{"paper_id":"31c9f2c5-18a2-453e-b601-0b5d6be95a4d","body_text":"1 \nHuman trunk embryoids with patterned anterior-posterior and dorsal-ventral body 1 \naxes: utility for understanding human development and disease 2 \nTianming Wu1*, Hao Yu1, Brian S.H. Wong1, Kexin Teng1, Weiman Xiang1, Ling Xu1,2, 3 \nJianan Zhang1, Angel Y.F. Kam1, Ethel S.K. Ng1, Joaquim Vong1, Jiannan Zhang3, Bo 4 \nGao1, Stephen K.W. Tsui1, Stephen Dalton1* 5 \n1. School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, 6 \nHong Kong SAR, China 7 \n2. School of Biomedical Sciences, The University of Hong Kong, Hong Kong SAR, 8 \nChina 9 \n3. Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, 10 \nCollege of Life Sciences, Sichuan University, Chengdu, China 11 \n*Correspondences: sdalton@cuhk.edu.hk and tianmingwu@cuhk.edu.hk 12 \n 13 \nSummary 14 \nHuman embryoid models enable mechanistic studies of development and disease. We 15 \ngenerated trunk embryoids from human pluripotent stem cells that recapitulate posterior 16 \ntrunk formation at Carnegie stage (CS) 8-10, with patterned anterior-posterior (A-P) and 17 \ndorsal-ventral (D-V) axes. These self-organizing structures comprise a ventral 18 \nnotochord, dorsal neural tube, floor plate and bilateral somites. Genetic and chemical 19 \nperturbations of SHH signaling confirmed the notochord’s central role in D-V patterning. 20 \nMoreover, VANGL1/2 loss-of-function mutations recapitulated mouse phenotypes, 21 \nincluding axial truncation and somite segmentation failure. This model enables detailed 22 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 2 \nstudy of key developmental events that underlie posterior trunk formation and provides 23 \na promising platform for human disease modeling. 24 \n 25 \nKeywords: pluripotent stem cells, embryoid, bipotent NMPs, notochord, A-P and D-V 26 \naxes, human trunk development 27 \n 28 \nIntroduction 29 \nUnderstanding the molecular and cellular aspects of peri- and post-implantation human 30 \nembryogenesis are fundamental for a better understanding of congenital disease, the 31 \napplication of human pluripotent stem cell (hPSC) towards regenerative medicine and 32 \npotentially, drug validation. Developing hPSC-derived embryoid models offers 33 \nopportunities to address these issues.1-3 Although success has been achieved in 34 \ndeveloping mouse embryoids that exhibit multi-axial and multi-tissue patterning along 35 \nthe anterior-posterior (A-P), dorsal-ventral (D-V) and left-right (L-R) body axes,4-6 36 \nattempts to generate multi-axial human trunk models suffer from several limitations. 37 \nInitial human trunk models using bipotent neuromesoderm progenitors (bi-NMPs) are 38 \nuni-axial and comprised of somite-only7-9 or neural tube-only10 structures. Recently 39 \ndeveloped coupled models11-13 are composed of a neural tube and somites but, are 40 \noften aberrantly structured (e.g. unilateral somites and twisted neural tube) and fail to 41 \nestablish a D-V body axis. Notably, these models lack a notochord and are heavily 42 \nbiased towards dorsal patterning.12,13 In contrast, trunk models that form a notochord-43 \nlike structure are ventrally-biased and lack a neural tube and somites.14 These 44 \nlimitations highlight the need for a human model that supports co-development of the 45 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 3 \nnotochord alongside bi-NMPs, together with the establishment of the A-P and D-V body 46 \naxes. 47 \n  Temporal signaling by WNT, NODAL, BMP, retinoic acid (RA), FGF and SHH specifies 48 \ntailbud bi-NMPs and notochord progenitor cells along the A-P and D-V axes.14-18 A 49 \nmajor challenge is to establish a concerted signaling environment that supports these 50 \nco-developmental events. We iteratively optimized conditions to generate human trunk 51 \nembryoid models (hTEMs) from bi-NMPs and notochord progenitors. hTEMs self-52 \norganize into notochord, dorsal neural tube, floor plate, and bilateral somites. Validation 53 \nof this was based on morphological, cellular and molecular criteria using spatial and 54 \nsingle-cell transcriptomics, live imaging and scanning electron microscopy (SEM). 55 \nComparative analyses showed that hTEMs recapitulate posterior trunk development 56 \nequivalent to that in human embryos at Carnegie stages (CS) 8-10. 57 \n  The utility of hTEMs for modeling human development was investigated by perturbing 58 \nnotochord identity and notochord-derived SHH activity. By this approach, the notochord 59 \nwas shown to be a major driving force for D-V axis establishment in the human trunk. 60 \nMoreover, hTEMs faithfully reproduced human neural tube defects (NTDs), as shown by 61 \nloss-of-function mutations in the planar cell polarity (PCP) genes, VANGL1 and 62 \nVANGL2. These findings highlight the versatility of trunk embryoids for understanding 63 \nhuman development and congenital disease and points towards additional applications 64 \nfor drug discovery and regenerative medicine.  65 \n 66 \nResults and Discussion 67 \nDorsally biased neural tube-somite coupled embryoids 68 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 4 \nWe first aimed to generate topographically coupled embryoids with A-P segmented 69 \nsomites flanking an elongating neural tube (hTEM.v1; Figures 1A and 1B). To achieve 70 \nthis, size-controlled 3D human embryonic stem cell (hESC) spheroids received WNT 71 \nagonist CHIR99021 (CHIR), FGF2, and inhibitors for BMP (LDN-193189 (LDN)) and 72 \nNODAL (SB-431542 (SB)) pathways. Resultantly, hTEM.v1 embryoids became 73 \nasymmetric and by day4, underwent mediolateral narrowing and axial elongation 74 \n(Figures 1B and S1A). Matrigel and retinal (RAL) promoted axial elongation and somite 75 \nsegmentations  at days 4-7 (Figures 1C, S1B, and S1C).8 6-8 pairs of bilaterally 76 \npositioned somites progressively formed along with gradual extension and closure of 77 \nthe neural tube (Figures 1D, S1D, and Video S1). By day 7, hTEM.v1 reached a mean 78 \nlength of 1857 μm (Figure 1C).  79 \n  Immunostaining of hTEM.v1 (days 2-4) revealed the following features indicative of  A-80 \nP patterning; (i) polarized expression of TBXT and CDX2 at the posterior end, (ii) 81 \nmutually exclusive positioning of central neural (SOX2) and mediolateral pre-somitic 82 \nmesoderm (PSM; TBX6) cells; (iii) A-P symmetry breaking, indicated by anterior somitic 83 \n(SIX1) cells undergoing an epithelial-to-mesenchymal transition (EMT; N-cadherin) and 84 \ncaudalized tailbud (CDX2) cells (Figures 1E, S1E-S1G). In total, these characteristics 85 \nare reminiscent of the highly mitotic (pH3-Ser10), A-P patterned human CS8 embryo 86 \n(Figure 1E).19,20 Upon further inspection of hTEM.v1 (days 4-7), SOX2+TBXT- neural 87 \ntubes were observed on both dorsal and ventral sides along the A-P axis (Figures 1F-88 \n1H and S1H). This is coincident with the absence of a SOX2-TBXT+ notochord 89 \nstructure (Figures 1E and S1I). This suggests that although the A-P axis had formed, D-90 \nV patterning in hTEM.v1 was not established. 91 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 5 \n  To understand D-V patterning defects in hTEM.v1, single-cell RNA sequencing 92 \n(scRNA-seq) analysis was performed (days 4/5/7). Clustering and hierarchical 93 \ntranscriptomic profiling identified 12 major cell identities, including two major continuums 94 \ndelineating somitogenesis and neural tube formation (Figures 1I, 1J, S1J, and S1K); (i) 95 \nthe somitic lineage contains NMP-Meso (TBXT, MSGN1), posterior PSM (HES7, TBX6), 96 \nanterior PSM (MESP2, RIPPLY2) and pan-somite cells (PAX3, SIX1, comprised of 97 \nearly-, middle- and late-somite (E-, M- and L-somite) subtypes along the time course; (ii) 98 \nthe neural lineage includes NMP-Neural (SOX2, NKX1-2), caudal neural plate 99 \nprogenitors (Caud. NP; MSX2, SOX2, NKX1-2) and neural tube cells (PAX6, HES5). 100 \nOverall, due to the absence of ventral somite compartments (PAX1, PAX9) or ventral 101 \nneural tube (NKX6-1) and floor plate (FOXA2), the spatiotemporal expression profile of 102 \nhTEM.v1 confirmed dorsally-biased cell identities in both the somitic and neural 103 \nlineages (Figure 1K). Immunostaining of transversely sectioned day-7 hTEM.v1 further 104 \nconfirmed the unrestricted distribution of dorsal BMP4/7 signals and over-expansion of 105 \ndorsal neural (PAX6) cells, with few to no ventral neural (NKX6-1) cells in the neural 106 \ntube region (Figure 1L). 107 \n  Conventionally, bi-NMPs are identified by the co-expression of SOX2 and TBXT,21-23 108 \nbut questions about their molecular identities remain unresolved.24-26 Here, we identified 109 \ndistinct NMP-Neural (SOX2high,TBXTlow) and NMP-Meso (SOX2low,TBXThigh) subtypes 110 \nthat exhibited contrasting expression patterns (Figures 1J and 1M). Additionally, they 111 \nexpressed differential levels of FGF3/4/8/17, WNT3A/5A/5B/8A, and BM2/4/7 (Figure 112 \nS1L), which are believed to be the driving force for bi-NMP fate bifurcations in human 113 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 6 \ntrunk formation.27 This suggests hTEM.v1’s potential to resolve the plasticity of bi-NMP 114 \nbifurcation. 115 \n  Then, we asked why notochord was absent from hTEM.v1. scRNA-seq combined with 116 \nin situ hybridization chain reaction (HCR) only detected few cells expressing notochord 117 \nmarkers (TBXT, NOTO, CHRD, SHH), possibly representing the ventral node (SHH) 118 \nsurrounded by PSM (TBX6, HES7) (Figures 1J, S1M, and S1N).28 In human embryos, 119 \ncorrect D-V axis specification is initiated by SOX2high/TBXTlow in the dorsal neural plate 120 \nand SOX2low/TBXThigh in the ventral notochordal plate.20,27 However, in day-4 hTEM.v1, 121 \nTBXT protein was restricted to the tailbud and was undetectable by day 7, while SOX2 122 \nexpression extended to both dorsal and ventral tube structures (Figures 1E, 1G, and 123 \nS1I).  The SOX2 over-expansion and neural tube duplication phenotype in hTEM.v1 124 \ncoincides with previously reported NTDs caused by mutations/misregulation of Tbxt in 125 \nmouse notochord progenitors.29,30 We, therefore, attributed the neural tube duplication 126 \nfrom hTEM.v1 to the misregulation of TBXT and consequently, the inability to specify 127 \nnotochord cells.  128 \n  Overall, bi-NMP-derived hTEM.v1 with spatially coupled neural tube structures and 129 \nflanking somite segments were generated. Notochord is well-known for its role in D-V 130 \npatterning of neural tube and somitic cells in the embryo.31-33 The absence of a 131 \nnotochord and SHH signaling along the A-P axis in hTEM.v1 explains the heavy 132 \ndorsalization observed. Moreover, it is noteworthy that we observed a subset of somitic 133 \ncells resembling the endotome (EBF2) (Figure 1J), a poorly understood somite 134 \ncompartment that contributes to the dorsal aorta, endothelium and hematopoietic stem 135 \ncells.13,34,35 The anteriorly positioned EBF2:mScarlet signal coincided with vascular 136 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 7 \nendothelial cells (SOX17, VE-cadherin) at the somite periphery (Figure S1O and Video 137 \nS1). These findings indicate that hTEM-based embryoids have utility for studying more 138 \nadvanced events related to later stages of gastrulation in humans, such as 139 \nsomitogenesis, neural tube morphogenesis and somite-derived vasculogenesis.27  140 \n 141 \nExogenous SHH activation induces ventral fates in trunk embryoids 142 \nAs notochord-derived SHH signaling is key to ventral patterning,33 we postulated that 143 \nSHH activation could establish a D-V axis and rescue the dorsal-biased defects in 144 \nhTEM.v1. To test this, day-4 hTEM.v1 embryoids were exposed to varied durations and 145 \nconcentrations of Smoothened agonist (SAG) (Figure S2A). SAG-treated hTEM.v1 146 \nexhibited elevated transcripts (e.g., ventral neural tube; NKX6-1. Sclerotome; PAX1/9) 147 \nof ventral identities by day 7, in a dose- and time-dependent manner (Figure S2B). A 148 \npulse of 100 nM SAG was chosen to generate hTEM.v2, because this condition 149 \nestablished D-V patterning in neural and somitic cells without affecting morphology or 150 \naxial elongation (Figures 2A-2D and S2B). Immunostaining of day-7 hTEM.v2 confirmed 151 \nthe emergence of ventral (NKX6-1) neural cells in both neural tubes along the A-P axis 152 \n(Figure 2E). Notably, neural tube duplication was not rescued in hTEM.v2 (Figure 2E). 153 \n  Through scRNA-seq analysis, we observed distinct ventral cell identities in hTEM.v2, 154 \nincluding ventral neural tube (NKX6-1, NKX2-8), floor plate (FOXA2), ventral somite 155 \n(SNAI2, TWIST1), syndetome (SCX), and sclerotome (PAX1/9, SOX9), alongside 156 \ndorsal (PAX6) neural and dorsal (PAX3) somite cells (Figures 2F-2H, S2C, and S2D). 157 \nHowever, notochord (NOTO, CHRD) cells remained limited in number (Figure 2G). This 158 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 8 \nconfirmed that dorsally-biased patterning in hTEM.v1 was switched to a D-V balanced 159 \npatterning in hTEM.v2, due to SAG-induced SHH activity. 