Two parallel neural ectoderm progenitors contribute to the developing brain

Lineage tracing studies of mouse embryos published in Nature Neuroscience support a model in which two parallel, lineage-restricted neural ectoderm progenitors — anterior neural ectoderm (forebrain/midbrain) and posterior neural ectoderm (hindbrain) — emerge simultaneously during gastrulation and give rise to the brain as a composite organ. Differentiating human pluripotent stem cells into anterior or posterior neural ectoderm-like cells showed each was lineage committed to forebrain/midbrain versus hindbrain fates and carried diverging chromatin landscapes, and the researchers further generated hindbrain rhombomere 5/6-specific motor neurons that had been difficult to produce in vitro. The authors postulate the dual progenitors may be evolutionarily conserved across 550 million years from hemichordates to mammals; sequencing datasets are available under NCBI GEO accessions GSE286146, GSE286147, GSE286148 and SuperSeries GSE286214.

Abstract When and how different brain regions diversify from one another remains unresolved. Does a common neural ectoderm progenitor generate the entire brain? Or do multiple neural ectoderm progenitors exist, each restricted to form specific brain regions? Here our lineage tracing studies of mouse embryos support the latter model. Two parallel brain progenitors emerge simultaneously during gastrulation: anterior neural ectoderm forebrain/midbrain progenitor and posterior neural ectoderm hindbrain progenitor . Differentiation of human pluripotent stem cells into anterior or posterior neural ectoderm-like cells revealed these were lineage committed to forebrain/midbrain versus hindbrain fates, respectively. They harbored diverging chromatin landscapes foreshadowing future forebrain/midbrain versus hindbrain identities. We further differentiated human pluripotent stem cells into hindbrain rhombomere 5/6-specific motor neurons, which were hitherto difficult to generate in vitro. Hence, we postulate the brain is a composite organ emanating from two lineage-restricted progenitors; these dual progenitors may be evolutionarily conserved across 550 million years from hemichordates to mammals. This is a preview of subscription content, access via your institution https://wayf.springernature.com?redirect uri=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41593-026-02433-7 Access options Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription 27,99 € / 30 days cancel any time Subscribe to this journal Receive 12 print issues and online access 269,00 € per year only 22,42 € per issue Buy this article - Purchase on SpringerLink - Instant access to the full article PDF. 39,95 € Prices may be subject to local taxes which are calculated during checkout Data availability Bulk-population RNAseq, scRNAseq and OmniATAC-sequencing datasets generated as part of this study are available at the NCBI Gene Expression Omnibus accession nos. GSE286146 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE286146 OmniATAC-sequencing , GSE286147 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE286147 bulk RNAseq and GSE286148 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE286148 scRNAseq . These datasets are grouped under SuperSeries accession no. GSE286214 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE286214 . An interactive web browser to explore scRNAseq data of the hPS cell-derived cell types generated in this study is available at https://anglohlabs.shinyapps.io/dundes jokhai lohlab/ https://anglohlabs.shinyapps.io/dundes jokhai lohlab/ . Source data https://www.nature.com/articles/s41593-026-02433-7 Sec65 are provided with this paper. Code availability Computational scripts used in this study to analyze sequencing datasets are available via GitHub at https://github.com/lohlaboratory/ane-pne https://github.com/lohlaboratory/ane-pne . References 1. Moens, C. B. & Prince, V. E. 