Segregation of Neural Crest Specific Lineage Trajectories from A

bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 available under aCC-BY 4.0 International license.

1 Segregation of neural crest speciﬁc lineage trajectories from a heterogeneous

2 neural plate border territory only emerges at neurulation.

1,2 2,3 2 1,∗ 3 Ruth M Williams , Martyna Lukoseviciute , Tatjana Sauka-Spengler , and Marianne E Bronner

4 Abstract The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harbouring neural crest and cranial placode progenitors. Here we profile avian epiblast cells as a function of time using single-cell RNA-seq to define transcriptional changes in the emerging ‘neural plate border’. The results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridisation. Developmental trajectory analysis shows that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined lineages.

5 Keywords: neural plate border, neural crest, single-cell, placode, Pax7, chick

6 Introduction

7 During gastrulation, the ectoderm layer of the chordate embryo becomes segregated into the neural plate and the 8 surrounding non-neural ectoderm. The neural plate ultimately generates the central nervous system (CNS), whereas 9 the surrounding non-neural ectoderm forms the epidermis of the skin as well as the epithelial lining of the mouth and 10 nasal cavities. At the interface of these tissues is a territory referred to as the ‘neural plate border’ which in vertebrates 11 contains precursors of neural crest, neural and placodal lineages (Ezin et al., 2009, Streit, 2002). Neural crest and 12 ectodermal placodes share numerous common features including the ability to migrate or invaginate and form multiple 13 cell types. During neurulation, neural crest cells come to reside within the dorsal neural tube where they undergo 14 an epithelial-to-mesenchymal transition (EMT). Subsequently they delaminate and migrate throughout the embryo, 15 settle at their ﬁnal destinations and differentiate into numerous derivatives including neurons and glia of the peripheral 16 nervous system as well as cartilage, bone and connective tissue of the head and face. Like neural crest cells, cranial 17 placode cells become internalised, and then differentiate into sensory neurons and sense organs (nose, ears, lens) of the 18 head. Aberrant neural crest or placode development causes a number of developmental disorders affecting craniofacial 19 structures (Siismets and Hatch, 2020, Vega-Lopez et al., 2018), the enteric nervous system (e.g. Hirschsprung’s disease) 20 (Butler Tjaden and Trainor, 2013) and the heart (e.g., Persistent Truncus Arteriosus; CHARGE syndrome) (Pauli et al., 21 2017, Gandhi et al., 2020). Furthermore, a number of malignancies, including melanoma, neuroblastoma and glioma, 22 are known to arise from neural crest derivatives (Tomolonis et al., 2018).

23 An ongoing question is whether individual neural plate border cells are specified toward a particular lineage (i.e., 24 neural crest, placode or CNS) or if they have the potential to become any cell type that arises from the border. It has 25 been suggested that progenitors of these different lineages may be regionalised within the neural plate border, with the 26 more lateral cells contributing to the placodes and the more medial region giving rise to neural and neural crest cells 27 (Schlosser, 2008). Such segregation has been proposed to result from the influence of graded expression of signalling 28 factors emanating from surrounding tissues, e.g. Wnts and FGFs, on multipotent neural plate border cells (Schille and 29 Schambony, 2017). Alternatively, individual neural plate border cells predetermined toward a particular lineage may be 30 intermingled within the border. A recent study in the chick (Roellig et al., 2017) showed that while medial and lateral 31 regions of the border can be discerned by lineage markers, there is significant co-expression of markers characteristic 32 of multiple lineages (neural crest, neural plate and placodal) across this region. Moreover, this overlap of lineage 33 markers within the neural plate border is maintained from gastrulation through neurulation suggesting that these cells

∗Lead and corresponding author: Marianne Bronner ([email protected]) 1California Institute of Technology, Division of Biology and Biological engineering, Pasadena, 91125, USA 2University of Oxford, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK 3Current address: Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

34 may maintain plasticity through neurulation stages. Thus, while some neural plate border cells may be predisposed 35 toward a particular fate, others retain the ability to generate multiple lineages. However, the timing at which neural 36 plate border cells emerge and become distinguishable from neural and non-neural ectoderm has not been conclusively 37 characterised, complicating the assessment of cell heterogeneity within the neural plate border territory.