160 \n  Attention was then turned to evaluate bi-axial patterning in hTEM.v2. Using Visium HD 161 \ntechnology, transverse and longitudinal sections of day-7 hTEM.v2 spanning the A-P 162 \naxis were acquired (Figures 2I, 2J, and S2E-S2H). To validate A-P axis formation, 163 \nspatially resolved developmental events were assessed, including symmetry breaking, 164 \nsomite segmentation and inter-tissue RA signalling crosstalk. As seen in human 165 \nembryos, the tailbud-to-hindbrain A-P patterning was evident by tailbud cells expressing 166 \nCDX2 and anterior neural cells expressing CRABP1 (Figure 2I).36 The somite 167 \ndetermination front was marked by co-expression of RIPPLY1 and LFNG in the PSM 168 \nregion (Figure 2I).37 Mutually expressed TBX18 (rostral) and UNCX (caudal) were 169 \nobserved (Figure S2I), indicative of somite segmentations.38,39 ALDH1A2 (RA synthesis) 170 \nexpression in somite and RARB (RA effector) expression in neural tube cells were 171 \nsuggestive of somite-neural tube crosstalk along the A-P axis (Figure 2I).40 The anterior 172 \nexpression of ALDH1A2 was opposed to posterior CYP26A1 (RA degradation) (Figure 173 \n2I), consistent with “source and sink” RA signaling patterns that are fundamental to A-P 174 \naxis establishment in chick and mouse embryos.41,42 As seen in previous scRNA-seq 175 \nresults (Figure 2G), few node-like (NOTO, FOXJ1) cells were found adjacent to bi-176 \nNMPs in the tailbud (SOX2, TBXT) (Figure 2I). Moreover, caudal expression of HOXC9 177 \nand anterior HOXC4 were noted (Figure 2I), indicating the presence of an emerging 178 \nHOX code.43 179 \n  Next, D-V patterning of neural tube structures in transversely sectioned hTEM.v2 was 180 \nconfirmed by visualizing the spatially restricted expression of roof plate (WNT1, MSX1), 181 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 9 \ndorsal neural tube (PAX6, DBX2) and ventral neural tube (OLIG2, NKX6-1) markers 182 \n(Figure 2J). Likewise, dorsal somite (PAX3, RDH10)44 and ventral somite markers 183 \n(TWIST1, COL1A1) exhibited a dorsolateral-ventromedial pattern within the bilateral 184 \nsomites (Figure 2J). Of note, endotome (EBF2) and endothelial cells (KDR) were found 185 \nin the lateral somite compartment, as observed in hTEM.v1 (Figures 2J and S1O). 186 \nThese results confirmed the embryo-like cell-cell organization along the D-V axis for 187 \nneural and somitic lineages (Figures 2K-2N). Important signals, including WNTs 188 \n(WNT1/3/3A/4), BMPs (BMP4/7), PDGF (PDGFA) and heregulin (NRG1) displayed 189 \ndorsally enriched expression patterns in hTEM.v2 (Figure 2M), similar to that observed 190 \nin the neural tube in vivo.45-48 However, the floor plate (FOXA1/2, SHH) identity was 191 \nunder-represented (Figures 2H and 2M), suggesting an incomplete D-V axis 192 \nestablishment in hTEM.v2. Although WNTs were expressed in hTEM.v2 and are known 193 \nto induce myogenesis in vivo and in vitro,46,49 the excessive expression of FRZB (water-194 \nsoluble WNT antagonist) from the ventral somite cells accounts for the absence of 195 \ndermomyotome and myogenic populations (Figures 2H, 2J, and 2N). 196 \n  Altogether, in the absence of a notochord, exogenous SHH activation established only 197 \na limited D-V axis in hTEM.v2 and failed to rescue neural tube duplication. These 198 \nobservations emphasize the critical role of the notochord in trunk development and for 199 \ncorrect D-V patterning in somitic and neural lineages.50,51 To establish a bi-axial 200 \nembryoid comparable to the posterior trunk in human embryos, the next challenge was 201 \nto establish suitable culture conditions that support co-development of notochord 202 \nprogenitor cells with bi-NMP descendants. 203 \n 204 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 10 \nCo-development of notochord, neural tube and bilateral somites  205 \nDuring gastrulation, the coordinated emergence of notochord with bi-NMPs depends on 206 \nsustained WNT signaling and temporal modulation of NODAL and BMP signaling 207 \nactivity.14,15,52 Following initial WNT activation in the anterior primitive streak (APS), 208 \nnotochord induction coincides with the activation of a NODAL autoregulatory loop, 209 \nincluding CER1 and/or LEFTY2.15,53 This precise modulation was not achieved in 210 \nhTEM.v1/2, where broad and persistent NODAL inhibition by SB disrupted the APS-211 \nderived notochord process. Notably, CER1 expression precedes notochord formation 212 \nand localizes to notochord adjacent APS, definitive endoderm (DE), visceral endoderm 213 \n(VE) and axial progenitor populations in gastrulating mouse54 and human20 embryos 214 \n(Figures S2J-S2L). Furthermore, the co-development of notochord and bi-NMPs are 215 \ninvolved in initiation of D-V axis establishment, which depends on opposing signals of 216 \nSHH and BMP2/4/7 that are also active within these early populations (Figures S2J–217 \nS2L).55,56  218 \n  Since these critical signals were not intact in hTEM.v1 (Figure S1L), we hypothesized 219 \nthat its notochord deficiency resulted from; (i) disruption of APS-derived notochord 220 \ninduction by early and persistent NODAL inhibition (SB); (ii) absence of opposing SHH 221 \nand BMP signals necessary for D-V patterning and notochord/bi-NMP specification.  222 \n  To test this hypothesis, we replaced SB with recombinant human CER1 for the first 24 223 \nhours of bi-NMP induction (Figure 3A). By day 2, it was evident that temporal NODAL 224 \nmodulation by CER1 followed by SB supported the balanced co-emergence of APS 225 \n(OTX2, EOMES), early notochord (NOTO, FOXA2), NMP-Meso (TBXT, TBX6) and 226 \nNMP-Neural (SOX2, NKX1-2) progenitors (Figures S3A and S3B). Further interrogation 227 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 11 \nof transcriptomic profiles in gastrulating mouse and human embryos revealed an array 228 \nof signaling pathways with synergistic and opposing activity including pathways of 229 \nSHH55,56, BMPs47,55-58, FGFs12,57,59-62 , and RA13,16,58,60 within notochord-adjacent 230 \npopulations, such as APS, DE, VE and axial progenitors (Figures S2J–S2L). Through 231 \nempirical testing, we established a cocktail comprised of SHH, BMP2/4/7 at days 2-4, 232 \nplus temporal FGF2/3/4/8b/17 and RA at day 2-3 (Figures 3A and S3C), which 233 \nsupported the coordinated progress of notochord (NOTO), somitic (TBX6) and neural 234 \n(NKX1-2) lineages to day 4 (Figure S3D). At day 7, markers (SHH, FOXA2) for 235 \nnotochord and floor plate were highly expressed in these embryoids in contrast to 236 \nhTEM.v1/2 (Figure S3E). Day-7 embryoids elongated to ~2 mm in length and formed 6–237 \n8 pairs of somites flanking a midline neural tube (Figures 3B, 3C, and S3G). We refer to 238 \nthese embryoids as hTEM.v3. 239 \n 240 \nMorphological characteristics of hTEM.v3 241 \nUsing a NOTO:mClover3 H9-hESC line, time-lapse imaging captured notochord 242 \nmorphogenesis in hTEM.v3 at days 3-4. This began with a salt & pepper pattern of 243 \nNOTO:mClover3 expression followed by axial elongation of NOTO expressing cells, 244 \nnear the caudal end (Figure S3H). This aligns with in vivo observations that Noto is 245 \nexpressed in the node and nascent notochord in mice.63 Meanwhile, NKX1-2:mScarlet+ 246 \ncells were progressively enriched along the midline of hTEM.v3, indicative of caudal 247 \nneural plate morphogenesis (Figure S3I).  248 \n 249 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 12 \n  In day-4 hTEM.v3, distinct FOXA2+ notochord formed along the midline, flanked by 250 \ncaudal PSM (TBX6) cells (Figure 3D). Immunostaining-HCR results further confirmed 251 \nthe embryo-like arrangement of a dorsally-localized caudal neural plate (SOX2, NKX1-252 \n2) and a ventrally localized notochord (transcripts of NOTO, CHRD SHH and proteins of 253 \nNOTO:mClover3, TBXT, FOXJ1, FOXA2) in hTEM.v3 (Figures 3E, 3F, S3I, and S3J). 254 \nThese features closely resemble the gastrulating human CS8-9 embryos.20,27 After day 255 \n4, hTEM.v3 exhibited embryo-like D-V arrangements of the neural tube, notochord and 256 \nbilateral somites. The emergence of a ventral notochord coincided with rescue of the 257 \nneural tube duplication defect seen in hTEM.v1/2 (Figure 3G).  258 \n  In mice, the node is located at the anterior tip of APS and is composed of columnar 259 \nepithelial cells in the dorsal region and teardrop-shape, ciliated cells in the ventral part.64 260 \nSEM imaging of transversely fractured day-4 hTEM.v3 revealed a group of ciliated, 261 \nsquamous cells on the ventral side near the posterior end, resembling the ventral node 262 \nstructure in embryos (Figure S3K). Confocal imaging further confirmed ciliated cells 263 \n(FOXJ1, ARL13B) residing in the presumptive node (TBXT) region (Video S2). At day 264 \n5.5, SEM of hTEM.v3 revealed a dorsally positioned lumen representing the neural 265 \ntube, a vacuolated and ventrally localized notochord and bilateral rosette patterns 266 \nformed by cells in the somites (Figure 3H). Consistent with observations in frog, rabbit 267 \nand chick65-67, the inner canal of hTEM.v3 notochord contains lipid droplets of varying 268 \nsizes, while the outer layer is rich in extracellular matrix (ECM) fibers, making notochord 269 \ncells structurally distinguishable from neural tube cells. Notochord cells in hTEM.v3 270 \nwere flattened, vacuolated and larger than neural tube cells, indicative of changes in the 271 \nnature of the cytoplasm during notochord maturation. This is reminiscent of 272 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 13 \nmorphological features of the chicken notochord at Hamburger & Hamilton stage 14.66 273 \nThe structural similarities between notochord structures in hTEM.v3 and frog, rabbit, 274 \nchick and mouse embryos, signifies the conservation of notochord development across 275 \nvertebrate species. It also validates the hTEM.v3 as a legitimate model for human trunk 276 \ndevelopment. 277 \n 278 \nCellular composition of hTEM.v3 279 \nscRNA-seq integration and clustering analysis of hTEM.v3 (days 3-7) identified 29 280 \nmajor cell types (Figures S3L and S3M). RNA velocity analysis further unveiled intricate 281 \ndevelopmental trajectories of the tailbud (CDX2, CYP26A1) stemming from notochord 282 \n(TBXThighSOX2low, NOTO, SHH), NMP-Meso (TBXThighSOX2low, MSGN1) and NMP-283 \nNeural (TBXTlowSOX2high, NKX1-2) (Figure 3I and 3J).20,27 Bi-NMPs diverge into two 284 \nstreams, including; (i) NMP-Meso → posterior PSM → anterior PSM → E-/M-Somite 285 \n(pan-somite) → L-Somite (somite compartments comprised of sclerotome, syndetome, 286 \nendotome, myogenic progenitors, and dermomyotome) and; (ii) NMP-Neural → Caud. 287 \nNP → E-Neural tube and E-Floor plate → Neural tube and Floor plate (Figures 3I and 288 \n3K). In contrast to hTEM.v2, distinct myogenic progenitors (PAX7), dermomyotome 289 \n(MYF5, MYF6) and floor plate (FOXA1/2, NKX2-8) cells were observed in hTEM.v3, 290 \nhighlighting the importance of notochord for control of cell fate specification along the D-291 \nV axis.  292 \n  To better understand human notochord development, sub-clustering, RNA velocity and 293 \npseudotime analyses were conducted on the ‘notochord’ subset from hTEM.v3 (Figures 294 \nS3N-S3P). Four cell subtypes delineating notochord maturation were identified (Figures 295 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 14 \nS3Q and S3R). In line with APS-derived notochord processes,15,17 the first subtype 296 \ndesignated as ‘node or notochord progenitors’ was marked by NODAL, WNT3A, 297 \nCYP26A1 and CDX2. The second and third subtypes were both marked by NOTO, 298 \nequivalent to newly generated notochord cells in vivo.63 The second subtype was 299 \nenriched for ‘nascent notochord’ (FOXA2, CHRD, SHH) markers,20 while the third 300 \nsubtype was signified by ‘ciliated notochord’ (FOXJ1, RFX2, TCTEX1D1) markers.68 In 301 \nthe fourth ‘mature notochord’ subtype, NOTO transcript was decreased associated with 302 \nupregulation of FOXA1, SOX9, NOG and SEMA3C.14 hTEM.v3 is therefore a platform 303 \non which the detailed processes of human notochord development and function can be 304 \nexplored.  305 \n 306 \nhTEM.v3 recapitulates key aspects of the trunk A-P axis  307 \nTo assess whether hTEM.v3 could model aspects of in vivo A-P axis at the multi-tissue 308 \nlevel. Pseudotime analysis was individually performed on bi-NMP-derived somitic and 309 \nneural lineages and notochord cells. We ordered cells according to the rank of 310 \npseudotime indices and inferred the A-P axis for each lineage (Figures S4A and S4B). 311 \nAs expected, expression of marker genes for respective lineage progenitors were 312 \nenriched at the inferred posterior end, whereas marker genes for differentiated tissues 313 \nwere highly expressed at the anterior end (Figure 3L).  314 \n  Having built the inferred A-P axis, we then assessed the spatial distribution of RA, 315 \nFGF, WNT and NOTCH pathways that are crucial for trunk development as observed in 316 \nvertebrate embryos.16,42,69,70 RA signaling is important for the balanced bi-NMP 317 \nspecification into somitic and neural lineages.71 It was therefore important to establish if 318 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 15 \nRA signaling was active in hTEM.v3. The expression of RA degradation enzyme 319 \n(CYP26A1) peaked in the posterior-most NMP-Neural and notochord progenitor cells, 320 \nwith RA receptor gamma (RARG) exclusively expressed in the NMP-Neural cells 321 \n(Figure 3M). Expression of RA synthesis genes (RDH10, ALDH1A2) were restricted to 322 \nanterior somitic cells and the positionally parallel expression of RARB was exclusive to 323 \nthe anterior neural cells, coinciding with the expression of PAX6 (Figures 3L and 3M).72 324 \nThe A-P patterned RA circuit genes confirms the integrity of RA signaling in hTEM.v3 as 325 \nobserved in mouse embryos (Figure 3N).16,70  326 \n   How FGFs exert differential roles in coordinating multi-tissue co-patterning along the 327 \nA-P axis remains unknown.59,70 Distinct expression patterns of FGFs were noted among 328 \ndifferent cell types in hTEM.v3. For example, FGF3/4/8/17/19 in posterior neural and 329 \nsomitic cells, FGF13 in anterior neural cells, FGF13/18 in anterior somitic cells, while 330 \nFGF8/17 were expressed throughout the notochord without A-P polarity (Figure S4C). 