Occluding Apical or Basal Signalling Interfaces of Organizer‐Like Cells Does Not Hinder Lineage Specification in Human Gastruloid Models
July 12
Live imaging studies have shown that epiblast-derived progenitors orient their trajectories toward organiser-like cells and transiently dwell on either the apical junctional belt or the basal protrusive zone during the first day of gastruloid development; both behaviours correlate with subsequent expression of germ-layer markers. This correlational evidence has promoted the view that physical access to these discrete signalling interfaces is a necessary gatekeeper of fate acquisition, a process that accelerates markedly after the 24-h stage. If correct, masking either interface while the organiser presents novel morphogen–receptor combinations should disrupt or attenuate lineage commitment.
To test this prediction experimentally, we tracked single-cell dynamics in 17- to 42-h gastruloids (N = 153) undergoing a rapid lineage-tagging assay under three conditions: (a) unperturbed interface, in which organiser cells were fully exposed; (b) apical shield, in which the tight junction belt was coated with an opaque PEG hydrogel; and (c) basal shield, in which the protrusive basal lamellipodia were enveloped by a black alginate cloak. Progenitors labelled at 24 h and beyond adopted endodermal or mesodermal transcriptional programmes, and, critically, they did so irrespective of shielding condition.
Correlational analyses further revealed that successful fate conversion aligned with directed migration—quantified as organiser-oriented displacement and organiser-target contact shifts—but not with dwell time on either the apical or basal membrane. These observations indicate that the capacity to couple morphogen identity with positional information emerges around the 24-h milestone and that, at this stage, progenitors optimise their behaviour through collective sensing and matrix exploration rather than prolonged engagement with any specific organiser interface. We discuss the ramifications for in vitro tissue engineering, highlighting that robust lineage instruction can be achieved in geometrically constrained microenvironments without the need for precise presentation of organiser polarity cues.