38 One study using an ex ovo culturing method of explants from the chicken embryos proposed that a pre-neural 39 plate border region is established as early as stage Hamburger and Hamilton (HH) 3 (Prasad et al., 2020). In addition, 40 an in vitro model to derive neural crest cells from human embryonic stem cells shows that pre-border genes can be 41 induced by Wnt signaling (Leung et al., 2016). However, these observations have not been thoroughly addressed 42 in vivo. Therefore, the questions of how and when neural plate border cells establish/retain multipotency and the 43 comprehensive gene dynamics underlying these processes remain open.

44 Single-cell RNA-sequencing (scRNA-seq) provides a unique platform to address the intriguing question of when 45 a neural plate border transcriptional signature arises in an in vivo context. To this end, we examined single-cell tran- 46 scriptomes from the epiblast of gastrulating to neurulating chick embryos to determine the time course of emergence 47 of the neural plate border. The chick represents an ideal system for these studies since avian embryos develop as a flat 48 blastodisc at the selected time points, highly reminiscent of human development at comparable stages. Interestingly, 49 our results show that the border is not transcriptionally distinct until the beginning of neurulation (HH7), when neural 50 plate border markers define a discrete subcluster of the ectoderm. Furthermore, RNA velocity measurements show that 51 segregation of the neural plate border commences at HH6, but is more profoundly underway at HH7, with definitive 52 neural crest clusters only emerging in the elevating neural folds. We further demonstrate that Pax7+ cells are not re- 53 stricted to a neural crest fate and are capable of giving rise to all derivatives of the neural plate border. The data also 54 reveal numerous novel factors dynamically expressed across the developing epiblast as well as revealing key drivers of 55 neural plate border trajectories. Taken together, the data reveal dynamic changes in an emerging neural plate border 56 that becomes progressively segregated into defined lineages, that is complete only late in neurulation.

57 Results

58 Single-cell analysis of the avian epiblast during gastrulation (HH4 – 5)

59 To resolve the transcriptional signatures of individual neural plate border cells in the context of the developing 60 embryo, we ﬁrst performed single-cell RNA-seq analysis of the epiblast of gastrulating chick embryos at stages HH4-5 61 (Hamburger and Hamilton, 1951). We used the Chromium 10X platform in order to recover a large number of cells, 62 thus proﬁling the majority of neural plate border cells. As a reference for emergence of the neural plate border, we 63 used Pax7 and Tfap2A transcription factors, both of which are well-established early markers of the neural plate border 64 (Basch et al., 2006, de Croze et al., 2011). Tfap2A demarcates the lateral aspect of the neural plate border from HH4 65 and is also expressed in the non-neural ectoderm (Figure 1A). Pax7 is progressively enriched in the medial border 66 region from HH5 (Figure 1D).

67 At HH4 (Figure 1A), we recovered 2398 cells from 8 embryos. Five distinct clusters (2064 cells) were resolved after 68 quality control processing (Figure 1B, Figure S1A) and annotated by marker genes identiﬁed by single-cell differential 69 expression (SCDE) analysis (Figure S1B/C). Focusing on the future neural plate border, two ectoderm clusters were 70 recovered (HH4-Cl0, HH4-Cl2), identiﬁed by the enrichment in Cldn1 expression (Figure S1C). HH4-Cl0 was enriched 71 for neural plate markers (Otx2 and Fgfr1), and featured low levels of Tfap2A and Dlx5 (Figure 1C). Signaling molecules 72 including Sfrp2 and Pdgfa were also enriched in HH4-Cl0. Sfrp2 and Fgfr1 are expressed across the neural plate, 73 extending to the neural plate border (Chapman et al., 2004, Lunn et al., 2007) and Pdgfa was expressed in the posterior 74 epiblast (Yang et al., 2008). bHLH transcription factor MafA, more commonly associated with pancreatic beta-cell 75 differentiation (Hang and Stein, 2011), but also reported in the developing chick neural plate (Lecoin et al., 2004), was 76 enriched in HH4-Cl0 (Figure S1C). HH4-Cl2 was less distinctive but enriched in genes associated with pluripotency, 77 such as Sall4, Tgif1, ElavL1 (Lee et al., 2015, Ye and Blelloch, 2014, Zhang et al., 2006), (Figure S1B/C) suggesting 78 these may represent residual epiblast stem cells.