331 \nLike FGFs, WNT ligands also showed A-P graded expression in somitic and neural 332 \nlineages. Canonical (WNT3A/8A) and noncanonical (WNT5A/5B) WNT ligands were 333 \nexpressed in the posterior-most bi-NMPs (Figure S4D). In contrast, WNT3A/5B were 334 \nexpressed throughout the notochord lineage. Gradients of CTNNB1 (WNT effector) in 335 \neach lineage was opposed to the posteriorly restricted WNT ligands, consistent with 336 \npolarized cell proliferation and movements during axial elongation. Anterior expression 337 \nof SFRP1/2 (WNT inhibitors) in both neural and somitic lineages were opposed to the 338 \nWNT ligands in bi-NMPs, consistent with a negative WNT feedback loop during trunk 339 \nformation in mice.1 How the interacting gradients of FGF and WNT signaling drive 340 \nnotochord formation, segmentation clock and neurogenesis in humans is not well 341 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 16 \nunderstood hTEM.v3 will be useful for addressing such important developmental 342 \nquestions. 343 \n  Next, we investigated the patterning of NOTCH signaling in hTEM.v3 as it is critical for 344 \naxial elongation.64,73 How NOTCH signaling participates in D-V patterning is poorly 345 \nunderstood, but hTEM.v3 is likely to be a useful tool to address this. In the somitic 346 \nlineage, anteriorly expressed NOTCH modulator LFNG was opposed the posterior 347 \nNOTCH ligands (DLL3) and its effector (HES7) (Figure S4E), mirroring the anterior-to-348 \nposterior somitogenesis.42 Although NOTCH receptors (NOTCH1/2/3) were lowly 349 \nexpressed in the neural and notochord lineages, NOTCH effectors (HES1/4) and their 350 \ntarget (CCND1) were highly expressed in anterior neural cells and throughout the 351 \nnotochord, respectively. This could explain the rapid morphogenesis and axial 352 \nelongation of neural tube and notochord after day 4 (Figures 3B and 3C).74,75 353 \nInterestingly, we noted the expression of a NOTCH coactivator MAML2 throughout the 354 \nnotochord (Figure S4E). The role of Maml2 is possibly involved in Sox9-dependent 355 \ninhibition of the WNT pathway in mouse sclerotome.76,77 hTEM.v3 offers a unique 356 \nopportunity to characterize this notochord-dependent NOTCH regulatory mechanism in 357 \nhuman ventral patterning. 358 \n  Collectively, hTEM.v3 faithfully recapitulates A-P axis development at multi-tissue 359 \nlevels, mirroring key signaling networks of early human embryogenesis. By achieving 360 \nhigh-fidelity reconstruction of essential signaling pathways, this model opens new 361 \navenues to interrogate spatiotemporal signaling crosstalk and cell-fate decisions at a 362 \nmulti-tissue level. 363 \n 364 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 17 \nPatterning of neural and somitic cells along the D-V axis 365 \nTo delineate D-V specifications in hTEM.v3, sub-clustering and RNA velocity analysis 366 \nwas performed on neural and somitic cells, respectively (Figures 4A and 4B). The 367 \nresultant UMAP demonstrated clear D-V patterning, evident by distinct transcriptomic 368 \nprofiles of dorsal/ventral neural cells-floor plate and somite compartments, respectively 369 \n(Figures 4C, 4D, S5A, and S5B). This high degree of complexity regarding D-V 370 \nspecifications was not seen in hTEM.v1/2 (Figure 4E). Immunostaining of transversely 371 \nsectioned day-6.5 hTEM.v3 confirmed D-V patterned dorsal (PAX6) and ventral (NKX6-372 \n1, OLIG2) neural cells along the neural tube (Figure 4F). These midline-positioned 373 \nneural cells were flanked by distinct somite compartments including the dorsal somite 374 \n(PAX3), dorsolateral dermomyotome (MYF5:mClover3), myogenic progenitors (PAX7), 375 \nlateral endotome (EBF2:mScarlet), ventromedial sclerotome (PAX1) and surrounding 376 \nvascular endothelial cells (SOX17) (Figures 4F and 4G).  377 \n  To understand the molecular mechanisms underlying D-V axis establishment, we 378 \nperformed Gene Ontology (GO) and pathway enrichment analysis on scRNA-seq data 379 \nfor hTEM.v3. First, SCENIC78 was used to generate a regulon module enrichment 380 \nheatmap illustrating representative transcription factor (TF) genes associated with all 381 \ncell types of hTEM.v3 (Figures 4H, 4I, and S5C). As expected, biological processes in 382 \nGO terms enriched for each regulon module were consistent with associated cell types 383 \n(Figure S3L, S5D, and S5E). For example, module M4 comprised of key TFs (PAX6, 384 \nIRX3, NKX6-2) important for regulating neural tube formation, was significantly enriched 385 \nin “GO:0001840 neural plate development”, was highly expressed in the “Neural tube”, 386 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 18 \nbut not in “NMP-Meso” or “L-Somite” clusters in hTEM.v3 scRNA-seq data (Figures 4I 387 \nand 4J).  388 \n  Regulon module-based gene regulatory network (GRN) analysis identified key GRNs 389 \n(NOTO, PAX6, NKX6-2, FOXA1/2) and their target networks that drive D-V axis 390 \nformation (Figures 4K and S5F). The NOTO GRN was enriched for SHH signaling 391 \n(GLIS1), TBXT, and cilia functions (TPPP3, SCG3, TCTEX1D1). Consistent with Noto’s 392 \nrole in mice,79 NOTO target genes were expressed in human 'nascent' and 'ciliated' 393 \nnotochord subtypes (Figure S3Q). The NKX6-2 GRN contained targets that balance 394 \nventral neural tube patterning, including interneuron (HES5)80 and motoneuron 395 \nspecifiers (OLIG1/2)81 (Figure S5F). Finally, FOXA1/2 GRNs 396 \nexhibited differential functions. FOXA1 targets were associated with floor plate (ARX)82 397 \npatterning and notochordal fluid trafficking (CFTR)83, whereas the FOXA2 targets 398 \ngoverned node formation (PPIL6)84, cilia function (CFAP43)85, and maintaining 399 \nnotochord structure (KRT8, EPCAM, FN1)63 (Figure S5F). The GRN analysis revealed 400 \nmultifaceted networks in ventral patterning, demonstrating the establishment of D-V axis 401 \nin hTEM.v3. 402 \n  Overall, hTEM.v3 self-organizes into notochord, neural tube and somitic tissues with 403 \nproper A-P and D-V patternings (Figures 3E-3H and 4A-4G). A key advance in hTEM.v3 404 \nis its ventral neural specification. Unlike hTEM.v2, which displayed elevated HES5 and 405 \nlow NKX6-2 expression accompanied with a floor plate deficiency (Figure 2H), hTEM.v3 406 \nexhibited distinct floor plate (ARX) and ventral neural (OLIG2) markers alongside 407 \nreduction in HES5 and elevated NKX6-2 (Figure 3K). Our GRN analysis suggests that 408 \nNXK6-2 acts as a pivotal node, connecting SHH signaling and Notch-mediated HES5 409 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 19 \nactivity to balance motoneuron (OLIG2)/interneuron (HES5) specification (Figures S5A 410 \nand S5F).86,87 These data suggest a previously undescribed SHH-NKX6-2-HES5 411 \nregulatory mechanism in human neural tube patterning. 412 \n 413 \nSpatially transcriptomic profile reveals embryo-like signatures of hTEM.v3 414 \nTo map spatial allocation of cells in hTEM.v3, we performed Visium HD on longitudinal 415 \nsections (days 4/6.5). Key cell types including notochord, NMP-Meso, NMP-Neural and 416 \nD-V fated neural and somitic cells were all identified (Figure S6A). However, full 417 \nrecovery of D-V fated cells from Visium HD data, especially in somite compartments, 418 \nwas difficult due to limited sections. We therefore performed cell type deconvolution 419 \n(RCTD)88 by leveraging the reference annotations from scRNA-seq data of hTEM.v3 420 \n(days 3-7) (Figure S6B). The resulting spatial UMAP exhibited embryo-like spatial 421 \npatterns that correspond to anatomical regions of the posterior trunk in human CS8-10 422 \nembryos (Figures 4L, S6C, and S6D).20,27,36 For example, by day 4, the Caud. NP 423 \n(NKX1-2, GBX2) extending anteriorly out of the tailbud was flanked by anterior PSM 424 \n(RIPPLY2, CER1)89 (Figure S6E). By day 6.5, neural tube and floor plate were specified 425 \nin the midline along the A-P axis and flanked by compartmentalized somites (Figures 4L 426 \nand S6D). Remarkably, NMP-Neural and NMP-Meso cell populations occupied distinct 427 \nregions in the day-4 tailbud (Figure 4L, S6C and S6E), representing an unprecedented 428 \nbi-layered structure of bi-NMPs in vitro. This bi-layered structure signifies the onset of 429 \nD-V patterning as observed in a spatially resolved human CS9 embryo.27 430 \n  Next, we verified the inferred A-P signaling gradients from spatially resolved ligand-431 \nreceptor gene expression. For example, the RA (RARG-CYP26A1), FGF (FGF8-432 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 20 \nFGFR1), WNT (WNT3A-FZD7) and NOTCH (DLL3-NOTCH1) ligand-receptor pairs 433 \nwere spatially enriched in the tailbud of hTEM.v3 (Figures 4L and 4M), consistent with 434 \ntheir inferred A-P distribution in Figures 3M and S4C-S4E. In addition, CellChat90 435 \npredicted tissue-tissue communications from scRNA-seq data that were also validated 436 \nby Visium HD data (Figure 4O). For example, PAX1 (target of SHH pathway) 437 \nexpression in the sclerotome region was shown to be in close proximity to SHH 438 \nexpression in the floor plate (Figure S6F), reflecting the SHH signaling range. The 439 \nanterior expression of ALDH1A2 in somites and posterior CYP26A1 in tailbud (CDX2) 440 \nwere evident of the RA signalling along A-P axis (Figures 3M and 4N). The detection of 441 \nRARB and CRABP1 (RA target) in anterior neural cells confirms the somite-to-neural 442 \nRA crosstalk has been established as in the spinal cord of human CS10 embryos 443 \n(Figure S6F).36 Moreover, CellChat-predicted PDGF (PDGFA-PDGFRA) signaling 444 \nbetween neural and somitic cells was revealed on the spatial UMAP (Figures 4N and 445 \n4O). This suggests a neural-to-somite regulatory mechanism in patterning the 446 \nsclerotome.91 These spatial data demonstrate hTEM.v3’s utility in studying advanced 447 \norganogenesis co-patterning.  448 \n  The emergent HOX expression is key to axial elongation and spatially aligned with 449 \nbody axes formations,92 we therefore evaluated the fidelity of HOX coding in hTEM.v3. 450 \nhTEM.v3 exhibited HOX collinearity in somitic lineage as observed in axialoids8 and 451 \nextended it to neural and notochord lineages, with over 50% of HOX genes displaying 452 \ncoordinated expression (Figure S4F). Tissue-specific expression patterns of HOX genes 453 \nwere also noted. For example, HOXA5 and HOXB5 were expressed in anterior somitic 454 \nand notochord lineages but not in the neural lineage. HOXA9 was restricted to the 455 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 21 \nCaud. NP and posterior PSM, but not expressed in notochord cells (Figure S4F), 456 \nconsistent with its initial expression in caudal neural plate region in mouse gastrulating 457 \nembryos.93 We next exemplified the spatiotemporal HOX coding with scaled expression 458 \nof HOXC genes from days 4-6.5 (Figures S6G and S6H). During axial elongation, a 459 \nclear anterior expansion of HOXC6 and HOXC8 expression accompanied the caudal 460 \ndownregulation of HOXC10, as observed in the human developing spinal cord.94 461 \n  Collectively, our spatial and scRNA-seq transcriptomic data establishes hTEM.v3 as a 462 \nhigh-fidelity model of human posterior axial development. This system recapitulates the 463 \nspatially patterned signaling pathways and tissue-tissue crosstalk of human posterior 464 \ntrunk. Importantly, hTEM.v3 demonstrated a spatiotemporal HOX coding alongside axial 465 \nelongation, thereby capturing the core signaling and transcriptional machinery for future 466 \ndevelopmental studies. 467 \n 468 \nDevelopmental staging of hTEM.v3 resembles primate CS8-10 embryos 469 \nNext, we sought to allocate the developmental lineages of hTEM.v3 to those involved in 470 \ntrunk formation in primates and mice. First, a embryogenesis scRNA-seq reference, 471 \nconsist of public datasets of human  (CS7/8/10)20,27,84 and cynomolgus monkey 472 \n(CS8/9/11)19 embryos, was created based on primate orthologues (Figure S7A). Consist 473 \nwith hTEM.v3, the notochord, NMP-Neural and NMP-Meso in human-monkey (H-M) 474 \nreference dataset showed well-matched expression profiles (Figure 3K and 5A). Next, 475 \nsub-clustering of caudal trajectories (caudal epiblast, caudal mesoderm, somitic 476 \nmesoderm and NMP) in mouse embryo (E7.0-E8.5) datasets19,54 identified previously 477 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 22 \nunresolved mouse NMP-Meso and NMP-Neural populations (Figure 5B), highlighting 478 \nthe evolutionary conservation in trunk formation between mice and primates. 479 \n  Next, we examined cellular composition similarities between hTEM.v3 (days 3-7) and 480 \nH-M (CS7-11) datasets using Seurat. Comparative projection of cell types showed that 481 \nhTEM.v3 accurately recapitulates H-M posterior trunk development including the 482 \nnotochord, bi-NMPs, somite and spinal cord (Figures S7B and S7C). Likewise, hTEM.v3 483 \nwas mapped to ortholog mouse trajectories of axial progenitors (notochord, caudal 484 \nepiblast, caudal mesoderm, somitic mesoderm, NMP), paraxial mesoderm and spinal 485 \ncord (Figures S7D and S7E). Collectively, an unsupervised cluster similarity analysis 486 \nsummarized the major trunk cell types conserved across hTEM.v3, H-M, and mouse 487 \nembryos (Figures 5C and S7F). Further comparative analysis aligned the 488 \ndevelopmental stages between hTEM.v3 and H-M, and hTEM.v3 and mouse, 489 \nrespectively (Figures 5D and S7G). We noted the correspondences of advanced cell 490 \ntypes, such as neurons, intermediate mesoderm and endothelial cells at post-491 \ngastrulation stages between datasets (Figure S7G). Moreover, genes important for 492 \nnotochord, bi-NMP specification and pathways for RA, FGF and WNT signaling 493 \nexhibited broad similarities across species (Figures 5E and S7H). One exception is the 494 \nabsence of Fgf17 expression in mouse notochord. In hTEM.v3 and H-M notochord 495 \npopulations, FGF17 expression is highly expressed, suggesting a divergence in 496 \nmechanisms of notochord progress and D-V establishment between mice and primates 497 \n(Figures 5A and 5E).  These findings establish that hTEM.v3 is suitable for identifying 498 \nnew mechanisms of human development. 499 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 23 \n  In summary, combined with the embryo-like morphological features of hTEM.v3 500 \n(Figures 3D and S3I), cross-species comparative analyses indicates that hTEM.v3 501 \naccurately models cellular and molecular aspects of trunk development in CS8-10 H-M 502 \nembryos. hTEM.v3 (days 3-7) equates approximately to E19-23 in humans, E20-24 in 503 \ncynomolgus monkeys and E7.5-8.5 in mice (Figure 5F). 