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Shimamura, K. & Rubenstein, J. L. Inductive interactions direct early regionalization of the mouse forebrain. Development 124 , 2709–2718 1997 . Acknowledgements We thank J. Li, B. Conklin, T. Anbarchian, R. Nusse, F. Suchy, H. Nakauchi, A. Elefanty, Ed. Stanley and E. Ng for sharing reagents. We also thank S. Vijayakumar, B. Woodruff, C. Anselmi and A. Voskoboynik for contributing to experiments, and W. Talbot, A. Zorn, K. Campbell, R. Krumlauf, P. Beachy, C. Shatz, J. Day, A. Pollen, T. Nowakowski, L. T. Ang and N. Uchida for advice. Infrastructure support was provided by V. Park, L. Ou, K. Lee, C. Carswell-Crumpton, P. Lovelace, L. Dunkin-Hubby and the Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stem Cell FACS Core Facility, Veterinary Service Center, Cell Sciences Imaging Facility and Diabetes Research Center. Funding This work was supported by the US National Institutes of Health grant nos. DP5OD024558 and P30DK116074 to K.M.L., DP2GM146258 and R00GM121852 to D.E.W., R01DK115728 to K.C.G., R01DE027538 to M.E.B., T32GM119995 to C.E.D., T32GM007365 and T32GM007790 to R.T.J., T32GM141828 to A.R. and F31DE031154 to H.A.U. , the US National Science Foundation grant no. IOS1656628 to C.J.L. ; the California Institute of Regenerative Medicine grant nos. TB1-01195 to R.E.A.S.-S. and EDUC2-12677 to A.S.P. ; the Spinal Muscular Atrophy Foundation K.M.L. ; the Stanford Maternal and Child Health Research Institute K.M.L. ; the Stanford Beckman Center K.M.L. ; the Stanford Ludwig Center K.M.L. ; the Siebel Stem Cell Institute K.M.L. ; the Stinehart-Reed Seed Grant K.M.L. ; Anonymous, Fickel and Gilbert families K.M.L. ; the Gatsby Charitable Foundation M.M. ; the Howard Hughes Medical Institute M.M. and K.C.G. ; the Ford Foundation, Stanford Graduate and Stanford DARE Fellowships C.E.D. ; the Stanford Medical Scholars Research Program R.T.J. , the Yale Saybrook College and Stacey Leondis Fellowships C.X. ; and the Stanford MCHRI and Dean’s Postdoctoral Fellowships Y.Q. . K.M.L. was supported as a Packard Foundation Fellow, Pew Scholar, Baxter Foundation Faculty Scholar, Human Frontier Science Program Young Investigator RGY0069/2019 and Anthony DiGenova Endowed Faculty Scholar. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Ethics declarations Competing interests Stanford University has filed patent applications related to neural differentiation, with K.M.L., R.E.A.S.-S., C.E.D., H.A. and R.T.J. listed as inventors. The other authors declare no competing interests. Peer review Peer review information Nature Neuroscience thanks the anonymous reviewer s for their contribution to the peer review of this work. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Extended data Extended Data Fig. 1 Definitive ectoderm and neural ectoderm marker expression. https://www.nature.com/articles/s41593-026-02433-7/figures/7 a Single-cell RNA-sequencing of E6.75 mouse embryos; analysis performed on a previously-published resource<sup 77 https://www.nature.com/articles/s41593-026-02433-7 ref-CR77 </sup . Embryonic cells are shown, with extraembryonic cells excluded Elf5 and Rhox5 log<sub 2</sub normalized expression = 0 . Otx2 is expressed in definitive ectoderm and pluripotent cells, consistent with previous studies<sup 4 https://www.nature.com/articles/s41593-026-02433-7 ref-CR4 , 32 https://www.nature.com/articles/s41593-026-02433-7 ref-CR32 </sup . Oct4 Pou5f1 is broadly expressed across all cell-types, consistent with past studies<sup 110 https://www.nature.com/articles/s41593-026-02433-7 ref-CR110 , 111 https://www.nature.com/articles/s41593-026-02433-7 ref-CR111 </sup . b Immunostaining of hPS cells before, or after, differentiation into definitive ectoderm for 24 hours. NANOG and SOX2 immunostaining was performed with H7 hPS cells. OTX2 immunostaining was performed with H1 hPS cells. Scale bar = 100 μm. Representative images from 2 biological replicates