79 To reﬁne our analysis of the prospective neural plate border, we next performed 10X single-cell RNA-seq on 80 dissected neural plate border regions from HH5 embryos (8 dissections) (Figure 1D) yielding 6363 cells and 6 clusters 81 (Figure 1E, Figure S1D). Prospective neural plate border cells were conﬁned to cluster HH5-Cl4, enriched for Pax7 82 and Tfap2A expression, but also featuring a neural marker Sox3, and transcription factor Gbx2, which has a known role 83 in neural crest induction in Xenopus (Li et al., 2009) (Figure 1F). While neural plate border markers were enriched in

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84 HH5-Cl4 cells, co-expression of neural and non-neural ectoderm factors here (Figure S1E/F) suggests this intermediate 85 region is not yet distinguishable as a unique entity.

A HH4 D HH5 * G HH6 * J HH7 * ** ** ** ** ** **

Merge Merge Merge * * * Tfap2A Tfap2A Tfap2A

Pdgfa Pax7 Pax7 Pax7 Tfap2A Tfap2A Pax7 Tfap2A Pax7 Tfap2A Pax7

Ectoderm/ B 5.0 Mitochondrial E H Cardiac K Neural plate Anterior LPM genes mesoderm Posterior Neural plate Ectoderm Pdgfa LPM Sox3 5.0 Sfrp2 MafA 5.0 Frzb Mitochondrial Olfm1 Paraxial Fgfr1 genes Cardiovascular Sox21 Tfap2A mesoderm LPM Paraxial 2.5 Pdgfa 5.0 progenitors Sox2/3 Endoderm Gbx2 0 mesoderm Otx2 0 Streak/ Hensens 1 Sox17 1 early mesoderm 0 2 FoxA2 Posterior Node 2 1 3 0 Dll1 3 Cxcr4 Node 2 LPM 4 1 Meis1 0.0 4 3 5 0.0 Neural plate/ 2 Cdh2 5 0.0 4 Endoderm 6 tube UMAP_2 UMAP_2 UMAP_2 3 UMAP_2 6 5 7 4 0.0 Cldn1 7 6 8 Primitive 8 9 Frzb 7 9 Endoderm streak Tfap2A 10 Head Dnmt3a 10 Sox17 Mesoderm Pax7 11 Epiblast stem cells FoxA2 Proliferating mesenchyme −2.5 Neural plate Otx2 −5.0 Non-neural Sall4 Cxcr4 Cdx4 −5.0 Pdgfa cells Ectoderm Sox2 Endoderm Hensens Id3 Mesoderm Six1 Gata2 Non-neural Tfap2A −5.0 Sox3 Node Tgif1 Tbxt Alx1 Lmo2 Ectoderm Dlx5 Twist1 Six3 ElavL1 Cdx4 Olfm3 Dlx5 Astl Gata5 FoxD3 −5.0 Pitx2 Gata3 Pax6 2064 cells 6363 cells Tcf15 4294 cells Bmp4 4000 cells −5 0 5 −10 −5 0 5 −5 0 5 10 −5 0 5 UMAP_1 UMAP_1 UMAP_1 UMAP_1