504 \n 505 \nChanges in notochord identity and SHH signaling switch D-V fates  506 \nNext, the utility of hTEM.v3 as a testbed for investigating human D-V patterning at the 507 \ngenetic level was explored. Focus was directed towards NOTO which is important for 508 \nnotochord function in mice by serving as a key regulator of Shh signaling in mice.20,63 509 \nLoss-of-function mutation of NOTO (NOTO-LOF) derived day-6 hTEM.v3 exhibited 510 \nirregular posterior morphology and shorter axial length (Figure S8A-S8C), consistent 511 \nwith the shortened tail phenotype in Noto-null mice.79 Comparative scRNA-seq analysis 512 \nbetween NOTO-LOF and wild-type (WT) revealed an expanded population of ‘mature 513 \nnotochord’ (FOXA1, NOG) cells in day-6 hTEM.v3 (Figures 6A–6C). This shift in 514 \nnotochord identity correlated with a pronounced upregulation of SHH expression and 515 \nventrally-biased fates (Figures 6C-6E). For example, cellular proportion of ventral 516 \nlineages (floor plate; FOXA2, NKX6-1. sclerotome; PAX1, PAX9) were upregulated at 517 \nthe expense of dorsal identities in neural tube (PAX6, MSX1) and somites (PAX3) 518 \n(Figures 6D, 6E, and S8D). Focusing on the neural tube, elevated SHH activity (SHH, 519 \nGLI1) in NOTO-LOF sharply suppressed PAX6 while inducing NKX6-1 (Figures 6E and 520 \n6F). Moreover, NOTO-LOF downregulated cilia-related genes (FOXJ1, RFX3, 521 \nTCTEX1D1) (Figure S8E), consistent with Noto’s conserved role in regulating 522 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 24 \nciliogenesis.79 These data demonstrate hTEM.v3 as a valuable testbed for genetic 523 \ndissection of human development and establish the role of NOTO in restraining SHH 524 \nactivity and safeguard nascent notochord identities. 525 \n  In contrast to the NOTO-LOF, exposure to SANT195 (smoothened antagonist, 250 nM 526 \nat days 4-6) led to a downregulation of ventral (PAX1, NKX6-1, FOXA2) markers 527 \nwithout negatively impacting dorsal (PAX3, PAX6) markers or axial elongation (Figures 528 \nS8B-S8D). scRNA-seq and immunostaining results confirmed that SANT1 treated 529 \nhTEM.v3 were dorsally-biased similar to hTEM.v1 (Figures 6E and 6F). This was 530 \nsupported by reduced ventral populations (e.g., floor plate, sclerotome) and a 531 \nconcordant reduction in the expression of ventral markers (NKX6-1/6-2, OLIG1/2, 532 \nFOXA1/2) (Figures 6D, 6E, and S8D). It is noteworthy that the proportion of notochord 533 \ncells and endogenous levels of SHH were not negatively affected in SANT1 (Figures 6C 534 \nand 6D), indicating that the dorsally-biased fates in SANT1 can be attributed to the 535 \nblockade of SHH signaling between notochord and its targets. Pharmacological 536 \ninhibition of the SHH pathway downregulates BMPs, VEGFs and KDR which control 537 \ndorsal aorta development and vasculogenesis.96 In support of this observation, 538 \nreductions in the proportions of endotome, endothelial cells and related gene 539 \nexpression (MEF2C, CD34, KDR) are noted in hTLE.v3 (Figures 6B, S8F, and S8G).  540 \n  Collectively, these observations show that by modulating notochord identity and 541 \nderived SHH signaling, hTEM.v3 embryoids can be patterned into ventrally-biased 542 \n(NOTO-LOF) or dorsally-biased (SANT1) fates. This responsiveness allows timed 543 \nperturbations in SHH to be assessed and enables human bi-axial development to be 544 \nexplored with temporal precision and quantifiable measurements. 545 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 25 \n 546 \nDisease modeling of axial truncation by knockout of VANGL1/2  547 \nWe next evaluated hTEM.v3 as a platform for the study of human congenital disease. 548 \nFor proof-of-concept, PCP signaling core members VANGL1 and VANGL2 were 549 \nselected, as mutations in VANGL1/2 lead to NTDs including spina bifida and congenital 550 \nvertebral malformations.97-99 As expected, VANGL1/2 expression was widespread 551 \nthroughout day-6.5 hTEM.v3, with VANGL2 enriched in neural tube and VANGL1/2 552 \nslightly enriched in caudal neural plate and PSM (Figures 6G and S8H). To model 553 \nVANGL-related defects in these tissues, loss-of-function mutations of VANGL1/2 H9-554 \nhESC lines were made, including VANGL1 -/- (VANGL1-LOF), VANGL2 -/- (VANGL2-555 \nLOF), and VANGL1 +/-; VANGL2 -/- (VANGL1/2-LOF) (Figure S8A).  556 \n  As in mice,99,100 VANGL-mutant derived hTEM.v3 exhibited axial truncations with 557 \ndifferent degrees of penetrance (Figures 6H and S8I). VANGL1/2-LOF displayed an 558 \nearly failure in mediolateral narrowing on day 4 and subsequently, severe dysregulation 559 \nof somitogenesis (no segmentation) and neural tube genesis (loss of a neural tube) 560 \n(Figure 6I). These observations are consistent with the well-established role of Vangl in 561 \nC&E movements and PCP signaling during axial elongation in zebrafish and mouse 562 \nmodels.99,100 Contrarily, VANGL1-LOF and VANGL2-LOF showed milder axial 563 \ntruncation defects compared to VANGL1/2-LOF, with VANGL2-LOF reproducing the no-564 \nsegmentation defect presented in VANGL1/2-LOF (Figure 6I).  565 \n  To further characterize the role of VANGL2 in trunk development, we performed a 566 \ncomparative scRNA-seq analysis of VANGL2-LOF and WT embryoids (Figure 6A). In 567 \nVANGL2-LOF, a sharp decrease in the proportion of neural tube cells was accompanied 568 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 26 \nby a marked increase in PSM, while the somite population remained unchanged (Figure 569 \n6J). The expanded PSM population and the caudal accumulation of TBXT protein in 570 \nVANGL2-LOF mirrored the observations of widened PSM and caudally restricted 571 \nexpression of Tbxt and Tbx6 in Vangl2 mutant mice (Figure 6I).99 Focusing on the loss 572 \nof somite segmentations in VANGL2-LOF, differential gene expression analysis 573 \nrevealed that ITGA5, a gene required for fibronectin (FN1) accumulation during somite 574 \nboundary formation and neural tube closure (Figure 6G),101,102 was downregulated in 575 \npan-somite cells (Figure S8J). This indicates the importance of stable interactions 576 \nbetween VANGL2 and integrins in somite segmentation and points towards an unknown 577 \nVANGL2-dependent mechanism that regulates ITGA5 expression.103 578 \n  Turning to VANGL2-related PCP components, PRICKLE1, DVL1/3, PTK7, CELSR1/3 579 \nand FZD6 were broadly expressed throughout the somite and neural tube trajectories in 580 \nday-6.5 hTEM.v3 (Figure 6G). Although VANGL2 protein is typically negatively 581 \ncorrelated with PRICKLE1/2 protein levels,104 VANGL2-LOF resulted in increased 582 \nPRICKLE1/2 transcript levels in PSM in VANGL2-LOF (Figures 6K, 6L and S8J). This 583 \nfinding points towards the existence of compensatory transcriptional mechanism within 584 \nthe PCP network in PSM cells. Additional PCP genes including CELSR3, DVL2/3, PTK7 585 \nwere concordantly upregulated with over-expanded UNCX (caudal polarity) expression 586 \nin PSM and pan-somite cell populations (Figures 6L and S8J). In contrast, VANGL2-587 \nLOF caused downregulation of PRICKLE2, CELSR3, DVL2 and PTK2 in neural cells, 588 \naccompanied by a shortened neural tube (Figures 6I and S8J), consistent with their 589 \nreported implication in human NTDs.105 This contrasting expression pattern in VANGL2-590 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 27 \nLOF indicates a differential role of VANGL2 in coordinating PCP components in; (i) 591 \nestablishing A-P polarity somite segmentations and, (ii) neural tube elongation.  592 \n  Current VANGL-related early congenital disease models are based solely on 593 \nobservations made in zebrafish and mice.99,106 Using VANGL1/2 knockouts, we 594 \ndemonstrated the potential for our multi-tissue, bi-axial embryoids to serve as a NTD 595 \nmodel and as a platform for developing genetic screens, therapeutic strategies, toxicity 596 \nand drug tests in a more human relevant context. 597 \n  Overall, by leveraging the co-development of notochord and bi-NMPs, embryo-like 598 \nstructures were generated that, at the morphologic and molecular level, recapitulating 599 \nA-P and D-V axes formations in the human posterior trunk. These embryoids are self-600 \norganized and non-integrated. This provides a valuable tool that meets the criteria2 for 601 \nbenchmarking early human embryogenesis and the development of human disease 602 \nmodels. This can also be a platform for future development of next-generation 603 \nembryoids that recapitulate additional aspects of human development. 604 \n 605 \nLimitation of this study 606 \nFew to no neural crest (SOX10), endoderm (HNF4A), cardiac mesoderm (HAND1) or 607 \nanterior neural ectoderm (OTX2) cells were detected in hTEMs. The neural tube 608 \nstructures in embryoids are believed to represent secondary neurulation, which 609 \naccounts for the caudal portion of the A-P axis.107 More advanced embryo models are 610 \nneeded to reconstitute the fully body axes of A-P, D-V and L-R. 611 \n 612 \nResource availability 613 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 28 \nThe raw and processed sequencing data in this study are available in the GEO 614 \nrepository GSE314260. Source code for analyzing the sequencing data is available at 615 \nhttps://github.com/tianmingwucuhk/hTEM. 616 \n 617 \nAcknowledgement 618 \nWe thank the Core Laboratories of School of Biomedical Sciences at the Chinese 619 \nUniversity of Hong Kong for the service of single cell sorting, histology sections, 620 \nconfocal imaging and SEM. This study was supported by grants to S.D. from the 621 \nResearch Grants Council of Hong Kong (General Research Fund) and the Hong Kong 622 \nJockey Club Charities Trust. S.D. is a Global Stem Scholar and Director of the JC 623 \nSTEM Lab of Stem Cells and Regenerative Medicine. 624 \n 625 \nAuthor contributions 626 \nConceptualization, T.-M.W. and S.D.; Sequencing data generation, T.-M.W, H.Y., and 627 \nJ.V.; Sequencing data analysis, T.-M.W, H.Y., B.S.-H.W., L.X., and S.K.-W.T.; 628 \nExperiments, T.-M.W., K.-X.T., W.-M.X., E.S.-K.N., A.Y.-F.K., and Jianan Z.; 629 \nInterpretation and discussion, T.-M.W., S.D., Jiannan Z., B. G.; Supervision, T.-M.W. 630 \nand S.D.; Manuscript writing, T.-M.W. and S.D.; Funding acquisition, S.D. 631 \n 632 \nDeclaration of interests  633 \nS.D. and T.-M.W. are the applicants and inventors on a patent filed by the Chinese 634 \nUniversity of Hong Kong under reference number 25/MED/1610. The rest authors 635 \ndeclare no conflicts of interests.  636 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 29 \n 637 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 30 \n 638 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 31 \nFigure 1 Generation of dorsally biased fates hTEM.v1  639 \n(A) Schematic of the hTEM.v1 generation. 640 \n(B) Representative images of hTEM.v1 from day 0 to day 7. Scale bars, 200 μm. 641 \n(C) Box plot of embryoid length over time from three independent biological replicates 642 \n(rep). Each dot represents an individual hTEM.v1 length measurement (n = 9-124 for 643 \neach time point). 644 \n(D) SEM snapshots of day-7 hTEM.v1 samples (n = 3) in longitudinal orientation from 645 \nventral (left), dorsolateral (middle) and transversely fractured views. nt., neural tube. 646 \nsm., somite.  647 \n(E) 3D projections of representative day-4 hTEM.v1 stained for NMPs (TBXT, SOX2), 648 \nPSM (TBX6), early unsegmented somite (SIX1), and mitotic cells (pH3-Ser10). Scale 649 \nbars, 100 μm. 650 \n(F) Immunofluorescence image of longitudinally sectioned day-6 hTEM.v1 showing the 651 \nneural tube (SOX2) and flanking pairs of somites (PAX3). Scale bar, 100 μm. 652 \n(G) Immunofluorescence images of a transversely sectioned day-7 hTEM.v1 showing 653 \nthe spatial organization of duplicated, epithelialized (N-cadherin) neural tube structures 654 \n(SOX2) relative to paired somites (PAX3).  655 \n(H) 3D projections of day-7 hTEM.v1 from the dorsal (left) and ventral (right) displaying 656 \nthe duplicated neural tubes (SOX2) with flanking somites (SIX1). Caudally positioned 657 \nNMPs (TBXT) were barely detectable on day 7. Scale bars, 100 μm. 658 \n(I) UMAP of integrated hTEM.v1 integration from day 4/5/7 (total of 47,908 cells). Cell 659 \ntype annotations are indicated below.  660 \n(J) UMAP showing the expression of selected cell types from hTEM.v1. 661 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 32 \n(K) Dot plot displaying the expression levels (color) and proportion (dot size) of marker 662 \ngenes for dorsal and ventral fates. Proportions below 5% were omitted. 663 \n(L) (Top) Both neural tube (SOX2, PAX3/6, BMP4/7) and somite cell (PAX3, BMP4/7) 664 \nidentities were dorsal-biased in day-7 hTEM.v1, (bottom) with few detectable ventral 665 \nneural tube (NKX6-1, arrowhead) cells. Scale bars, 100 μm. 666 \n(M) Density plot showing the progress of NMP-neural, NMP-Meso and Node-like cells 667 \nover time.   668 \n 669 \n 670 \n 671 \n 672 \n 673 \n 674 \n 675 \n 676 \n 677 \n 678 \n 679 \n 680 \n 681 \n 682 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 33 \n 683 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 34 \nFigure S1 Characterization of hTEM.v1, related to Figure 1 684 \n(A) SEM image showing the size contrast between a representative day0-hESC 685 \nspheroid (top) and day-4 hTEM.v1 (below).  686 \n(B) Representative images of day-6 hTEM.v1 with or without 4% Matrigel. Scale bars, 687 \n200 μm.  688 \n(C) Box plot showing length measurements of day-6 hTEM.v1 with (n = 15) or without (n 689 \n= 6) 4% Matrigel. Data are presented as mean ±standard deviation. 690 \n(D) Number of somite pairs in hTEM.v1 over days 5-7. Each dot represents an 691 \nindividual hTEM.v1 randomly chosen from 3 biological replicates. Vertical lines 692 \nrepresent mean values.  