Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia

Hox genes are highly conserved, master regulators of spatial patterning in the embryo, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of the small and large intestines early in embryogenesis, which ultimately leads to their specialization in both form and function. While the midgut forms villi, the hindgut develops flat, brain-like sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the TGFβ pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme; together, these features lead to hindgut surface buckling. TGFβ, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional genetic identity that direct posterior intestinal morphogenesis. To more specifically identify which genes are commonly differentially expressed within the subepithelial mesenchyme of the midgut with Hoxd13 misexpression and the hindgut, relative to the midgut, a spatial sequencing approach called Light-seq was employed (Kishi et al., 2022). In situ reverse transcription was performed in midgut, hindgut, and RCAS-Hoxd13 tissue sections on slides. Light-directed crosslinking was used to iteratively barcode manually defined regions--the subepithelial mesenchyme, inner longitudinal muscle layer, and inner superficial side of the circumferential muscle layer. Barcoded cDNA was then extracted from slides and used for downstream qPCR amplification and library preparation. Each sequenced library contained reads from all three compartments. Downstream barcode parsing then enabled assignment of each read to the tissue layer of interest. Though three layers were barcoded and sequenced, only the inner mesenchyme layer was used for differential gene expression analysis comparing conditions (midgut, hindgut, midgut + Hoxd13).