C Tfap2A Dlx5 F Tfap2A Pax7 I Tfap2A Pax7 L Tfap2A Pax7

2.5 2.0 2.0 3 3 2.0 2 2 1.5 1.5 1.5 1.5 2 1.0 2 1.0 1.0 1.0 1 1 0.5 1 0.5 0.5 1 0.5 0.0 0 0.0 0 0 0.0 0 0.0

Otx2 Fgfr1 Gbx2 Sox3 Sox2 Dlx5 Sox2 Dlx5

2.5 3 3 2.5 2.0 2.0 2.0 2.0 2 1.5 1.5 2 2 1.5 1.5 2 1.0 1.0 1.0 1 1.0 1 1 1 0.5 0.5 0.5 0.5 0 0.0 0 0.0 0.0 0 0.0 0

Figure 1: Single-cell RNA-seq of avian epiblast from HH4 through HH7. (A) Tfap2A expression at HH4, detected by HCR. (B) UMAP plot depicting 5 clusters resolved from 2064 epiblast cells at HH4. (C) Feature plots of selected genes in HH4 clusters. (D) Pax7 and Tfap2A expression at HH5 detected by HCR. (E) UMAP plot depicting 8 clusters resolved from 6363 cells from dissected neural plate border regions at HH5, black dotted region in (E). (F) Feature plots of selected genes in HH5 clusters. (G) Pax7 and Tfap2A expression at HH6 detected by HCR. (H) UMAP plot depicting 12 clusters resolved from 4294 epiblast cells at HH6. (I) Feature plots of selected genes in HH6 clusters. (J) Pax7 and Tfap2A expression at HH7 detected by HCR. (K) UMAP plot depicting 11 clusters resolved from 4000 epiblast cells at HH7. (L) Feature plots of selected genes in HH7 clusters.

86 Single cell analysis of the avian epiblast during neurulation (HH6 - 7)

87 During the process of neurulation in amniote embryos, the neural plate gradually folds inwards on itself, the border 88 edges elevate forming the neural folds which, as neurulation progresses, fuse at the dorsal midline to form the neural 89 tube. In the chicken embryo, this process is completed by HH8. Therefore, we performed single cell analysis at HH6 90 and HH7 to capture changes in the neural plate border as a function of time. While expression of Pax7 was detectable 91 but low during gastrulation stages (HH4/5), its expression is strongly enhanced by HH6/7 (Figure 1G/J), when this 92 transcription factor marks more medial neural plate border cells.

93 Single-cell analysis at HH6 showed increased complexity revealing 12 distinct clusters from 4294 cells (Figure 1H, 94 Figure S2A). A neural plate cluster (HH6-Cl8) was characterised by neural genes including Sox2/3, Hes5 and Otx2 95 (Figure 1I, Figure S2B/C) as well as neural crest regulators FoxD3, Ednrb and Gbx2 (Figure S2B), whereas Tfap2A 96 and Dlx5 were enriched in the cluster HH6-Cl10. Pax7 was heterogeneously detected at the interface of HH6-Cl8 and 97 HH6-Cl10 (Figure 1I), indicating both these clusters harbour neural plate border cells.

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98 At HH7 (Figure 1J), we identiﬁed 11 clusters from 4000 cells (Figure 1K, Figure S2D). Cells of the neural plate 99 appeared in HH7-Cl3 and were distinct from the non-neural ectoderm cells found in HH7-Cl9 (Figure 1K/L). In 100 addition to neural markers detected at earlier stages (Otx2, Sox2, Sox3), other neural/neural crest genes featured in HH7- 101 Cl3, including Zeb2 and Zic2 (Figure S2E/F). Additional factors, such as Pax6, a crucial regulator of eye development 102 (Liu et al., 2006) featured in the cluster HH7-Cl9 (Figure S2E). Zfhx4 (Figure S2E), a zinc ﬁnger transcription factor 103 previously observed at later stages in the neural crest and neural tube (Williams et al., 2019) was featured across 104 HH7-Cl3 and HH7-Cl9 clusters.

