The RNA-binding protein Squid regulates embryonic midgut development via Axin alternative splicing in Bombyx mori

The insect midgut is crucial for digestion and nutrient absorption, but its embryonic development is poorly understood. Here, we show that the RNA-binding protein Squid is essential for embryonic midgut epithelium development in the model insect, Bombyx mori. CRISPR/Cas9-mediated knockout of Squid causes embryonic lethality, with mutants exhibiting disorganized midgut epithelium, lipid droplet accumulation, and impaired nutrient absorption. Integrated RNA sequencing (RNA-seq) and RNA-immunoprecipitation sequencing (RIP-seq) analyses reveal that Squid directly regulates the alternative splicing of Axin, a key Wnt/β-catenin pathway component. In Squid-depleted embryos, Axin splicing shifts from the long (Axin-L) to a short isoform (Axin-S), as confirmed by RT-qPCR. Consequently, β-catenin protein levels are significantly reduced in the midgut epithelium. Overexpression in BmN cells confirms that Axin-L, but not Axin-S, elevates β-catenin. Overall, this study uncovers a critical post-transcriptional pathway wherein Squid ensures proper midgut development by regulating Axin alternative splicing to fine-tune β-catenin levels.