693 \n(E) Top row, 3D projection of day-2 hTEM.v1 showing polarized patterns of bi-NMPs 694 \n(SOX2, TBXT, CDX2). Bottom row, complementary TBX6+ PSM and SOX2- regions.  695 \n(F) Immunofluorescence images of longitudinally sectioned day-4 hTEM.v1 showing A-696 \nP patterned bi-NMPs (TBXT, SOX2), neural plate (SOX2), PSM (TBX6), and flanking 697 \nsomitic (SIX1) cells.  698 \n(G) 3D projection of A-P symmetry breaking of day-4 hTEM.v1 with posterior tail bud 699 \n(CDX2) and anterior somitic cells (N-cadherin). Scale bar, 100 μm.  700 \n(H) Immunofluorescence image showing the duplication of the neural tubes with flanking 701 \nsomites in transversely sectioned day-7 hTEM.v1.  702 \n(I) 3D projections of day-4 hTEM.v1 showing the duplication of neural plate (SOX2) 703 \nstructures along A-P axis. TBXT was absent from the presumed ventral side and 704 \nrestricted to the posterior-most region. nt., neural tube. sm., somite.  705 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 35 \n(J) Heatmap of scRNA-seq showing clusters of transcripts associated with cell identities 706 \nof hTEM.v1 (days 4/5/7) as seen in Figure 1I.  707 \n(K) Changes in cell type composition in hTEM.v1 over days 4-7. Colors for cell types are 708 \nin consistent with (J). 709 \n(L) UMAP showing transcript levels for FGF, BMP and WNT family members in 710 \nhTEM.v1 (days 4/5/7). 711 \n(M) HCR-IF images of day-4 hTEM.v1 showing the node-like (SHH, arrowhead) 712 \nstructure adjacent to PSM (HES7, TBX6).  713 \n(N) Counts of the node-like cells from hTEM.v1 scRNA-seq data, based on detected 714 \nmarker genes at listed days. 715 \n(O) Left, 3D projection showing anteriorly positioned vascular endothelial cells (SOX17). 716 \nRight, Immunofluorescence of vascular endothelial cells (SOX17, VE-cahderin) at the 717 \nouter layer of epithelialized somites (N-cadherin).  718 \nAll scale bars in the immunofluorescence images are 100 μm. 719 \n 720 \n 721 \n 722 \n 723 \n 724 \n 725 \n 726 \n 727 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 36 \n 728 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 37 \nFigure 2 SHH activation by SAG caused D-V specifications in neural tube and 729 \nsomite without a notochord 730 \n(A) Schematic of hTEM.v2 generation.  731 \n(B) Representative images of hTEM.v2 over days 5-7. Scale bars, 200 μm. 732 \n(C) Bar plot showing length measurements comparing hTEM.v1 to hTEM.v2 over days 733 \n5-7. Each dot represents a length measurement of individual hTEM.v1/2 embryoids at 734 \nshown time point (n = 14-57 for each group from same batch). n.s., no statistical 735 \nsignificance.  736 \n(D) qPCR results showing dorsal and ventral marker gene expression in hTEM.v2. Data 737 \nare presented as mean ±standard deviation. Data were reproduced twice. 738 \n(E) 3D projection of day-7 hTEM.v2 showing dorsal and ventral neural tubes (SOX2) 739 \nflanked by paired somites (SIX1). Arrowhead, somites. Dashed line, duplicated neural 740 \ntubes. Scale bar, 100 μm.  741 \n(F) UMAP showing identified cell types from hTEM.v2 (days 5/7, total of 16,535 cells). 742 \nDay-4 hTEM.v1 scRNA-seq data was integrated for accurate trajectory annotations. 743 \n(G) UMAP showing the expression of indicated lineage markers in hTEM.v1. 744 \n(H) Dot plot showing the expression profile reflecting somite compartments and neural 745 \ntube D-V patterning, induced by SAG treatment. Proportions below 5% were omitted. 746 \n(I-J) Spatial gene expression patterns in longitudinal (I) and transverse (J) day-7 747 \nhTEM.v2 sections. The major tissue trajectories were consistent with those shown in 748 \nFigures S2E and S2G. 749 \n(K-L) Schematic diagram of D-V patterning of the neural tube (K) and adjacent 750 \ncompartmentalization of the somite (L). 751 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 38 \n(M-N) Dot plots showing the expression profiles of D-V patterns of neural tube (M) and 752 \nsomite (N) cells from transversely sectioned day-7 hTEM.v2 Visium HD data. The listed 753 \nclusters were consistent with those in Figure S2G. 754 \n 755 \n 756 \n 757 \n 758 \n 759 \n 760 \n 761 \n 762 \n 763 \n 764 \n 765 \n 766 \n 767 \n 768 \n 769 \n 770 \n 771 \n 772 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 39 \n 773 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 40 \nFigure S2 Characterization of hTEM.v2, related to Figure 2 774 \n(A) Experimental design for timely and varied SAG concentrations, based on the 775 \nhTEM.v1 protocol. 776 \n(B) qPCR showing expression levels of marker genes for indicated cell types. Sample 777 \nindices are the same as those in (A). Data are presented as mean ±standard deviation. 778 \nData were reproduced twice. 779 \n(C-D) Numbers of D-V patterned cells from somitic (C) and neural (D) clusters in 780 \nhTEM.v2 scRNA-seq data. Dorsal somite marker; PAX3. Ventral somite markers; 781 \nTWIST1, SCX, PAX1. Dorsal neural tube marker; PAX6. Ventral neural tube markers; 782 \nNKX6-1, NKX2-8, FOXA2. 783 \n(E-F) UMAP (E) and spatial UMAP (F) showing identified cell types from transversely 784 \nsectioned day-7 hTEM.v2 (n = 4). Transverse samples 1-4 are sections from posterior 785 \nto anterior positions. 786 \n(G-H) UMAP (G) and spatial UMAP (H) showing identified cell types from longitudinally 787 \nsectioned day-7 hTEM.v2 (n = 4). 788 \n(I) Spatially expressed TBX18 (anterior) and UNCX (posterior) indicating somite 789 \nsegmentation in longitudinal section of day- 7hTEM.v2 (Visium HD sample 1). Signals of 790 \nTBX18 and UNCX were quantified and scaled along the A-P axis. 791 \n(J) UMAP trajectory showing notochord emergence in E7.25-7.5 mouse embryos (E-792 \nMTAB-6967).  793 \n(K) Dot plot showing the expression of signals emanating from indicated tissues during 794 \nearly mouse embryogenesis (E-MTAB-6967).  795 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 41 \n(L) Dot plot showing the expression of signals emanating from the indicated tissues in 796 \nthe human CS8 embryo (HRA005567). Plot was generated using the online tool in 797 \nhttp://cs8.3dembryo.com/.  798 \n 799 \n 800 \n 801 \n 802 \n 803 \n 804 \n 805 \n 806 \n 807 \n 808 \n 809 \n 810 \n 811 \n 812 \n 813 \n 814 \n 815 \n 816 \n 817 \n 818 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 42 \n 819 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 43 \nFigure 3 Co-development of notochord, NMP-Neural, NMP-Meso and subsequent 820 \nneural and somitic lineages in hTEM.v3 821 \n(A) Schematic of hTEM.v3 generation.  822 \n(B) Representative images of hTEM.v3 at day 4 (left) and day 7 (right). 823 \n(C) Boxplot showing length measurements of hTEM.v3 from days 0-7 (n = 21-84 for 824 \neach time point) from 9 independent biological replicates (rep). Each dot represents an 825 \nindividual hTEM.v3 at shown time point. 826 \n(D) Immunofluorescence image showing the A-P elongating notochord (FOXA2), caudal 827 \nand bilateral PSM (TBX6) and apical junctions (Phalloidin) within anterior cells. Scale 828 \nbar, 100 μm. 829 \n(E) HCR 3D projection showing the D-V layers of caudal neural plate cells (NKX1-2) 830 \nand notochord cells (CHRD) in day-4 hTEM.v3. Scale bar, 100 μm. 831 \n(F) HCR image showing transcripts of key genes (NOTO, SHH, FOXA2) expressed in 832 \nnotochord cells in day-4 hTEM.v3. The posterior node area was zoomed. Yellow arrow, 833 \ntriple positive. White arrow, double positive. Scale bar, 100 μm. 834 \n(G) Top, Immunofluorescence images of transversely sectioned day-5.5 hTEM.v3 835 \nshowing dorsally positioned neural tube (SOX2) and ventrally positioned notochord 836 \n(NOTO:mClover3). Arrowhead, SOX2+NKX6-1+ ventral neural cells. Bottom, D-V 837 \npatterned neural tube (SOX2) and ventral notochord (TBXT) and bilateral somites (ZO-838 \n1). nt., neural tube. sm., somite. noto., notochord. Scale bars, 100 μm. 839 \n(H) SEM images of day-5.5 hTEM.v3 (n = 3). Wholemount view (top left), transversely 840 \nfractured viewpoint (top-middle). Zoomed views of the transversely fractured sample 841 \nshowing; somite (top-right), neural tube (bottom-left), notochord with lipids (yellow 842 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 44 \narrows) and vacuoles (blue arrows) (bottom middle) and ECM fibers (white arrow, 843 \nbottom-right). 844 \n(I) RNA velocity analysis based on the UMAP of hTEM.v3 (days 3-7) scRNA-seq 845 \nintegration (total of 100,370 cells). Black arrows indicate calculated differentiation 846 \ndirections. Yellow arrows indicate the two major developmental streams stemming from 847 \nbi-NMPs. 848 \n(J) Differential expression of key genes to distinguish tailbud (CDX2, CYP26A1)-derived 849 \nNMP-Neural (SOX2highTBXTlow, NKX1-2), NMP-Meso (SOX2lowTBXThigh, MSGN1) and 850 \nnotochord (NOTO, SHH). 851 \n(K) Dot plot showing the expression of identified cell type markers in hTEM.v3. 852 \nProportions below 5% were omitted. 853 \n(L) Normalized expression of respective lineage marker genes along the pseudotime 854 \ninferred A-P axis. Data are shown as coloured smooth spline with standard deviation in 855 \ngrey shade. Vertical black lines at the bottom of the plots show cells co-expressing 856 \nSOX2 and TBXT.  857 \n(M) Heatmap showing scaled expression of genes in the RA pathway along the 858 \npseudotime inferred A-P axis. 859 \n(N) Schematic diagram of RA signaling along the A-P axis of hTEM.v3.  860 \n 861 \n 862 \n 863 \n 864 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 45 \n 865 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 46 \nFigure S3 Experimental design, morphological and cellular composition of 866 \nhTEM.v3, related to Figure 3 867 \n(A) Experimental design to test the conditions of timely BMP and NODAL inhibition for 868 \nnotochord cells and bi-NMP induction. 869 \n(B) qPCR results showing marker levels of bi-NMPs, notochord and anterior primitive 870 \nstreak under test conditions shown in (A). Data are presented as mean ±standard 871 \ndeviation. Results were reproduced more than three times. 872 \n(C) Experimental design to test combinations of BMPs, FGFs and SHH for concordant 873 \nmaintenance and differentiation of notochord, NMP-Neural and NMP-Meso. 874 \n(D) qPCR showing expression of indicated lineage markers for notochord (NOTO), 875 \nNMP-Neural (NKX1-2), NMP-Meso (TBX6) at day 4, from conditions listed in (C). Data 876 \nare presented as mean ±standard deviation. Results were reproduced more than three 877 \ntimes. 878 \n(E) qPCR results comparing expression levels of dorsal (PAX6) and ventral (NKX6-1, 879 \nSHH, FOXA2) markers in neural and notochord cells in day-6 hTEM.v3. Test conditions 880 \nare shown in (C). Data are presented as mean ±standard deviation. Results were 881 \nreproduced more than three times. 882 \n(G) Dot plot showing numbers of segmented somite pairs in hTEM.v3 at days 5-7. 883 \n(H) Representative images showing the caudal enrichment of NOTO:mClover3+ 884 \nnotochord cells in the midline of hTEM.v3 at days 3-4.  885 \n(I) Representative images showing the gradual enrichment of NKX1-2:mScarlet+ caudal 886 \nneural plate progenitor cells in the midline of hTEM.v3 at days 3-4. 887 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 47 \n(I-J) Immunofluorescence images of transversely sectioned day-5.5 hTEM.v3 exhibiting 888 \nthe bi-layered organization of dorsal neural plate (SOX2) and ventral notochord (TBXT, 889 \nNOTO, FOXJ1, FOXA2).  890 \n(K) SEM images of day-4 hTEM.v3 (n = 2); dorsal view. Regions of interest (R1/2/3) 891 \nwere dash lined. Zoomed views of R1+R2 and R3 are displayed, highlighting the neural 892 \nplate and the ciliated ventral node.  893 \n(L) UMAP showing identified cell types from hTEM.v3 scRNA-seq integration (days 3-7, 894 \ntotal of 100,370 cells).  895 \n(M) Changes in cell type composition of hTEM.v3 from day 3-7. Colors of cell types 896 \ncorrespond to those in (L). 897 \n(N-P) RNA velocity (N), sub-clustering (O), and pseudotime (P) analysis on notochord 898 \ncells (n = 888) subset from hTEM.v3 scRNA-seq dataset in (L).  899 \n(Q) UMAP showing expression of markers for indicated notochord subtypes. 900 \n(R) Relative proportions of notochord subtypes identified from (N). 901 \n 902 \n 903 \n 904 \n 905 \n 906 \n 907 \n 908 \n 909 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 48 \n 910 \n 911 \n 912 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 49 \nFigure S4 A-P organization of cell types and gene expression profiles inferred 913 \nfrom scRNA-seq integration of hTEM.v3 (days 3-7), related to Figure 3. 914 \n(A-B) Pseudotime analysis showing the progression of somitic (A) and neural lineages 915 \n(B). Cells analyzed here were subset from the hTEM.v3 (days 3-7) scRNA-seq dataset 916 \nin Figure S3L.  917 \n(C-F) Heatmap showing scaled expression of FGF (C), WNT (D), NOTCH (E) pathways 918 \nand the HOX (F) genes along pseudotime inferred A-P axis.  919 \n 920 \n 921 \n 922 \n 923 \n 924 \n 925 \n 926 \n 927 \n 928 \n 929 \n 930 \n 931 \n 932 \n 933 \n 934 \n 935 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 50 \n 936 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 51 \nFigure 4 Molecular profiling of D-V organization and spatial patterning for 937 \nhTEM.v3 938 \n(A-B) RNA velocity analysis of neural (A) and somitic (B) lineage subclusters from 939 \nhTEM.v3 (day 3-7) scRNA-seq integration in Figure S3L. To distinguish somite 940 \ncompartments, regression was performed on TBX18 and UNCX to remove somite A-P 941 \npatterns before re-clustering.  942 \n(C-D) Density plot showing trajectories of D-V patterned neural (C) and somitic (D) 943 \nlineages in hTEM.v3. Dorsal neural markers; PAX6, DBX2, and IRX5. Ventral neural 944 \nand floor plate markers; NKX6-1, FOXA2 and SHH. pan- and dorsal somite; PAX3. 945 \nDermomyotome; MYF5. Endotome; EBF2. Syndetome; SCX. Sclerotome; PAX1. 946 \nMyogenic progenitors; PAX7. 947 \n(E) Comparison of hTEM.v1/2/3 cell types to contrast the presence or absence of D-V 948 \nfates. 