in 1 experiment. c Flow cytometry of H1 hPS cells differentiated into definitive ectoderm for 24 hours. The percentage of SOX2 + NANOG- definitive ectoderm cells is shown. Each dot represents 1 independent differentiation experiment, with 3 experiments shown. d Flow cytometry of HES3 MIXL1-GFP hPS cells differentiated into definitive ectoderm day 1 , anterior neural ectoderm day 2 , posterior neural ectoderm day 2 , border ectoderm day 2 , surface ectoderm day 1 , or mid primitive streak<sup 30 https://www.nature.com/articles/s41593-026-02433-7 ref-CR30 </sup day 1, as a positive control to induce MIXL1 expression . Negligible numbers of MIXL1+ primitive streak cells emerged after ectoderm differentiation, reaffirming the suppression of spurious primitive streak differentiation. Representative data from 2 biological replicates in 1 experiment. e In situ staining of E7.5 mouse embryo, showing overlap of Sox2 and Gbx2 expression in pNE. Scale bar = 25 μm. Representative images from 40 embryos from 10 experiments. f Single-cell RNA-sequencing of E7.5 mouse embryos; analysis performed on a previously-published resource<sup 77 https://www.nature.com/articles/s41593-026-02433-7 ref-CR77 </sup . Neural ectoderm cells identified by log<sub 2</sub normalized expression of Sox2 0.5, and Nanog, Tfap2a, Rhox5, Foxj1 , and Elf5 = 0 are shown. g Intracellular flow cytometry for SOX2 protein expression in H1 hPS cells, prior to or after differentiation into anterior neural ectoderm or posterior neural ectoderm for 2 days. The percentage of SOX2+ cells, and median fluorescence intensity MFI of SOX2 expression, are both shown. Representative data from 2 biological replicates in 1 experiment. h Immunostaining of H1 hPS cells differentiated into definitive ectoderm for 24 hours, followed by differentiation into aNE or pNE for 24 hours. Scale bar = 100 μm. Representative images from 2 biological replicates in 1 experiment. Extended Data Fig. 2 Posterior neural ectoderm induction. https://www.nature.com/articles/s41593-026-02433-7/figures/8 a Western Blot to detect the expression of GBX2 protein in H1 hPS cell-derived day-2 aNE or day-2 pNE. VINCULIN protein serves as a loading control. Images from 2 biological replicates in 1 experiment. b β-CATENIN immunostaining of H1 hPS cells that were differentiated into various cell-types. First, hPS cells were differentiated into definitive ectoderm for 24 hours, and then treated with posterior neural ectoderm-inducing media for 24 hours A-83-01 1 μM + LDN193189 100 nM + TTNPB 50 nM + FGF2 20 ng/mL + XAV939 1 μM + Thiazovivin 1 μM , or as a positive control to induce nuclear β-CATENIN, modified posterior neural ectoderm media lacking WNT inhibitor and including a WNT agonist for 24 hours A-83-01 1 μM + LDN193189 100 nM + TTNPB 50 nM + FGF2 20 ng/mL + CHIR99021 2 μM + Thiazovivin 1 μM . Alternatively, as a positive control to induce nuclear β-CATENIN, hPS cells were differentiated into mid primitive streak, with WNT agonist-containing media Activin A 30 ng/mL + BMP4 40 ng/mL + CHIR99021 6 μM + FGF2 20 ng/mL for 24 hours. Nuclear β-CATENIN was absent from hPSC-derived posterior neural ectoderm, and either weakly or strongly positive in the two WNT agonist-treated cell populations. Scale bar = 100 μm. Representative images from 2 biological replicates in 2 experiments. Extended Data Fig. 3 Differentiation of hPSCs into anterior neural, posterior neural, border or surface ectoderm. https://www.nature.com/articles/s41593-026-02433-7/figures/9 a Summary of the hPSC differentiation strategy described in this study. b Top: In situ hybridization of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into aNE or pNE for 24 hours. Scale: 100 μm. Bottom: Immunostaining of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into border or surface ectoderm for 24 hours. Scale: 100 μm. Percentages of cells expressing a given marker are shown. Representative images from 2 biological