Timed binary WNT activity is sufficient to model the early human neural axis
Abstract
The vertebrate nervous system develops into functionally distinct domains along the body axis[1]. Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear [2,3], leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing [4-11], yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three 'first-order' domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested 'second-order' domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.
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BibTeX
@misc{hackland2026timed,
title = {{Timed binary WNT activity is sufficient to model the early human neural axis}},
author = {J.O.S. Hackland and G. Anand and R. Vijayakumar and X. Wang and V.D. Bocchi and E.S. Rowland and D. Yang and Y. Wu and S. Mehta and A. Zhong and E. Grossi and R.P. Koche and V. Kirigin Callaú and R.M Walsh and A. Evangelisti and Y. Fan and E. Bernstein and R.J. Garippa and L.A. Stamp and M.M. Hao and T. Zhou and L. Studer},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.17.759682},
url = {https://www.biorxiv.org/content/10.64898/2026.10.17.759682v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Timed binary WNT activity is sufficient to model the early human neural axis
AU - J.O.S. Hackland
AU - G. Anand
AU - R. Vijayakumar
AU - X. Wang
AU - V.D. Bocchi
AU - E.S. Rowland
AU - D. Yang
AU - Y. Wu
AU - S. Mehta
AU - A. Zhong
AU - E. Grossi
AU - R.P. Koche
AU - V. Kirigin Callaú
AU - R.M Walsh
AU - A. Evangelisti
AU - Y. Fan
AU - E. Bernstein
AU - R.J. Garippa
AU - L.A. Stamp
AU - M.M. Hao
AU - T. Zhou
AU - L. Studer
PY - 2026
DA - 2026/10/17
PB - bioRxiv
DO - 10.64898/2026.10.17.759682
UR - https://www.biorxiv.org/content/10.64898/2026.10.17.759682v1
AB - The vertebrate nervous system develops into functionally distinct domains along the body axis[1]. Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear [2,3], leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing [4-11], yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three 'first-order' domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested 'second-order' domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.
SN - 2692-8205
ER -