105 Overall, across the stages analysed, we observed a progressive refinement of transcriptional signatures in individual 106 ectoderm clusters reflecting neural versus non-neural lineages. While we identified markers of the neural plate border 107 within multiple clusters, the border itself, surprisingly, is not distinguishable as a unique entity. Highlighting the het- 108 erogeneity of cells within the neural plate border, defined by combinatorial gene signatures shared with the surrounding 109 neural plate and non-neural ectoderm.

A Neural plate HH5-Cl4 C Caudal neural HH6-Cl8+Cl10 E HH7-Cl3+Cl9 Otx2 Msx1 Neural plate border Sox2 Wnt5A/B Pax7 Sox21 Caudal neural Hes5 Non-neural Tfap2a 2 Gbx2 ectoderm Dlx5 HoxB1 Tfap2a Bmp4 Caudal neural Msx1 Cdx2 Dlx5 2 2.5 Znf703 Msx2 Gbx2 BMP4 Six1 Gata2/3 0 0 0 0 0.0 1 0 1 1 2 2 2 3 4 Mid-neural Rostral Lmo1 −2.5 neural Sox21 −2 −2 Six3 Frzb Cyp26a1 Non-neural Dlx2 Sox2 ectoderm Neural plate Otx2 Tfap2a Notochord Otx2 progenitors Tfap2c Sox2 Dlx5 −5.0 FoxD3 −4 Six3 Shh −4 664 cells 373 cells 1077 cells Chrd −2.5 0.0 2.5 −4 −2 0 2 4 −3 0 3

B Sox2 Tfap2A D Sox2 Tfap2A F Pax7 Msx1

2.5 2.0 1.5 2.0 3 1.5 1.5 1.5 1.0 1.5 2 1.0 1.0 1.0 1.0 0.5 1 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0

Dlx5 Msx1 Dlx5 Msx1 Bmp4 Tfap2A

3 2.5 2.5 2 2.0 2.0 2.0 2 2 1.5 1.5 1.5 1 1.0 1.0 1 1.0 1 0.5 0.5 0.5 0 0.0 0 0 0.0 0.0

Pax7 Bmp4 Pax7 Bmp4 Tfap2B Dlx5

2.0 1.5 1.6 1.5 1.5 1.0 2 1.2 1.0 1.0 1.0 0.8 0.5 0.5 1 0.5 0.4 0.5 0.0 0.0 0.0 0.0 0

Figure 2: Subclustering ectoderm clusters extracted from whole epiblast data. (A) UMAP plot depicting 3 clusters resolved from HH5-Cl4. (B) Feature plots of selected genes in HH5 subclusters. (C) UMAP plot depicting 3 clusters resolved from HH6-Cl8 and HH6-Cl10. (D) Feature plots of selected genes in HH6 subclusters. (E) UMAP plot depicting 5 clusters resolved from HH7-Cl3 and HH7-Cl9. (F) Feature plots of selected genes in HH7 subclusters.

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110 Subclustering reveals progressive transcriptional segregation of ectoderm cells

111 As the chick embryo undergoes gastrulation, ectodermal cells that will become neural, neural plate border, placode 112 or epidermis remain in the upper layer of the embryo (epiblast), while mesoendodermal cells ingress and internalise at 113 the primitive streak and Hensen’s node. To further resolve the transcriptional complexity of the developing neural plate 114 border, we extracted and subclustered the ectodermal clusters at stages HH5, HH6 and HH7. These are designated 115 HH5-Cl4, HH6-Cl8/10 and HH7-Cl3/9 (Figure 1E, H, K).