949 \n(F) Immunofluorescence images of transverse (left) and longitudinal (right) sections of 950 \nday-6.5 hTEM.v3 showing the presence of D-V patterning in the neural tube structure 951 \nalong D-V and A-P axes. Dorsal neural marker; PAX6. Ventral neural and floor plate 952 \nmarkers; OLIG2 and NKX6-1. Scale bars, 100 μm. 953 \n(G) Immunofluorescence images of transverse sections of day-6.5 hTEM.v3, showing 954 \nsomite compartments along D-V axis. pan- and dorsal somite, PAX3. Endotome, 955 \nEBF2:mScarlet. Dermomyotome, MYF5:mClover3. Myogenic progenitors, PAX7. 956 \nEndothelial cells, SOX17. Scale bars, 100 μm. 957 \n(H) Circular heatmap showing the regulon representing TF genes in the indicated cell 958 \ntypes from hTEM.v3. 959 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 52 \n(I) Heatmap (left) of regulon modules based on cell type associated regulon activities in 960 \nFigure S5C, with the corresponding GO terms related to each regulon module 961 \nhighlighted (right). 962 \n(J) Heatmap showing the enrichment of regulon modules across listed cell types. L- 963 \nSomite, late somitie, including sclerotome, syndetome, dermomyotome, myogenic 964 \nprogenitors and endotome cells from hTEM.v3 scRNA-seq data in Figure S3L. 965 \n(K) NOTO gene regulatory networks in hTEM.v3. The top 15 interacting genes are 966 \ndisplayed. 967 \n(L) Spatial UMAP of RCTD cell type annotations in day-4 and day-6.5 hTEM.v3 (n = 8 968 \neach). Colors for annotation and cell types displayed are consistent with those in Figure 969 \nS6B-S6D. 970 \n(M-N) Spatial gene expression patterns in longitudinally sectioned day-4 (M) and day-971 \n6.5 (N) hTEM.v3. Dashed lines, areas for neural plate (day 4) and neural tube (day 6.5). 972 \n(O) Bubble plot showing the selected ligand-receptor interactions between indicated cell 973 \ntypes from hTEM.v3. Dot color represents the normalized sum of expression level of 974 \nligand in source cells and interacting receptor in targeting cells. 975 \n 976 \n 977 \n 978 \n 979 \n 980 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 53 \n 981 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 54 \nFigure S5 Molecular profiles of hTEM.v3, related to Figure 4 982 \n(A-B) Normalized gene expression profiles of listed neural (A) and somitic (B) lineages 983 \nfrom hTEM.v3, reflecting the D-V axis formation in hTEM.v3.  984 \n(C) Heatmap showing regulon activities represented by selected transcription factors 985 \n(TFs) in 500 randomly sampled cells in hTEM.v3.  986 \n(D) UMAP showing the regulon module activity over trajectories for hTEM.v3.  987 \n(E) Gene Ontology (GO) enrichment analysis based on top ranking genes enriched in 988 \nregulon modules from Figure 4I.  989 \n(F) Gene regulatory networks of PAX6, NKX6-2, FOXA1 and FOXA2 in hTEM.v3. Top 990 \n15 interacted genes were displayed. 991 \n(G) Cell-cell communication patterns grouped by cell types from hTEM.v3 (days 3-7; 992 \nleft). Significantly enriched signaling pathways belonging to each pattern (right). 993 \n 994 \n 995 \n 996 \n 997 \n 998 \n 999 \n 1000 \n 1001 \n 1002 \n 1003 \n 1004 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 55 \n 1005 \n 1006 \n 1007 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 56 \nFigure S6 Spatially resolved hTEM.v3 cell type organization using Visium HD, 1008 \nrelated to Figure 4. 1009 \n(A) UMAP showing the primary cell types identified from Visium HD results of day-4 and 1010 \nday-6.5 hTEM.v3. 1011 \n(B) UMAP showing the RCTD cell types using reference annotations from hTEM.v3 1012 \nscRNA-seq data in Figure S3L. To reduce annotation complexity and to discern 1013 \nannotation colors in RCTD, ‘E-Somite’ and ‘M-Somite’ from scRNA-seq reference were 1014 \ncombined as ‘pan-Somite’ in RCTD. ‘E-Neural tube’ and ‘Neural tube’ from scRNA-seq 1015 \nreference were merged as ‘Neural tube’ in RCTD, ‘E-Floor plate’ and ‘Floor plate’ from 1016 \nscRNA-seq reference were merged as ‘Floor plate’ in RCTD. Colors and annotations 1017 \nare listed at right side.  1018 \n(C-D) Spatial UMAP showing the RCTD annotated cell types from Visium HD resolved 1019 \nfrom day-4 (C) and day-6.5 (D) hTEM.v3. Colors and annotations are from (B). 1020 \n(E-F) UMAP and spatial expression patterns of indicated genes in longitudinally 1021 \nsectioned day-4 (E) and day-6.5 (F) hTEM.v3. Dashed lines, the neural plate (day 4) 1022 \nand neural tube (day 6.5) structures. 1023 \n(G-H) Spatial expression of HOXC6 and HOXC8 at day 4 (G) and day 6.5 (H) in 1024 \nhTEM.v3. Signals of HOXC family genes were quantified and scaled along the direction 1025 \nof P-A at the right side. 1026 \n 1027 \n 1028 \n 1029 \n 1030 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 57 \n 1031 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 58 \nFigure 5 Developmental staging of hTEM.v3 resembles primate CS8-10 embryos 1032 \n(A) Top, sub-clustering UMAP revealing the notochord, NMP-Neural and NMP-Meso 1033 \npopulations from human (CS7/8/10) and monkey (CS8/9/11) embryo integration 1034 \ndataset. Bottom, differential gene expression patterns delineating these cell types. 1035 \n(B) Sub-(top-right) and re-(top-right) clustering UMAPs revealing the notochord, NMP-1036 \nNeural and NMP-Meso from mouse embryo dataset (E7.0 to E8.5). Bottom, differential 1037 \ngene expression patterns delineating these cell types. 1038 \n(C) Heatmap of correlation co-efficient among cell types from human, monkey, mouse 1039 \nand hTEM.v3. The correlation was calculated and summarized from Figure S7F.  1040 \n(D) Sankey diagram showing relationships between selected cell types in hTEM.v3, 1041 \nhuman (CS7/8/10), monkey (CS8/9/11) and mouse embryos (E7.0 to E8.5).  1042 \n(E) Gene expression profiles for key transcription factors and key signaling pathways 1043 \n(SHH, WNT, BMP, NODAL, and FGF), in cells forming the posterior trunk in hTEM.v3, 1044 \nhuman, monkey and mouse embryo datasets. 1045 \n(F) Cartoon summary of similarities across human, monkey, mouse embryos with 1046 \nhTEM.v3 in developmental stages. 1047 \n 1048 \n 1049 \n 1050 \n 1051 \n 1052 \n 1053 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 59 \n 1054 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 60 \nFigure S7 Cross-species comparison between hTEM.v3 and embryos from 1055 \nhuman, monkey, and mice, related to Figure 5 1056 \n(A) Ortholog UMAP trajectory of organogenesis in early human (CS7/8/10) and monkey 1057 \n(CS8/9/11) embryos. See ‘Methods’ for published human and monkey embryo scRNA-1058 \nseq datasets. 1059 \n(B) Left, hTEM.v3 projection onto public human-monkey (H-M) embryo integration 1060 \ndataset. Middle, H-M cell types overlaid by hTEM.v3. Colors and annotations for H-M 1061 \nare consistent with those in (A). Right, hTEM.v3 cell types overlapping with H-M. Colors 1062 \nand annotations for hTEM.v3 are consistent with Figure S3L. 1063 \n(C) Sankey plot showing a detailed cell type overlay between hTEM.v3 and H-M 1064 \nembryos dataset. Colors and annotations for H-M are consistent with those in (A).  1065 \n(D) Top-left, UMAP showing cell types in mouse embryos (E7.0-E8.5). Top-right, UMAP 1066 \nshowing hTEM.v3 projection on mouse embryo data (E7.0-E8.5). Bottom-left, mouse 1067 \ncell types overlaid by hTEM.v3. Bottom-right, hTEM.v3 cell types overlapping with 1068 \nmouse embryos. Colors and annotations for hTEM.v3 are consistent with Figure S3L. 1069 \n(E) Sankey plot showing the detailed cell type overlay between hTEM.v3 and mouse 1070 \nembryo dataset. Colors and annotations for mouse embryos are consistent with those in 1071 \n(D).  1072 \n(F) Heatmap showing cluster similarities in listed cell types between hTEM.v3, H-M, and 1073 \nmouse embryos, respectively. The highest correlations are highlighted by black boxes. 1074 \nThe second highest is in red box. 1075 \n(G) Sankey diagram showing relationships between selected cell types in hTEM.v3 with 1076 \nhuman (CS7/8/10), monkey (CS8/9/11) and mouse embryos (E7.0-E8.5).  1077 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 61 \n(H) Heatmap showing the scaled expression pattern of genes involved in RA, FGF and 1078 \nWNT signaling pathways in indicated cell types across datasets of hTEM.v3 and 1079 \nembryos. 1080 \n 1081 \n 1082 \n 1083 \n 1084 \n 1085 \n 1086 \n 1087 \n 1088 \n 1089 \n 1090 \n 1091 \n 1092 \n 1093 \n 1094 \n 1095 \n 1096 \n 1097 \n 1098 \n 1099 \n 1100 \n 1101 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 62 \n1102 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 63 \nFigure 6 SHH signaling modulation and disease modeling using hTEM.v3 1103 \n(A) UMAP of integration of respective day-6 hTEM.v3 scRNA-seq data from WT (14,462 1104 \ncells), NOTO-LOF (16,081 cells), SANT1 (250 nM, days 4-6, 14,502 cells), and 1105 \nVANGL2-LOF (16,504 cells). 1106 \n(B) UMAP showing the re-analysis of notochord cells from WT, NOTO-LOF, and SANT1 1107 \nin (A). Pseudotime scores reflect the developmental direction indicating notochord 1108 \nmaturation. 1109 \n(C) UMAP showing expression of marker genes indicating notochord maturation. 1110 \n(D) Changes in cellular composition from NOTO-LOF (top) and SANT1 treatment 1111 \n(bottom) in contrast to WT. Dots represents log2(NOTO-LOF/WT or SANT1/WT) fold 1112 \nchanges for listed cell types.  1113 \n(E) Heatmap showing changes in the expression of key genes in regulation of 1114 \nnotochord development, neural tube D-V patterning, FGF and NOTCH signaling upon 1115 \nNOTO-LOF and SANT1 treatment. 1116 \n(F) Whole-mount immunostaining of dorsal (PAX6) and ventral (NKX6-1) fate changes 1117 \nin neural tube (SOX2) in WT, NOTO-LOF and SANT1 treated day-6 hTEM.v3. nt., 1118 \nneural tube. Scale bars, 100 μm.   1119 \n(G) Spatial expression patterns of representative PCP genes in day-6.5 hTEM.v3 1120 \n(sample 2 in Figure 4L). Dashed lines, areas of neural tube. Black arrows, somite 1121 \nsegments. 1122 \n(H) Comparison of length measurements using hTEM.v3 between WT, VANGL1-LOF, 1123 \nVANGL2-LOF and VANGL1/2-LOF (n ≥ 21 in each group at different times). ****p 1124 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 64 \nvalues < 0.0001 were calculated using students’ t-test. n.s., no statistical significance. 1125 \nResults were reproduced in more than 3 biological replicates.  1126 \n(I) Whole-mount Immunostaining of day-6 hTEM.v3 showing defects in neural tube 1127 \nelongation (SOX2) and somite (SIX1) segmentation using WT, VANGL1-LOF, VANGL2-1128 \nLOF and VANGL1/2-LOF lines. Arrow heads, somite segment. nt., neural tube. Scale 1129 \nbars, 100 μm. 1130 \n(J) Changes in cellular composition in VANGL2-LOF vs WT. Dots represents 1131 \nlog2(VANGL2-LOF/WT) fold changes for listed cell types.  1132 \n(K) Sub-clustering of PSM, pan-Somite, Caud.NP and Neural tube from VANGL2-LOF 1133 \nand WT samples shown in (A). 1134 \n(L) UMAP showing PCP gene expressions involved VANGL2-related human neural tube 1135 \ndefects. 1136 \n 1137 \n 1138 \n 1139 \n 1140 \n 1141 \n 1142 \n 1143 \n 1144 \n 1145 \n 1146 \n 1147 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 65 \n 1148 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 66 \nFigure S8 Morphological and molecular changes upon genetic and chemical 1149 \nperturbations in hTEM.v3, related to Figure 6 1150 \n(A) Schematic showing the CRISPR/Cas9 design and genotyping validations of NOTO-1151 \nLOF, VANGL1-LOF, VANGL2-LOF, and VANGL1/2-LOF hESC lines. 1152 \n(B) Representative images of day-6 hTEM.v3 in NOTO-LOF or, SANT1 treatment (250 1153 \nnM, days 4-6). Scale bars, 200 μm. 1154 \n(C) Comparison of length measurements of day-6 hTEM.v3 between WT, NOTO-LOF 1155 \nand SANT1 treatment (250 nM, days 4-6) (n = 6-8 each group). *p value < 0.05 and 1156 \n****p value < 0.0001 was calculated by Student’s t test. n.s., no significance. The results 1157 \nwere reproduced in more than 3 biological replicates. 1158 \n(D) qPCR showing changes in D-V patterning in WT, NOTO-LOF and SANT1 1159 \ntreatments (100 nM or 250 nM at days 4-6) in day-6 hTEM.v3. Data are presented as 1160 \nmean ±standard deviation. Results were reproduced twice. 1161 \n(E-G) Violin plot showing changes in expression of genes related to notochord, 1162 \nendotome and endothelial cells in NOTO-LOF or, SANT1 treatment (250 nM, days 4-6).  1163 \n(H) Violin plot showing expression of VANGL1 and VANGL2 across listed cell types in 1164 \nhTEM.v3 dataset from Figure S3L. 1165 \n(I) Representative bright field images of day-6 hTEM.v3 in VANGL1-LOF, VANGL2-1166 \nLOF, or VANGL1/2-LOF. Scale bars, 200 μm. 1167 \n(J) Scaled expression levels of key genes involved in axial elongation, PCP and 1168 \nNOTCH signaling in day-6 hTEM.v3 dataset from Figure 6A. 1169 \n 1170 \n 1171 \n 1172 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 67 \nMethods 1173 \nCulture of human ES and iPS cells 1174 \nThe following human ESC and iPSC lines were used: wild type H9-hESC (Sex: female, 1175 \nWiCell, WAe009-A), K3-iPSC (Sex: male)108 generated from human neonatal foreskin 1176 \nfibroblasts (ATCC, PCS-201-010), H9-hESC line carrying IRES-mClover3 allele in the 3’ 1177 \nUTR of PAX3 and IRES-mScarlet allele in the 3’ UTR of EBF2 (PAX3:mClover3; 1178 \nEBF2:mScarlet dual reporter), H9-hESC line carrying IRES-mScarlet allele in the 3’ 1179 \nUTR of NKX1-2 (NKX1-2:mScarlet reporter), H9-hESC line carrying IRES-mClover3 1180 \nallele in the 3’ UTR of PAX3 and IRES-mScarlet allele in the 3’ UTR of NOTO 1181 \n(NOTO:mClover3 reporter), H9-hESC line carrying IRES-mClover3 allele in the 3’ UTR 1182 \nof MYF5 (MYF5:mClover3 reporter), and H9-hESC derived NOTO-LOF, VANGL1-LOF, 1183 \nVANGL2-LOF, and VANGL1/2-LOF (Figure S8A). H9-hESC lines and K3-iPSCs were 1184 \nroutinely cultured and passaged as described previously.109,110 Briefly, 5 × 104/cm2 1185 \ncells were seeded onto culture-treated petri dishes coated with 1:200 diluted Geltrex 1186 \n(Thermo, A1413302). The basic culture media is in-house prepared using DMEM/F-12 1187 \nw/o glutamine (Thermo, 21331020 or Servicebio, G4514), supplemented with 0.5% 1188 \nProbumin (Sigma, 821001), 1x Antibiotic-Antimycotic (Thermo, 15240062), 1x MEM 1189 \nNEAA (Thermo, 11140050), 1x Trace Elements A/B/C (Corning), 64 μg/mL Ascorbic 1190 \nacid magnesium (TCI, A2521), 10 μg/mL Transferrin (Athens Research and 1191 \nTechnology), and 1 x GlutaMax (Thermo, 35050061). To maintain pluripotency of 1192 \nhESCs and hiPSCs, the basic culture media is completed by addition of 10 ng/mL 1193 \nHeregulin β1 (Qkine, QK045), 10 ng/mL Activin A (Qkine, QK001), 8 ng/mL FGF2-G3 1194 \n(145aa) (Qkine, QK052) and 200 ng/mL IGF-1 LR3 (Qkine, QK041). The complete 1195 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 68 \npluripotency maintenance medium is abbreviated as HAIF. hESC lines and K3-iPSCs 1196 \nwere cultured in HAIF with media changes every 24 hours at 37 °C in 5% CO2 and 1197 \npassaged at 90% confluency using Accutase (Thermo, 00-4555-56). 