Tissue-level integration overrides gradations of differentiating cell identity in beetle extraembryonic tissue

During animal embryogenesis, one of the earliest specification events distinguishes extraembryonic (EE) from embryonic tissue fates: the serosa in the case of the insects. While it is well established that the homeodomain transcription factor Zen1 is the critical determinant of the serosa, subsequent realization of the tissue’s identity has not been investigated. Here, we examine serosal differentiation in the beetle Tribolium castaneum based on quantification of morphological and morphogenetic features, comparing embryos from a Tc-zen1 RNAi dilution series, where complete knockdown results in amnion-only EE tissue identity. We assess features including cell density, tissue boundary morphology, and nuclear size as dynamic readouts for progressive tissue maturation. While some features exhibit an all-or-nothing outcome, other key features show dose-dependent phenotypic responses with trait-specific thresholds. Collectively, these findings provide nuance beyond the known status of Tc-Zen1 as a selector gene for serosal tissue patterning. Overall, our approach illustrates how analysis of tissue maturation dynamics from live imaging extends but also challenges interpretations based on gene expression data, refining our understanding of tissue identity and when it is achieved.