116 HH5-Cl4 resolved into 3 closely related clusters (Figure 2A). Neural plate (Sox2, Otx2, Sox21, Six3) subcluster 117 (HH5-sub-1; Figure S3A) versus the non-neural ectoderm subcluster (Tfap2A, Dlx5; HH5-sub-2) were clearly de- 118 lineated (Figure 2B). The third subcluster, HH5-sub-0, contained cells expressing caudal neural plate (Msx1, Pdgfa, 119 Wnt8A, Pou5f3) and caudal epiblast genes (Tbxt, Cdx2/4, Meis1/2; Figure S3A/B). Pax7+ cells were most prominent 120 in HH5-sub-0 (caudal) but a small portion of HH5-sub-2 (non-neural ectoderm) cells also expressed Pax7 (Figure 2B). 121 HH5-sub-0 cells co-expressed other neural plate border genes Tfap2A, Dlx5, Msx1 and Bmp4 with Pax7, these genes 122 were also present in other clusters (Figure 2B).

123 Similar signatures were found at HH6 (Figure 2C/D) but with increasing transcriptional segregation. Here, the 124 neural plate was represented by HH6-sub-1 (Sox2, Otx2, Six3; Figure 2D, Figure S3C). Non-neural ectoderm markers 125 (Tfap2A, Dlx5) were enriched in HH6-sub-0 (Figure 2D). Pax7+ cells clustered within the HH6-sub-2 (Figure 2D), 126 together with caudal epiblast markers (Tbxt, Cdx2/4) (Figure S3C); however, Pax7 expression also featured in HH6- 127 sub-0 and HH6-sub-1, with Pax7+ cells positioned at the interface of the 3 subclusters (Figure 2D). These Pax7+ 128 cells at the clusters’ interface also expressed Tfap2A, Msx1, Dlx5 and Bmp4 which were enriched in other clusters, as 129 observed at HH5. This demonstrates that the neural plate border cluster is not yet distinct and indicates that neural 130 plate border cells share signatures with other tissues at this stage (Figure 2D).

131 Further transcriptional reﬁnements occurred between the HH5 and HH6 ectoderm subclusters. Irf6 which was 132 broadly present across HH5 subclusters, was now enriched in HH6-sub-0 (non-neural ectoderm), along with Grhl3 133 which was present in a small portion of HH5-sub-2 (non-neural ectoderm; Figure S3B/D). FoxD3 expression was 134 barely detectable in cells of HH5-sub-1 (neural) but was clearly enriched in HH6-sub-1 (neural) (Figure S3B/D). Gbx2 135 was broadly expressed across caudal epiblast and non-neural ectoderm subclusters (HH5-sub-0, HH5-sub-2 and HH6- 136 sub-0, HH6-sub-2), while Znf703 was restricted to more caudal epiblast cells (HH5-sub-0 and HH6-sub-2) (Figure 137 S3B/D). Gata2 and Gata3 were found in a portion of HH5-sub-2 cells but their expression was expanded across HH6- 138 sub-0 (Figure S3B/D). The placode marker Six1 was found at low levels across all subclusters at HH5, but at HH6, Six1 139 was restricted to only a portion of HH6-sub-0, potentially to cells within a placode progenitor niche (Figure S3B/D).

