Wt1 splice isoforms bias lineage programmes during formative pluripotency

ABSTRACT How transcriptional asymmetry first arises in the epiblast prior to gastrulation remains a central question in mammalian development. During the transition from naïve to formative pluripotency, epiblast cells acquire responsiveness to lineage-inducing cues while already expressing lineage-associated transcriptional programmes. The molecular basis of these asymmetries and whether they reflect stochastic variation or regulated lineage-associated bias remain unclear. Here, using a targeted CRISPRa screen, we identify Wt1 (Wilms tumor 1) as a previously unrecognized regulator of lineage-associated transcriptional programmes during formative pluripotency. Wt1 is transiently induced during formative pluripotency in vitro and in the pre-gastrulation epiblast in vivo , with peak expression coinciding with the emergence of lineage-associated transcriptional biases. Precocious Wt1 induction overrides the naïve transcriptional network and advances cells toward a post-implantation epiblast identity, whereas Wt1 -deficient cells remain capable of naïve exit but show impaired acquisition of formative and lineage-associated transcriptional programmes. Genome-wide binding analyses during formative state acquisition show that WT1 engages active regulatory elements of the emerging post-implantation gene regulatory network together with core formative transcription factors, including FOXO1, OTX2 and OCT4. Direct comparison of all four major Wt1 splice isoforms identifies KTS splice status as the major determinant of divergent anterior/neuroectodermal and posterior/mesodermal transcriptional programmes in vitro . In the E5.5 epiblast, Wt1 expression and splice composition align with lineage-biased transcriptional states, linking isoform usage to anterior transcriptional biases in vivo . Isoform-dependent gene expression modules are conserved in human pluripotent cells, indicating that this regulatory logic is preserved across species. Together, our findings indicate that lineage-associated transcriptional programmes diverge during formative pluripotency prior to gastrulation and identify alternative splicing as a mechanism that tunes lineage-associated transcriptional bias.