Tissue-specific I-Smad mechanisms revealed by structure–function analysis in Drosophila

Inhibitory Smads (I-Smads) regulate TGF-β/BMP signaling through multiple distinct mechanisms, but whether different tissues preferentially employ specific mechanisms remains unknown. To address this question, we performed structure–function analyses of the Drosophila I-Smad, Dad, and its vertebrate orthologs Smad6 and Smad7 in neural and wing tissues, measuring outputs of BMP signaling in vivo. We identified a 24–amino acid putative DNA-binding domain within the MH1 domain of Dad that is essential for inhibitory function in wing tissue but unessential in neural tissue. Structural analyses revealed that ΔDNA-binding domain disrupts a β-hairpin structure homologous to R-Smad DNA-binding regions. We also found that Dad requires an intact MH1 domain to disrupt wing development, whereas either MH1 or MH2 can independently disrupt BMP signaling in motor neurons. These findings support a model where Dad functions through MH1-mediated transcriptional regulation in wing primordium, but through multiple mechanisms in neurons. Comparative analysis revealed that vertebrate I-Smad orthologs also show tissue-specific activity patterns, with structural predictions suggesting that Smad6 retains ancestral DNA-binding capacity, whereas Smad7 has evolved enhanced MH2-mediated functions. These results reveal context-dependent mechanisms of I-Smads that further the understanding of TGF-β/BMP pathway regulation. © 2026, Life Science Alliance, LLC. All rights reserved.