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

## TL;DR

The RNA-binding protein Squid is crucial for midgut development in Bombyx mori by regulating Axin splicing and β-catenin levels.

## Contribution

This study identifies a novel post-transcriptional regulatory pathway involving Squid and Axin in insect midgut development.

## Key findings

- CRISPR/Cas9 knockout of Squid leads to embryonic lethality and disorganized midgut epithelium in Bombyx mori.
- Squid regulates Axin alternative splicing, shifting from Axin-L to Axin-S, which reduces β-catenin levels in the midgut.
- Axin-L, but not Axin-S, elevates β-catenin levels when overexpressed in BmN cells.

## Abstract

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.

The RNA-binding protein Squid modulates embryonic midgut development by post-transcriptionally regulating Axin alternative splicing to maintain β-catenin levels in Bombyx mori.