Analytical Pipeline and Orthology References for: Structural and Temporal Constraints Prevent Enucleation in Avian Erythrocytes

Abstract The failure of human induced pluripotent stem cell (iPSC)-derived erythroblasts to undergo terminal enucleation remains a fundamental bottleneck in the manufacture of transfusable blood. To elucidate the regulatory constraints of nuclear extrusion, we generated a high-resolution single-cell atlas of avian (chicken) erythropoiesis—a natural model of the nucleated state—and integrated it with primary human and enucleation-deficient iPSC datasets. Comparative transcriptomics and phylogenetic reconstruction identified a dual-layered evolutionary blockade in the avian lineage: a structural layer characterized by a proteolytically resistant "MRM–H" patch in the Vimentin (VIM) head domain, and a temporal layer defined by a heterochronic delay in the niche-dependent TGFB1–SRF signaling axis. In contrast, placental mammals share a conserved VIM cleavage hotspot (QDS motif) and a synchronized bioenergetic surge. Crucially, we found that iPSC-derived erythroblasts successfully mimic the mammalian program of VIM downregulation, yet remain arrested due to an attenuated TGFB1–SRF axis and the absence of coordinated RHOA/CASP3 mechanical pulses. To functionally validate these constraints, we employed a sequential pharmacological strategy in-ovo to bypass the avian structural lock. Destabilization of the VIM scaffold initiated cellular rounding and nuclear condensation but resulted in an arrested, pre-extrusion state. Our findings demonstrate that while vimentin disassembly is a mandatory structural prerequisite for enucleation, it is insufficient for nuclear expulsion without the precise temporal synchronization of niche-derived signaling. These results delineate the regulatory requirements for terminal erythropoiesis and identify specific molecular targets to overcome maturation arrest in lab-grown blood.