140 At HH7, ectodermal cells formed 5 discrete clusters (Figure 2E), suggesting lineage segregation was ongoing at 141 this stage. Here neural plate border markers Pax7, Msx1 and Bmp4 were found in a niche of cells within HH7-sub-1, 142 which broadly expressed Tfap2A and other non-neural ectoderm markers (Figure 2F, Figure S3E). Neural crest genes 143 (Tfap2B, Draxin, Snai2) were arising in the Pax7+ domain (Figure S3F). HH7-sub-3 subcluster was characterised by 144 neural markers; Six3, Otx2, Sox21 and Sox2 (Figure S3E). Some HH7-sub-1 cells also expressed placode markers 145 (Dlx5, Pax6, Six1/3) but these cells did not co-express Pax7 (Figure 2F; Figure S3E/F). Importantly, this reﬂects 146 segregation of the neural plate border into medial (Pax7+) and lateral (Tfap2A+) regions, as well as highlighting the 147 overlap and combination of genes co-expressed across these regions. HH7-sub-1 also featured Irf6 expression, and 148 Grhl3 was present in a subset of cells which co-expressed Irf6 and Tfap2A (Figure S3F). Another subset of HH7-sub-1 149 cells expressed Pax6, possibly already delineating prospective lens placode at this stage (Figure S3F), as these cells 150 co-expressed Six3, known to activate Pax6 by repressing Wnt signaling (Liu et al., 2006). HH7-sub-0 and HH7-sub-2 151 were also characterised as neural plate subclusters and could be resolved by axial markers, whereby mid-neural plate 152 markers Lmo1 and Nkx6.2 were found in HH7-sub-0 and more caudally expressed genes Cdx2/4, Hes5, Znf703 were 153 found in HH7-sub-2 (Figure S3E/F). Cells in HH7-sub-4 were enriched for Shh, Chrd and FoxD3, suggesting they 154 were notochord progenitors (Figure S3E).

155 In summary, analysis of our single cell HH5 and HH6 transcriptomes did not identify a distinct cell cluster marked 156 by known neural plate border genes; rather cells expressing these markers were distributed across neural and non-neural 157 clusters. Our analysis reveals the complex heterogeneity of the developing neural plate border and shows neural plate 158 border cells are not distinct as a transcriptionally separate group of cells until HH7 when they also begin to segregate 159 into presumptive medial and lateral territories. Moreover, many factors are shared between the neural plate border 160 and other ectodermal cells as the neural plate border is progressively established from the neural plate and non-neural 161 ectoderm.

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162 Validation of gene expression using Hybridisation Chain Reaction reveals dynamic in vivo expression patterns of novel

163 neural plate border genes.

164 We validated the in vivo expression pattern of intriguing genes identiﬁed in our single-cell datasets, using ﬂuores- 165 cent /textitin situ hybridisation (Hybridisation Chain Reaction, HCR) (Choi et al., 2018), which enables simultaneous 166 expression analysis of multiple transcripts. Embryos from HH4 through HH10 were screened to glean the time course 167 of gene expression.

168 We identiﬁed several new genes in the ectoderm, many of which persisted into neural or non-neural tissues. At 169 HH4 the chromatin remodeler, Ing5, Astacin-like metalloendopeptidase, Astl, and neuronal navigator 2, Nav2 were 170 enriched in the ectoderm cluster (HH4-Cl0) (Figure 3A, Figure S1C). At stages HH4-HH6 Ing5 was expressed across 171 the neural plate border, whereas Astl expression was predominantly detected in the posterior neural plate border (Figure 172 3B, Figure S4A). While both genes overlapped with Tfap2A, Ing5 expression spread into the neural plate whereas Astl 173 expression extended more laterally into the non-neural ectoderm (Figure 3B’). At HH8- Astl expression continued in 174 the developing neural plate border and surrounding non-neural ectoderm, and also the neural plate (Figure 3C, 3C’). 175 Ing5 was also detected in the neural plate border and in the neural folds (Figure 3C). By HH8, Astl expression was 176 prominent in the neural folds, most strongly in the posterior hindbrain (Figure 3D, 3D’). Ing5 was detected along the 177 neural tube at HH8 (Figure 3D, 3D’); however, by HH10 Ing5 transcripts were no longer detectable. Astl expression 178 was also broadly decreased at HH10, where activity was restricted to a small region of the neural tube in the hindbrain 179 and the emerging otic placodes (Figure S4A). Consistent with these observations Astl and Ing5 were detected in HH5- 180 Cl4 and HH6-Cl10 (Astl)/HH6-Cl8 (Ing5). Astl was seen in HH7-Cl9, Ing5 was not differentially expressed at this 181 stage but was present in HH7-Cl3 (Figure S4B).

