Differential paracrine sensing in limb-bud progenitors: influence of tissue relatedness and morphogenetic context

During embryogenesis, cells extract crucial positional information by monitoring the signalling exchanges occurring among neighbouring tissues. Depending on the molecular context—such as immunogenetic relatedness of the signal source and the nature of the morphogenetic activity—the recipient’s calcium dynamics, migratory behaviour and stress-pathway activation can differ markedly. Moreover, these responses may vary according to each cell’s own niche identity. Using live limb-bud organoids containing multiple developmental “generations” of mesenchymal progenitors, we perfused time-fractionated secretomes generated by isogenic or allogenic donors engaged in one of four morphogenetic states: collective migration, mechanical collision, quiescence, or cooperative junctional remodelling. We quantified calcium-spark frequency, directional motility and ROS-linked stress signalling in the recipients. In parallel, we assessed how a progenitor’s baseline autocrine output and gap-junctional activity within its native organoid correlated with these readouts. Progenitors displayed significantly higher calcium-spark density when exposed to isogenic secretomes than to allogenic ones, an effect most pronounced in posterior, niche-restricted (functionally subordinate) cells with intrinsically low oxidative stress reactivity. Autocrine blebbing rates were elevated in younger progenitors encountering allogenic cues, but not isogenic cues, and in cells characterised by high baseline self-signalling and minimal junctional coupling. With respect to context, secretomes derived from migratory or collision phases elicited stronger calcium activation and motility than those from quiescent or remodelling phases. Collectively, limb-bud progenitors cultured in multi-generational organoids exhibit a selective drive to harvest paracrine information from related tissues; this sensitivity is modulated by niche position and inherent capacity to withstand morphogenetic stress.