Gastruloids develop the three body axes in the absence of extraembryonic tissues and spatially localised signalling

Key results

1. 01

   ShownAfter a 48-72h pulse of the Wnt agonist CHI99201 (Chi), 120h Gastruloids show a posterior T/Bra and Cdx2 tip and an orthogonal dorsoventral-like axis. Lateral Smad2,3 reporter asymmetry hints at a left-right axis and a node-like region.

   Immunostaining and reporters at 120h after aggregation (AA): T/Bra and Cdx2 at the tip with a shallow TLC2 Wnt gradient; Sox1::GFP and Sox2 neural tissue on one side, clustered Nodal::YFP opposite; Smad2,3 activity turns bilaterally asymmetric over time. Shaken cultures reach 160h AA, exceed 1mm and contain a Sox17-high, Sox2-low tube. · 2.1 Axial organisation in Gastruloids; Figure legends

2. 02

   ShownGastruloids are built only from embryonic, epiblast-like cells. They lack extraembryonic gene expression, BMP signalling and early GATA6, so their axes form without extraembryonic tissue; 48h and 72h AA approximate E6.0 and E7.0.

   qRT-PCR (~64 Gastruloids per time-point) at 48h AA detects Fgf4, Fgf5, Axin2, Wnt3, Nodal, Cripto and low Lefty1, but not BMP4, Dkk, Furin, Lrp2 or Dab, with very low Cerberus; the Smad1,5,7 BMP reporter is silent under Chi yet responds to exogenous BMP; GATA6::H2B-Venus is absent for the first 72h, appearing at ~88h. · 2.3 Extraembryonic tissues are not required for…; 5.4 Quantitative RT-PCR; Figure legends

3. 03

   ShownT/Bra polarises spontaneously in neutral N2B27 medium. A Wnt/beta-catenin pulse (Chi or Wnt3a) during 48-72h amplifies, sharpens and stabilises this pole and drives elongation, and the timing of exposure is critical.

   T/Bra::GFP is weak and partly biased at 24h AA and more polarised at 48h; Chi or Wnt3a at 48-72h gives tighter fluorescence distributions than DMSO, and Hu Moment PCA separates elongated Chi/Wnt3a Gastruloids from spherical controls on PC1. Chi at 24-72h yields multiple T/Bra foci, whereas exposure at 48-96h tends to abolish polarisation and elongation. · 2.2 Wnt/β-catpole of expression is less…; 2.6 Wnt/β-catenin can generate multiple axes in…; Figure legends

4. 04

   ShownNodal/ALK4 signalling is required to start T/Bra expression, whereas raised Nodal after 48h AA restrains elongation, so T/Bra induction and axial extension can be uncoupled.

   SB431542 (SB43) at 48-72h abolishes T/Bra::GFP and elongation (Gastruloids stay spherical), and Chi co-treatment does not rescue it; SB43 at 24-48h before Chi delays and destabilises T/Bra but leaves elongation intact or enhanced; Nodal at 48-72h, alone or with Chi, raises T/Bra yet leaves Gastruloids spheroid or ovoid. · 2.4 Nodal signalling promotes T / Bra expression; Figure legends

5. 05

   ShownSpatially uniform Nodal rescues symmetry breaking in Nodal-null Gastruloids, so a localised Nodal source is not needed for AP polarisation, provided Nodal is supplied before the Wnt pulse.

   Nodal^(-/-) Gastruloids stay spherical or ovoid and ~90% form small bulbous protrusions by 120h AA; Chi (48-72h) alone raises elongation to ~50% but fails to upregulate T/Bra and Cdx2; 100 ng/ml Nodal at 24-48h before Chi restores T/Bra and Cdx2 patterning and lifts the elongated, protrusion-free class (0 to 50%); later Nodal windows are less effective. · 2.7 A polarised source of Nodal signalling is…; Figure legends