replicates in 2 experiments. Extended Data Fig. 4 Lineage tracing of neural ectoderm cells within mouse embryos. https://www.nature.com/articles/s41593-026-02433-7/figures/10 a In situ staining of E9.5 Gbx2-CreER ; ROSA26-tdTomato mouse embryo that was not exposed to 4OHT. This negative control revealed minimal Gbx2-CreER -driven recombination in the absence of 4OHT. Scale bar = 500 μm. Image of 1 embryo from 1 experiment. b E7.0 Gbx2-CreER ; ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos were exposed to 1 mg 4OHT intraperitoneally delivered into pregnant females to label Gbx2 <sup +</sup pNE with tdTomato. Subsequently, in situ staining was performed on E8.25 embryos to visualize pNE-derived progeny in relation to the expression of Wnt1 which marks the midbrain/hindbrain boundary and Gbx2 which marks the hindbrain . Scale bar = 100 μm. Representative images from 18 embryos from 4 experiments. c E7.5 Sox2-CreER ; Confetti mouse embryos were exposed to 0.5 mg 4OHT intraperitoneally delivered into pregnant females to label single E7.5 Sox2 <sup +</sup neural ectoderm progenitors with one of three detectable fluorescent colors red, cyan, or yellow . Subsequently, E9.5 embryos were immunostained to visualize neural ectoderm-derived cell clusters and where they resided in relation to the forebrain, midbrain, and hindbrain; photos of three independent embryos are shown left . The forebrain/midbrain domain was molecularly determined by Otx2 immunostaining left . Quantification of the locations of all neural ectoderm-derived cell clusters analyzed in this study, totaling 494 cell clusters from 16 embryos from 7 experiments right . Scale bar = 100 μm. d Detailed analysis of the spatial position of a E7.5 Sox2 <sup +</sup neural ectoderm-derived cell cluster relative to the midbrain/hindbrain boundary. E7.5 Sox2-CreER ; Confetti mouse embryos were exposed to 0.5 mg 4OHT intraperitoneally delivered into pregnant females to label single E7.5 Sox2 <sup +</sup neural ectoderm progenitors with one of three detectable fluorescent colors red, cyan, or yellow . Subsequently, E9.5 embryos were immunostained to visualize neural ectoderm-derived cell clusters and where they resided in relation to the forebrain, midbrain, and hindbrain. The forebrain/midbrain domain was molecularly determined by Otx2 immunostaining. This is a higher magnification image of an embryo shown in Extended Data Fig. 4c https://www.nature.com/articles/s41593-026-02433-7 Fig10 , which was taken from 1 experiment. Asterisks indicate autofluorescence from red blood cells RBCs . Scale bar = 100 μm. Extended Data Fig. 5 Differentiation of hPSCs into forebrain or midbrain progenitors. https://www.nature.com/articles/s41593-026-02433-7/figures/11 a qPCR of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with the following WNT pathway modulators for 48 hours: WNT inhibitor XAV939, 1 μM , control media no WNT modulator, depicted as “-” , RSPO2 25 nM , RSPO2 + WNT3A increasing concentrations of 10, 100 or 1000 ng/mL , RSPO2 + NGS WNT-Fc increasing concentrations of 0.03, 0.3 or 3 nM , or CHIR99021 increasing concentrations of 0.75, 1.5 or 3 μM . For WNT3A or NGS WNT-Fc treatment conditions, RSPO2 was added to increase cell-surface levels of FRIZZLED receptors, thereby enhancing cellular responsiveness to these WNT pathway agonists<sup 112 https://www.nature.com/articles/s41593-026-02433-7 ref-CR112 </sup . NGS-WNT-Fc refers to an Fc-tagged synthetic WNT pathway agonist that acts by heterodimerizing FZD and LRP6 receptors<sup 112 https://www.nature.com/articles/s41593-026-02433-7 ref-CR112 </sup . Heatmap depicts row Z-score, calculated across the 12 treatment conditions from the mean normalized expression of 2 biological replicates from 1 experiment. b In situ staining of an E8.5 Tcf/Lef:H2B:Venus reporter mouse embryo<sup 82 https://www.nature.com/articles/s41593-026-02433-7 ref-CR82 </sup for Foxg1 forebrain marker and Venus indicating