1198 \nGeneration of CRISPR/Cas9 knock-in fluorescent reporter H9-hESC lines   1199 \nTo generate knock-in fluorescent reporter lines from H9-hESCs, we utilized the 1200 \nhomology directed repair approach based on Cas9/gRNA introduced double strand 1201 \nbreak. The px330 vector expressing human codon-optimized Cas9 and sgRNA was 1202 \nobtained from Addgene. Oligos for sgRNA targets were individually cloned into px330 1203 \nusing restriction enzyme BbsI. Homology directed repair (HDR) donor arms flanking the 1204 \ninternal sequences of ribosome entry site (IRES) and fluorescent protein coding 1205 \nsequence (mClover3 or mScarlet) was synthesized and cloned into a pUC57 vector by 1206 \nBGI. The px330 with gRNAs targeting the gene of interest, homology donor arm vector 1207 \nand corresponding surrogate reporter vector (pRGS, PNA Bio) (vector ratio of 2:3:1, 1208 \ntotal of 9 μg vectors per 3 x 106 cells) were electroporated into H9-hESCs using a Neon 1209 \nelectroporation system (voltage = 1050 V, width = 30 ms, pulse = 2 cycles) (Thermo, 1210 \nMPK10025). To enhance cell viability, 1x CEPT111 (in-house preparation) was used 1211 \nduring electroporation and FACS sorting. Two days after electroporation, single cells 1212 \nwere isolated by FACS sorted and plated on pMEF (Sigma, PMEF-NL-P1) coated 96-1213 \nwell plates based on pRGS surrogate reporter signals. Single cell derived clones were 1214 \nscreened by genotyping PCR, expanded in HAIF and subject to hTEM protocols. 1215 \nTo generate CRISPR/Cas9 knock-out H9-hESC lines, gRNAs targeting upstream and 1216 \ndownstream regions of exon(s) were used together with a surrogate reporter vector. 1217 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 69 \nElectroporation and single cell clone screening procedures were the same as 1218 \ngenerating knock-in cell lines. All oligos for sgRNA targets are listed in the Table S1. 1219 \n 1220 \nGeneration of human trunk embryoid models (hTEMs) 1221 \nRoutinely 2D passaged hPSCs (H9-hESC lines and K3-iPSC) at 80% confluency were 1222 \ndissociated using Accutase for 5 min at 37 °C and counted by a hemocytometer. To 1223 \nstart 3D spheroid formation, a total of 1-2 million cells in 5 mL of HAIF medium with 10 1224 \nμM Y-27632 (Aladdin) were seeded per well of an ultra-low attachment 6-well plate 1225 \n(Thermo, 174929). The plate was then placed on an orbital shaker (Thermo, 88881104) 1226 \nat 110-120 rpm at 37 °C in 5% CO2. The next day, a media change consisting of HAIF 1227 \nwith 10 μM Y-27632 was applied. hPSC spheroids were allowed to form at size of 200-1228 \n220 μm in diameter within 40 hours of shaking. To generate hTEMs, hPSC spheroids 1229 \nwere collected with a wide-bore 1 ml tip and transferred into a 1.5 mL Eppendorf tube, 1230 \nthen washed with N2B27 basal medium. N2B27 basal medium comprises a 1:1 mix of 1231 \nDMEM/F12 and Neurobasal A (Thermo, 21103049) supplemented with 1×B27 (Thermo, 1232 \n17504001), 1× N2 (Thermo, 17502048), 1.5× GlutaMAX, 1×MEM NEAA, 1x Sodium 1233 \nPyruvate (Thermo, 11360039), 1x Antibiotic-Antimycotic, and 64 μg/mL Ascorbic Acid 1234 \nMagnesium. The HAIF primed hPSC spheroids (day 0 of hTEM) were then individually 1235 \ntransferred to a well of an ultra-low attachment 96 well plates (Thermo, 174929), subject 1236 \nto hTEM protocols.  1237 \nFor hTEM.v1, hPSC spheroids (day 0) were induced by culture in bi-NMP induction 1238 \nmedia composed of N2B27 basal media and supplements of 10 μM CHIR-99021 1239 \n(CHIR) (MCE, HY10182), 500 nM LDN-193189 (LDN) (Tocris, 1517128), 10 μM SB-1240 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 70 \n431542 (SB) (Aladdin, S125924), and 20 ng/mL FGF2-G3. 48 hours later, N2B27 basal 1241 \nmedia was replenished. At day 4, media is replaced by N2B27 basal media 1242 \nsupplemented with 4% Geltrex (Thermo, A1413302) of Matrigel (Mogengel, 827775) 1243 \nand 1 μM all-trans retinal (RAL) (Aladdin, A122355) to support somitogenesis and 1244 \nneural tube elongation.  1245 \nFor hTEM.v2, 100 nM SAG (Aladdin, S872455) was added at day 4-5 based on the 1246 \nhTEM.v1 protocol, followed by replenishment of N2B27 basal medium containing 4% 1247 \nMatrigel and 1 μM RAL at day 5.  1248 \nFor hTEM.v3, the hTEM.v1 day 0-2 protocol is modified by replacing SB with 20 ng/ml 1249 \nhuman recombinant CER1 (MCE, HY-P7822) for the first 24 hours. At day 2-3, media 1250 \nwas replenished with N2B27 basal medium supplemented with 0.2 ng/mL SHH (R&D, 1251 \n8908-SH), 2 ng/mL BMP4 (R&D, 314-BP), 1 ng/ml BMP2 (Qkine, QK007), 0.5 ng/ml 1252 \nBMP7 (R&D, 354-BP), 8ng/ml FGF2-G3, 4 ng/ml FGF3/4/8b/17 (MCE, HY-1253 \nP700065/HY-P7014/HY-P70533/HY-P700060), 1 nM all-trans retinoic acid (RA) 1254 \n(Aladdin, R106320) and 0.2% Matrigel. At day 3-4, medium was changed to N2B27 1255 \nbasal medium supplemented with 0.4 ng/ml SHH, 0.5 ng/ml BMP2/4/7, 10 ng/ml 1256 \nWNT5A (R&D, 645-WN) and 0.4% Matrigel. At days 4-7, medium was replaced with 1257 \nN2B27 medium containing 4% Matrigel and 200 nM RAL. 10 ng/ml WNT3A (R&D, 1258 \n5036-WN) and 0.2 ng/ml Heregulin β1 were included at days 4-7 to enhance neural 1259 \ntube genesis. 1260 \nAll hTEM cultures were limited to 7 days due to accumulated cell death in the anterior 1261 \nregion and ceased axial elongation. 1262 \n 1263 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 71 \nInclusion criteria of hTEM embryoids 1264 \nAll hTEM embryoids were collected using wide-bore tips from ultra-low attachment 96 1265 \nwell plates within 7 days of culture. Embryoids were inspected under a phase contrast 1266 \nmicroscope, based on morphometric features resembling human CS8-10 embryos. At 1267 \nday 3-4 (equivalent to CS8), embryoids undergo uniaxial symmetry breaking and 1268 \nbecome oval shaped. Embryoids with cylindrical morphology and visible mediolateral 1269 \nnarrowing near the caudal end were selected for further use. The neural plate is to be 1270 \nvisible as a groove along the midline extending from the posterior end and flanked by a 1271 \nshaded area anterior to the tailbud, reflecting the PSM cells (Figures 1B and 3B). If 1272 \nusing the NOTO:mClover3 or NKX1-2:mScarlet reporter line, polarized and caudal 1273 \naccumulation of NOTO+ or NKX1-2+ cells at day 3 and axial distribution of these cells in 1274 \nthe midline at days 4-5 (Figures S3F and S3G) were expected. This is equivalent to the 1275 \nC&E movements of caudal trunk progenitors in natural CS8 human embryos. Embryoids 1276 \nwith a correct body plan require culture in media supplemented with 4% Matrigel over 1277 \ndays 4-7 to allow somitogenesis and neural tube morphogenesis. At days 4-7 1278 \n(equivalent to CS9 to CS10) the following criteria were applied: (1) somites were to be 1279 \nclearly segmented after day 5; (2) presence of a lumen representing the closed neural 1280 \ntube was observed along the midline from hTEMs (Figures 1B, 2B, and 3B); (3) a single 1281 \nbody axis with less-dense tailbud cell populations on the posterior end and high-dense 1282 \nunsegmented somite cells surrounding the neural tube tip on the anterior end. At day 4, 1283 \n~70% of the embryoids with a correct body plan were collected for analysis or subject to 1284 \nfurther Matrigel culture. At days 5-7, ~30% of the hTEM.v1/2 and ~20% of hTEM.v3 1285 \nsatisfied the criteria specified above. 1286 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 72 \n 1287 \nMorphometric feature measurements 1288 \nThe length of hTEMs were analyzed using ImageJ. To calibrate the length in pixels, a 1289 \nstandard ruler is utilized to set the scale of images with the same magnification. The 1290 \nanterior-most and posterior-most points of the embryoids were set as start and end 1291 \npoints for measurements. The length was measured along a custom defined midline 1292 \nalong the direction of axial elongation. The counting of somite pairs was based on their 1293 \norder from the posterior end. The presence of two rows of somite segments flanking a 1294 \nneural tube structure was confirmed before counting all somite pairs along the A-P axis.  1295 \n 1296 \nRNA extraction & RT-qPCR analysis 1297 \nhTEMs RNA was extracted using E.Z.N.A. MicroElute Total RNA Kit (OMEGA, R6831-1298 \n02). 500 ng of total RNA was reverse transcribed into cDNA using iScript Reverse 1299 \nTranscription Supermix (Bio-rad, 1708841) according to manufacturer’s instructions. 1300 \nQuantitative real-time PCR was performed using Taq Pro HS Universal Probe Master 1301 \nMix (Vazyme, QN113-01) on QuantStudio 7 Pro Real-Time PCR System (Applied 1302 \nBiosystems, A43183). Expression levels for each gene re normalized to 18S rRNA 1303 \ncheck italics as an endogenous control using the ΔΔCt method. Taqman probes used in 1304 \nthis study are listed in the Table S1. 1305 \n 1306 \nScanning electron microscopy (SEM)  1307 \nEmbryoids were washed with DPBS to remove Matrigel, then fixed with 4% PFA for 30 1308 \nminutes at room temperature. Next, samples were treated with 1% osmium tetroxide in 1309 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 73 \ndistilled water for 1 hour and then washed three times with distilled water for 10 minutes 1310 \neach. Samples were then dehydrated by serial washes in 70% to 100% ethanol. Critical 1311 \npoint drying (CPD) was carried out using a Tourimis Samdri (PVT30) Critical Point 1312 \nDryer. To prepare samples for imaging, an 80:20 platinum/palladium sputtering process 1313 \nwas conducted with rotation, depositing a 3 nm conductive layer using the Quorum 1314 \nQ150T Automatic Coating System. Finally, SEM images were acquired using a Hitachi 1315 \nSU8010 cold-field emission scanning electron microscope at 9.8 kV. 1316 \nWhole-mount hTEM immunostaining and imaging 1317 \nhTEM embryoids were transferred to 1.5 ml Eppendorf tubes, fixed in 4% 1318 \nparaformaldehyde (PFA)/DPBS at RT for 1 hr and then washed with 1x DPBS at 10 min 1319 \nintervals for 30 min to remove residual PFA. For antigen retrieval, samples were 1320 \nimmersed in warm 0.5% SDS/DPBS (preheated at 55 °C) for 15 mins, followed by 1321 \nincubation with 0.5% Trition X-100 in DPBS for 30 min. After blocking samples with 1322 \nDuolink blocking solution (Sigma) for 30 min at room temperature, samples were 1323 \nincubated with primary antibodies, diluted in MAXbind staining medium (Active Motif) 1324 \novernight at 4 °C. Next, samples were washed three times with 0.04% Tween-20/DPBS 1325 \n(5 min each) and then incubated with DAPI  (TCI, 1ug/ml) and Alexa Fluor secondary 1326 \nantibodies (Thermo, 1:500 dilution) in MAXbind staining medium for 2 h at room 1327 \ntemperature. This was followed by one wash in 1 ml MAXwash washing medium (Active 1328 \nMotif) and two washes in 0.04 % Tween-20/DPBS (5 mins each) at room temperature. 1329 \nOptionally, a clearing procedure with Optimus Clearing Solution was used.112 Finally, 1330 \nsamples were individually transferred into chamber slides (iBidi, 81811), cured by 1331 \nProLong Gold antifade mountant (Thermo, P36934) overnight before imaging. All 1332 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 74 \nsamples were imaged and analysed using a BZ-X All-in-one inverted fluorescent 1333 \nmicroscope (Keyence) or SP8 inverted confocal microscope (Leica). Antibodies used in 1334 \nthis study are listed in the Key Resources Table.  1335 \nCryosectioning of hTEMs 1336 \nhTEM embryoids were washed twice with ice-cold DPBS to remove Matrigel and fixed 1337 \nby 4% PFA, then dehydrated in 30% sucrose (Aladdin, S112234) overnight at 4°C. 1338 \nSamples were then transferred into a 1cm x 1cm cryomold, positioned and embedded in 1339 \nOCT compound (Tissue-tek, 4583). OCT embedded molds were snap frozen in dry ice 1340 \nand 95% isopentane (Macklin, I813377) and transferred to -80°C overnight before 1341 \nsectioning. The frozen and fixed samples were sectioned using a cryostat microtome 1342 \n(Leica, CM1950) at 10 μm. Sections were kept at -20°C before further analysis. All 1343 \nbuffers in contact with hTEM embryoids were pre-treated with RNaseOUT (Thermo, 1344 \n10777019) to preserve RNA integrity for Visium HD process. 