Matrix Metalloproteinase-2 Deficiency Impairs Eye Development in Crispant Zebrafish

Matrix metalloproteinase-2 (Mmp2, also known as gelatinase A) is a zinc-dependent endopeptidase, and mutations in human MMP2 can lead to some human diseases. However, the underlying function and mechanism of mmp2 during eye development are still unclear. The present study generated mmp2 crispant zebrafish by CRISPR/Cas9 system. Whole-mount in situ hybridization (WISH), immunofluorescence, qRT-PCR, and transcriptomic and metabolomic analyses were performed. Results showed that mmp2 knockdown led to small eyes at 48–96 hpf (hours post-fertilization). WISH and qRT-PCR showed that mmp2 knockdown at 72 hpf (hours post-fertilization) significantly decreased the expression of cone-specific markers (opn1sw1, opn1sw2, opn1mw1, and opn1lw1), the rod-specific marker rhodopsin, retinal cell-specific markers (gnat1 and gnat2), and lens-specific markers (crybgx and crygm2c) in the retina. Immunofluorescence showed increased apoptosis (TUNEL+ cells), ectopic localization of the neuronal differentiation marker HuC/D at the ciliary marginal zone, and decreased photoreceptor cells (Zpr1) and photoreceptor (RCVRN) fluorescence. Transcriptomic analysis indicated that 520 differentially expressed genes were enriched in phototransduction and retinol metabolism. Metabolomics analysis indicated that mmp2 knockdown led to abnormal lipid metabolism and amino acid metabolism. Integrated omics analysis revealed shared pathways involving PPAR signaling and nucleotide sugar metabolism, while correlation analysis highlighted strong associations between eye-development-related genes (e.g., gnat1, gnat2, opn1sw1) and specific metabolites such as 2-palmitoyl-rac-glycerol and 1-hexadecyl-sn-glycero-3-phosphocholine. Collectively, these findings demonstrate that Mmp2 is essential for normal eye development in zebrafish, and its deficiency disrupts retinal cell survival, differentiation, and metabolic homeostasis, providing new insights into the molecular mechanisms underlying Mmp2-associated ocular abnormalities.