WNT transcriptional response . This disclosed a posteriorizing gradient of WNT signaling, with minimal WNT signaling in Foxg1 <sup +</sup forebrain, but high WNT signaling in the midbrain. Representative image from 15 embryos from 2 experiments. Extended Data Fig. 6 Lineage commitment of hPSC-derived anterior and posterior neural ectoderm. https://www.nature.com/articles/s41593-026-02433-7/figures/12 a scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk indicates that the legend is identical to that shown in Fig. 4c https://www.nature.com/articles/s41593-026-02433-7 Fig4 , and is reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type. b H1 hPSCs were differentiated into definitive ectoderm for 24 hours, and then subsequently differentiated into either anterior neural ectoderm aNE or posterior neural ectoderm pNE within an additional 24 hours. hPSC-derived aNE and pNE cells were then dissociated and then replated in either forebrain-, midbrain-, or hindbrain-inducing signals for 48 hours, and then immunostaining was performed. Scale bar = 50 μm. Representative images from 4 biological replicates from 4 experiments. Extended Data Fig. 7 Accessible chromatin landscapes and lineage commitment of hPSC-derived anterior vs. posterior neural ectoderm. https://www.nature.com/articles/s41593-026-02433-7/figures/13 a H1 hPSCs were differentiated into either anterior or posterior neural ectoderm for 2 days. They were then respectively challenged with either forebrain-, midbrain-, or hindbrain-inducing signals for 4 days. Immunostaining was performed on day 6 of differentiation. Scale = 100 μm. Representative images from 2 biological replicates from 1 experiment. b H1 hPSCs were differentiated into either aNE or pNE within 2 days; pNE was fluorescently labeled, and then aNE uncolored and pNE dye-labeled were mixed. Cocultures were treated with either forebrain-, midbrain-, or hindbrain-inducing signals for 4 days, prior to immunostaining that is, immunostaining was performed on day 6 of hPSC differentiation . Quantification indicates the percentage of pNE cells that is, dye-labeled that express a given marker protein. aNE: day-2 anterior neural ectoderm. pNE: day-2 posterior neural ectoderm. Representative images from 4 biological replicates from 2 experiments. c OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE. A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types false discovery rate FDR < 0.05 . Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. d OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE that were challenged with forebrain-inducing signals for 2 additional days 1 day of XAV939 1 μM + LDN193189 100 nM + FGF2 20 ng/mL , followed by 1 day of XAV939 1 μM and LDN193189 100 nM . A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types FDR < 0.05 . Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. e OmniATACseq and RNAseq of hPSC-derived aNE and pNE that were treated with either forebrain- or hindbrain-inducing signals for 48 hours. Data from 3 biological replicates from 1 experiment. f OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE that were challenged with hindbrain-inducing signals CHIR99021 750 nM + FGF2 20 ng/mL for 2 additional days. A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types FDR < 0.05 . Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. g Single-cell RNA-sequencing of H7 hPSCs differentiated into day-1 definitive ectoderm, day-2 anterior neural ectoderm, day-2 posterior neural ectoderm, day-2 border ectoderm, or day-2 surface ectoderm. Colors denote differentiation conditions. Fzd5 , Lhx5 , and Fezf1 have previously been associated with anterior neural identity in vivo at later developmental stages<sup 113 ref-CR113 , 114 ref-CR114 , 115 https://www.nature.com/articles/s41593-026-02433-7 ref-CR115 </sup , whereas Meis2 has been previously associated with posterior neural identity in vivo <sup 116 https://www.nature.com/articles/s41593-026-02433-7 