1345 \nTime-lapse imaging of hTEMs 1346 \nBright-field and fluorescent images of live hTEMs were taken with a BZ-X All-in-one 1347 \ninverted fluorescent microscope (Keyence) in the ‘Time lapse capture’ mode using a 1348 \n10× plan objective. Images were captured using the z-stack mode at a step depth of 6-1349 \n10 μm, spanning a total of ~100 μm. For time-lapse imaging, the incubator module was 1350 \nset at 37 °C and 5% CO2 and images taken every 30  min or 60 min. To generate the 1351 \ntime-lapsed video, stacked snapshots within the best focus range from each time point 1352 \nwere used. 1353 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 75 \n 1354 \nIn situ hybridization chain reaction (HCR) 1355 \nhTEM embryoids were fixed in 4% PFA for 30 min at room temperature (RT), followed 1356 \nby one wash with ice-cold DPBS. Before HCR, fixed samples were pre-treated with 1357 \n0.5% SDS/DPBS for 15 min at RT, washed twice with 0.5% Triton X-100/DPBS and 1358 \nwashed three times in 0.1% Tween-20/DPBS for 5 min each wash. HCR was performed 1359 \nfollowing manual instructions (Molecular Instruments). In brief, samples were incubated 1360 \nin hybridisation buffer (HB) for 5 min at RT, then for 30 min at 37 °C. Probes were 1361 \nprepared at 8 nM in HB and incubated for 30 min at 37 °C before use. Samples were 1362 \nthen incubated with probes for 12-16h on a thermal cycler (Bio-rad) at 37 °C. The next 1363 \nday, samples were washed with probe wash buffer (WB) three times for 15 min each at 1364 \n37 °C, then with 5x SSCT (5x SSC and 0.1% Tween-20 diluted in UltraPure Water) 1365 \nthree times for 15 min each at RT. Samples were then pre-amplified in probe 1366 \namplification buffer for >30 min at RT. Amplifier hairpins (h1 and h2) were prepared by 1367 \nheating at 95 °C for 90 sec separately followed by cooling down for 30 min at RT in 1368 \ndark. h1 and h2 were then mixed at 6 nM in amplification buffer and incubated with 1369 \nsamples for 12-16 hrs at RT in the dark. Before imaging, samples were washed three 1370 \ntimes in SSCT for 15 min, and stained with DAPI. All HCR images were acquired and 1371 \nprocessed with a Nikon Ti2E inverted microscope. All HCR probes with associated 1372 \nhairpins are listed in the Table S1. 1373 \nSingle-cell RNA-seq  1374 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 76 \nhTEMs (hTEM.v1; day3 = 35, day 4, n = 31, day 5, n = 22, day7, n = 11. hTEM.v2; day 1375 \n5, n = 22, day 7, n = 16; hTEM.v3; day 3, n = 35, day 4, n = 23, day 5, n = 12, day 6, n = 1376 \n9, day 7, n =14. NOTO-LOF; hTEM.v3 day 6, n = 11. SANT-1 (250 nM at days 4-6) 1377 \nhTEM.v3 day 6, n = 8. VANGL2-LOF; hTEM.v3 day 6, n =18.) were washed with N2B27 1378 \nbasal medium twice and then dissociated using 1:1 TrypLE (Thermo) and Accutase at 1379 \n37 °C for 10-15 min. Single cells were counted and adjusted to 106/ml in 1 ml N2B27 1380 \nbasal medium, filtered through a 40 μm cell strainer and loaded onto Chromium Single 1381 \nCell 3’ Library and Gel Bead Kit v3.1 or v4 (10× Genomics). Following the cDNA-1382 \namplification reaction, quality control and quantification was performed on the Agilent 1383 \n4200 Tapestation using the High Sensitivity D5000 kit (Agilent Technologies). Illumina 1384 \nsequencing libraries were constructed by fragmentation, end repair, A-tailing and 1385 \ndouble-sided size selection, adaptor ligation and sample-index PCR. Quality control and 1386 \nquantification of final libraries were performed on the Agilent 4200 Tapestation using the 1387 \nD1000 kit (Agilent Technologies) and Qubit 4 (Thermo Fisher Scientific). Libraries were 1388 \nthen sequenced on NextSeq 2000 (Illumina) with a customized sequencing run format 1389 \nuntil sufficient saturation was reached. 1390 \n 1391 \nPre-processing of scRNA-seq reads 1392 \nSingle-cell RNA-seq data generated in this study: 1393 \nAll sequencing reads in this study were mapped to the reference genome GRCh38 1394 \nusing cellranger (v9.0.1) with default parameters. Quality control and downstream 1395 \nanalysis were performed with in R (v4.3.3) with Seurat (v4.3.0.1). For each dataset, 1396 \ncells with fewer than 200 genes expressed or > 5% expressed mitochondrial genes 1397 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 77 \nwere removed. Doublets wre identified and removed using DoubletFinder (v2.0.4) 1398 \nimplemented in scutilsR (v0.1.1). Next, ambient RNA contamination was estimated, 1399 \ncounts were corrected using celda (v1.18.2) implemented in scutilsR (v0.1.1). Cells with 1400 \nless than 200 genes expressed after count correction were filtered out. Cell cycle phase 1401 \nscores were estimated using the corrected counts and these features were regressed 1402 \nout prior to dimension reduction and UMAP construction using Seurat. 1403 \nPublic Single-cell RNA-seq data: 1404 \nPublic and pre-processed datasets from E-MTAB-6967 (human CS7), HRA005567 1405 \n(human CS8), GSE155121 (human CS10), GSE193007 (Cynomolgus monkey 1406 \nCS8/9/11), E-MTAB-6967 (E7.0 to E8.5 with all known lineages within this period) were 1407 \ndownloaded and included for cross-species analysis. Data from human and monkey 1408 \nembryos were integrated based on ortholog using biomaRt (v2.62.0) and Seurat’s 1409 \ndefault integration pipeline. Cell-cycle genes were regressed out using the matrix 1410 \ngenerated by ScaleData function, followed by dimension reduction, UMAP visualization, 1411 \nand clustering. Cells belonging to extra-embryonic tissues were excluded from 1412 \ndownstream analysis to reduce the complexity of annotation and data visualization 1413 \n 1414 \nDataset integration and batch effect correction: 1415 \nTo analyze scRNA-seq datasets created in this study and public H-M embryo data 1416 \ngenerated on different platforms, Seurat, BBKNN or Harmony were used for integration 1417 \nand batch effect correction, based on best performance. Each dataset from different 1418 \nexperiments (this study or public) is considered a batch and contains at least one 1419 \nshared cell type. Briefly, log-normalized and scaled matrices from each dataset were 1420 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 78 \nintegrated using the best-performed approaches. Harmony (v1.0.1) was used for 1421 \nhTEM.v1 (days 4/5/7) and hTEM.v2 (days 5/7) data integration. BBKNN (v1.1.1) was 1422 \nused for hTEM.v3 (days 3-7) data integration. The reciprocal PCA from Seurat was 1423 \nused to integrate ortholog human (CS7/8/10) and monkey (CS8/9/11) embryo datasets. 1424 \nThe integration performance is justified given that the gene-expression profiles are from 1425 \nwell-studied cell populations that closely match with the identified cell type datasets 1426 \nreported here.  1427 \n 1428 \nRNA velocity analysis 1429 \nFor RNA velocity analysis, the raw fastq sequence data using scvelo (v0.2.5) were 1430 \nfirstly reanalyzed to obtain the count matrices containing the spliced and unspliced 1431 \nreads, followed by filtering out cells not subjected to UMAP projection and clustering 1432 \nanalysis. Then, RNA velocity was analyzed with velocyto (v0.17.17) in the Python 3.7 1433 \nenvironment. Parameters were min_shared_counts= 50 and n_top_genes= 2000 for 1434 \nscv.pp. filter_and normalized function, n_pcs= 30 and n_neighbors= 30 for scv.pp. 1435 \nmoments function. Mode was set to be stochastic when computing velocities. The 1436 \nvelocity was projected to the UMAP generated previously. 1437 \n 1438 \nPseudotime analysis 1439 \nBefore pseudotime analysis, the scaled matrix in Seurat was converted to the h5ad 1440 \nformat using coverFormat function in sceasy (v0.0.7) package. Palantir (v1.3.3) was 1441 \nthen used with default parameters in python (v3.9). Note that markers (NKX1-2 for 1442 \nneural, TBX6 for somitic and NODAL for notochord lineage) for differentiation initiation 1443 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 79 \nof each lineage were predetermined for each lineage. The resulting pseudotime scores 1444 \nwere displayed based on UMAP embeddings from sub-clustering of each lineage to 1445 \nelucidate developmental dynamics. The A-P axis inference is based on transcriptomic 1446 \nprofile of each lineage through time and cell fate specifications in a pseduotime ranked 1447 \nmanner. 1448 \n 1449 \nSCENIC analysis 1450 \nSCENIC (v1.3.1) analysis was performed on the integrated hTEM.v3 (days 3-7) scRNA-1451 \nseq data using R (v4.3.3) and Python (v3.13.2) with arboreto (v0.1.6). Firstly, SCENIC 1452 \nwas performed based on motif annotations (hgnc v9) and cisTarget resources of human 1453 \n‘hg38_refseq-r80_500bp_up_and_100bp_down_tss.mc9nr.feather’ and ‘hg38_refseq-1454 \nr80_10kb_up_and_down_tss.mc9nr.feather’ following default pipeline in 1455 \nhttps://htmlpreview.github.io/?https://github.com/aertslab/SCENIC/blob/master/inst/doc/1456 \nSCENIC_Running.html.  Next, the predicated regulon activity scores (RAS) represented 1457 \nby major transcription factors were associated to cell types of hTEM.v3 and grouped by 1458 \nhierarchical clustering to generate the regulon module enrichment heatmap. To this 1459 \nheatmap, correlation matrix was calculated using scaled cell type-associated RAS and 1460 \nclustered by using ‘method = pearson’ from clusterProfiler (v4.14.4) in R. Genes from 1461 \neach regulon module were subject to Gene Ontology analysis and visualized using 1462 \nigraph (v2.0.3) in R. 1463 \nCell-cell communication analysis  1464 \nCellChat (v2) was used to analyze intercellular communication following cluster 1465 \nannotation. The normalized gene expression matrix from the Seurat object was supplied 1466 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 80 \nas input and processed with default parameters. The CellChatDB. human database was 1467 \nused to infer the probability of cell-cell communication with “type = \"truncatedMean\", 1468 \ntrim = 0.1”. Significant ligand–receptor pairs (p < 0.05) were identified and assigned to 1469 \nsignaling pathways. For visualization of overall communication patterns, the summed 1470 \nexpression profiles of ligands and receptors were integrated with the predicted 1471 \ncommunication probability. 1472 \n 1473 \nCross-species cell type projection and comparative analysis 1474 \nSingle-cell transcriptomic datasets were obtained from CS8-CS11 cynomolgus monkey 1475 \nembryos and E7.0-E8.5 mouse embryos, together with the corresponding cell 1476 \nannotations. Gene symbols from monkey and mouse were converted to their human 1477 \northologues using the biomaRt package. To map hTEM.v3 cells onto UMAPs of H-M 1478 \nand mouse embryos, cells of hTEM.v3 were down-sampled to 20,000 and then 1479 \nprojected using reference mapping approach described in Seurat pipeline 1480 \nhttps://satijalab.org/seurat/articles/integration_mapping. Briefly, the conserved label 1481 \nanchors between selected objects were identified using FindTransferAnchors function, 1482 \nfollowed by MapQuery function. The overlapped cell types between objects were 1483 \ndetermined by using TransferData function. Cross-species cell type projection was 1484 \nsubsequently visualized in UMAP and Sankey plot to assess cell type conservation 1485 \namong datasets. ClusterSimilarity was used to determine the pairwise correlation co-1486 \nefficient across cell types from hTEM.v3 and embryos. 1487 \n 1488 \nPreparation of Visium HD spatial gene expression assay 1489 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 81 \nPFA fixed and OCT frozen sectioning (hTEM.v2 and hTEM.v3) slides were prepared as 1490 \ndescribed in the previous section. Sectioning follows the Visium HD Fixed Frozen 1491 \nTissue Preparation Handbook (10x Genomics, CG000764) for the workflows. 1492 \nSequencing was conducted by the Single Cell & Spatial Omics Core, School of 1493 \nBiomedical Sciences at The Chinese University of Hong Kong 1494 \n(https://www3.sbs.cuhk.edu.hk/en/core_laboratories/single-cell-omics-core/.). 1495 \n 1496 \nProcessing of Visium HD data and visualization 1497 \nRaw hTEM.v2 and hTEM.v3 Visium HD sequencing reads were mapped to the 1498 \nreference genome using spaceranger (v3.1.3) with default settings then, processed and 1499 \nanalyzed with Seurat (5.2.1) running on R (4.4.2). For quality control, spots containing 1500 \nless than 700 total UMI counts were filtered out, as these spots represent areas outside 1501 \nof the actual tissue. Similarly, low-quality spots containing less than 200 total UMI 1502 \ncounts and spots containing > 5% mitochondrial counts were filtered out. Following 1503 \nquality control, 3091 spots of transversely sectioned and 39212 spots of longitudinally 1504 \nsectioned day-7 hTEM.v2 were retained with a median transcript read out of 4180 and 1505 \n2122 unique molecular identifiers (UMI), respectively. For the spatial transcriptome of 1506 \nhTEM.v3, a total of 34,884 (median UMI 2,360) and 58,646 (median UMI 1,260) spots 1507 \nwere retained from day 4 and day 6.5 sections, respectively.  1508 \n  Filtered read counts were then processed following the standard Seurat protocol 1509 \n(https://satijalab.org/seurat/articles/visiumhd_analysis_vignette). Briefly, samples were 1510 \nnormalized and scaled, followed by dimension reduction using PCA and UMAP. The 1511 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 82 \nFindNeighbors function was followed by RunUMAP function with 50 dimensions. The 1512 \nprimary clusterings of Visium HD data were identified via the FindClusters function for 1513 \ncrude cell type annotations. Unsupervised clustering was conducted using a bin size of 1514 \n8 μm and Seurat default settings. All other parameters used for each function were kept 1515 \nat default values. 1516 \nTo deconvolute the spot-level data for accurate cell type annotations, the Robust Cell 1517 \nType Decomposition (RCTD) approach from Seurat was applied using the reference cell 1518 \ntypes from integrated hTEM.v3 (days 3-7) scRNA-seq data. RCTD clusters were 1519 \nannotated by leveraging the cell type reference in Figure S3L. To visualize spatial 1520 \nUMAP of marker gene expression, customized scripts were devised using 1521 \nSpatialDimPlot function in Seurat for displaying weight normalized read counts listed in 1522 \nFigure 4L-4N, 6G, S5E-S5H. 1523 \n 1524 \nQuantification and statistical analysis 1525 \nAll statistical results and graphs were generated by Graphpad Prism. The numbers of 1526 \nsamples and types of statistical analyses are given in the figure captions 1527 \nand results sections. 1528 \n 1529 \n 1530 \n 1531 \n 1532 \n 1533 \n 1534 \n 1535 \n 1536 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted December 22, 2025. ; https://doi.org/10.64898/2025.12.20.695666doi: bioRxiv preprint \n\n 83 \nReferences 1537 \n 1538 \n1. Miao, Y., and Pourquié, O. (2024). 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