. Increased expression of P53 and P21 was accompanied by reduced CBX2 and H2AK119ub enrichment at their promoters, suggesting a direct epigenetic role for CBX2 in repressing pro-apoptotic genes. In parallel, cbx2 knockdown disrupted canonical Wnt/β-catenin signaling through the upregulation of APC, a negative regulator of the pathway, leading to reduced expression of Wnt target genes. This indicates that CBX2-mediated repression at the apc gene locus is essential for maintaining the balance between Wnt and RA signaling during embryonic development. To further validate these findings, a stable cbx2 mutant zebrafish line was generated using a dual-guide CRISPR/Cas9 strategy. Sequence analysis confirmed compound deletions within exons 2 and 3, resulting in a premature stop codon and truncation of the protein. The ongoing characterization of heterozygous and homozygous mutant lines will enable long-term studies on the developmental and epigenetic consequences of CBX2 loss, complementing the morpholino-based findings. Collectively, these results identify cbx2 as a critical epigenetic regulator that integrates PRC1-mediated chromatin repression with RA and Wnt signaling to ensure proper embryonic patterning in zebrafish. By maintaining RA homeostasis and preventing inappropriate activation of pro-apoptotic and differentiation programs, CBX2 safeguards the coordinated development of multiple organ systems. The generation of a cbx2 mutant line provides a powerful model for future studies to dissect the chromatin-level mechanisms underlying CBX2-mediated regulation and its relevance to congenital malformations and human developmental disorders.