ref-CR116 </sup . Data from 1 biological replicate per cell-type. Extended Data Fig. 8 Differentiation of hPSCs into dorsal forebrain and ventral forebrain progenitors within 7 days. https://www.nature.com/articles/s41593-026-02433-7/figures/14 scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk indicates that the legend is identical to that shown in Fig. 4c https://www.nature.com/articles/s41593-026-02433-7 Fig4 , and is reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type. Extended Data Fig. 9 Differentiation of hPSCs into dorsal and ventral forebrain progenitors, and subsequently, dorsal and ventral hindbrain neurons. https://www.nature.com/articles/s41593-026-02433-7/figures/15 a scRNAseq of H7 hPSC-derived dorsal forebrain, ventral forebrain, dorsal hindbrain and ventral hindbrain populations on day 14 of differentiation. An asterisk indicates images identical to those shown in Fig. 5d https://www.nature.com/articles/s41593-026-02433-7 Fig5 , which were reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type. b scRNAseq of H7 hPSC-derived dorsal forebrain populations on day 14 of differentiation reveals expression of Cajal-Retzius neuron markers<sup 117 https://www.nature.com/articles/s41593-026-02433-7 ref-CR117 </sup . Data from 1 biological replicate per cell-type. c scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation reveals expression of cortical interneuron markers. Data from 1 biological replicate per cell-type. d scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation reveals expression of hypothalamus-associated markers. hPSC-derived groups I and II express PROX1 ref. <sup 118 https://www.nature.com/articles/s41593-026-02433-7 ref-CR118 </sup , LHX5 ref. <sup 119 https://www.nature.com/articles/s41593-026-02433-7 ref-CR119 </sup , and ADCYAP1 ref. <sup 120 https://www.nature.com/articles/s41593-026-02433-7 ref-CR120 , 121 https://www.nature.com/articles/s41593-026-02433-7 ref-CR121 </sup , which are expressed in vivo by various regions of the hypothalamus, including the preoptic area, paraventricular nucleus, and supraoptic nucleus. hPSC-derived group I is additionally defined by OTP , the archetypic transcription factor required for the development of paraventricular and supraoptic nuclei<sup 122 https://www.nature.com/articles/s41593-026-02433-7 ref-CR122 </sup . Instead, hPSC-derived group II is additionally defined by POU4F1 BRN3A and EBF1 , and may correspond to an early Pou4f1 + Ebf1 + Pomc - hypothalamic lineage in vivo <sup 123 https://www.nature.com/articles/s41593-026-02433-7 ref-CR123 </sup . hPSC-derived group II also expresses ADCYAP1R1 the ADYCAP1 receptor , which is expressed by multiple brain regions, including the preoptic area, paraventricular nucleus, and supraoptic nucleus<sup 124 https://www.nature.com/articles/s41593-026-02433-7 ref-CR124 </sup . Finally, hPSC-derived group III is demarcated by ISL1 and POMC , which are expressed within the arcuate nucleus of the hypothalamus<sup 64 https://www.nature.com/articles/s41593-026-02433-7 ref-CR64 </sup . Data from 1 biological replicate per cell-type. e scRNAseq of H7 hPSC-derived ventral hindbrain populations on day 14 of differentiation reveals expression of multiple ventral hindbrain neuron subtype markers left , whose identities were extrapolated from ventral spinal cord neuron subtype markers<sup 125 https://www.nature.com/articles/s41593-026-02433-7 ref-CR125 , 126 https://www.nature.com/articles/s41593-026-02433-7 ref-CR126 </sup . Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies right <sup 125 https://www.nature.com/articles/s41593-026-02433-7 ref-CR125 </sup . The box reflects the neuron subtypes putatively produced in our in vitro differentiation system. Data from 1 biological replicate per cell-type. f scRNAseq of H7 hPSC-derived dorsal hindbrain populations on day 14 of differentiation reveals expression of multiple dorsal hindbrain neuron subtype markers left . Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies right . The box reflects the neuron subtypes putatively produced in our in vitro differentiation system. Data from 1 biological replicate per cell-type. Extended Data Fig. 10 Functional assessment of hPSC-derived dorsal and ventral hindbrain neurons. https://www.nature.com/articles/s41593-026-02433-7/figures/16 a Day 28 H1 hPSC-derived ventral hindbrain neurons transduced with AAVdj- CAG-bReaChES-eYFP were stimulated with either prolonged 1 second , or pulses of 5 ms, 5 Hz , 560 nm light, and electrophysiological recording was performed to detect action potentials. Representative data from 4 cells from 1 experiment. b Electrophysiological activity of H1 hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing voltage-dependent K<sup +</sup left or Na<sup +</sup right channel currents elicited by depolarization of the holding potential from -90 mV to 50 mV in voltage-clamp mode. Representative data from 4 cells in 1 experiment. c Current-clamp characterization of H1 hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing action potentials elicited by injection of pulsed 500 pA, 5 ms, 10 Hz, left or prolonged 50 pA, 1.5 seconds, right currents in current-clamp mode. Representative data from 4 cells in 1 experiment. d Live Ca<sup 2+</sup imaging of WTC11 AAVS1-CAG-GCaMP6f hPSCs differentiated into dorsal hindbrain neurons for 30 days, which exhibited spontaneous Ca<sup 2+</sup transients as assessed within individual cells left and across the culture right . Scale bar = 50 μm. Representative data from 7 single cells in 1 experiment. Supplementary information Supplementary Information download PDF https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM1 ESM.pdf Supplementary Figs. 1–10 and Representative flow cytometry gating schema. Supplementary Tables 1 and 2 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM3 ESM.xlsx Supplementary Table 1 a list of experimental reagents used in this study and Table 2 a list of qPCR primer sequences used in this study . Source data Source Data Fig. 1 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM4 ESM.xlsx qPCR datapoints for Fig. 1. Source Data Fig. 2 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM5 ESM.xlsx qPCR datapoints for Fig. 2. Source Data Fig. 3 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM6 ESM.xlsx qPCR datapoints for Fig. 3. Source Data Extended Data Fig. 1 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM7 ESM.xlsx Flow cytometry datapoints for Extended Data Fig. 1. Source Data Extended Data Fig. 2 download PDF https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM8 ESM.pdf Raw western blot gels for extended Data Fig. 2. Source Data Extended Data Fig. 5 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM9 ESM.xlsx Flow cytometry datapoints for Extended Data Fig. 5. Source Data Extended Data Fig. 7 download XLSX https://media.springernature.com/original/springer-static/esm/art%3A10.1038%2Fs41593-026-02433-7/MediaObjects/41593 2026 2433 MOESM10 ESM.xlsx Differentially accessible genomic regions for Extended Data Fig. 7. A list of genomic regions with differential accessibility in OmniATACseq datasets obtained from hPS cell-derived aNE, hPS cell-derived pNE, and aNE and pNE populations challenged with either forebrain telencephalon -inducing signals or hindbrain-inducing signals. Rights and permissions Springer Nature or its licensor e.g. a society or other partner holds exclusive rights to this article under a publishing agreement with the author s or other rightsholder s ; author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Jokhai, R.T., Dundes, C.E., Ahsan, H.S. et al. Two parallel neural ectoderm progenitors contribute to the developing brain. Nat Neurosci 2026 . https://doi.org/10.1038/s41593-026-02433-7 - Received: - Accepted: - Published: - Version of record: - DOI: https://doi.org/10.1038/s41593-026-02433-7