182 Nav2, detected in ectoderm clusters HH4-Cl0, HH5-Cl4, HH6-Cl10 and HH7-Cl9 was expressed across the ecto- 183 derm and neural plate at HH5–HH7, where it over-lapped with the neural marker Sox21 (Figure S4C/D), continuing 184 into the neural folds at HH8 where Nav2 was particularly prominent in the hindbrain (Figure S4C). At HH9 Nav2 185 expression was restricted to rhombomeres 2 and 4, whereas Sox21 was more broadly expressed along the neural tube 186 axis (Figure S4C).

187 We identified a number of transcription factors in HH6-Cl8 (neural) and HH6-Cl10 (non-neural ectoderm). The 188 latter was characterised by Tfap2A enrichment and also harboured Grhl3 and Irf6 (Figure 3E). In situ analysis showed 189 Grhl3 was specifically expressed in the neural plate border, most strongly in the posterior region at HH6 and HH8- 190 (Figure S4E, Figure 3F). Irf6 was expressed in the neural plate border as well as the surrounding non-neural ectoderm at 191 HH6 and HH8- (Figure S4E, Figure 3F). Furthermore, we observed significant cellular co-expression of all three factors 192 in the neural plate border (Figure 3F’). At HH8+, Irf6 was restricted to the dorsal neural tube including premigratory 193 neural crest cells, Grhl3 was also detected here at lower levels but was present in the emerging otic placode (Figure 194 3G). By HH9+ Grhl3 levels were broadly diminished but remained in the developing otic placode. Irf6 was maintained 195 in the neural tube during neural crest delamination and also enriched in the otic placode (Figure 3H).

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A Ing5 B HH5 B’ C HH8- C’

Merge B’ 2.5 2.0 C’ 1.5 1.0 HH4 0.5 0.0 Merge Ing5 Ing5 Merge Astl Ing5 Tfap2A HH8 D’ Astl D

D’ Astl

5 4 3 HH4 2 1 0 Tfap2A Astl Tfap2A Merge Astl Ing5 Tfap2A

E Grhl3 F HH8- F’ G HH8+ G’

Merge G’

F’

HH6 0.0 Merge Grhl3 Grhl3 Merge Irf6 Grhl3 Tfap2A Irf6 H HH9+ H’

H’

Irf6

HH6 0.5 0.0 Tfap2A Irf6 Tfap2A Merge Irf6 Grhl3 Tfap2A

Figure 3: HCR validation of novel genes identified from whole epiblast data. (A) Feature plots of Ing5 and Astl in HH4 data. (B) Whole mount HCR shows co-expression of Ing5 and Astl with neural plate border marker Tfap2A at HH5. (B’) Transverse cryosection of (B) (dashed line). Dashed boxed region shown at high magnification in right panel. (C-D) Whole mount HCR shows co-expression of Ing5 and Astl with neural plate border marker Tfap2A at HH8- (C) and HH8 (D). (C’-D’) Transverse cryosections of (C) and (D) respectively. (E) Feature plots of Grhl3 and Irf6 in HH6 data. (F) Whole mount HCR shows co-expression of Grhl3 and Irf6 with neural plate border marker Tfap2A at HH8-. (F’) Transverse cryosection of (F) (dashed line). Dashed boxed region shown at high magnification in right panel. (G-H) Whole mount HCR shows co-expression of Grhl3 and Irf6 with neural plate border marker Tfap2A at HH8+ (G) and HH9+ (H). (G’-H’) Transverse cryosections of (G) and (H) respectively.

196 In the ectodermal subclusters, we identified a number of novel factors including Wnt pathway genes. In HH6- 197 sub-2, for example, we identified enrichment of Wnt signaling ligands Wnt5A, Wnt5B, Wnt8A (Figure S3C) as well 198 as Wnt processing factors Sp5 and Sp8 (Figure 4A/E). Conversely Wnt antagonists Sfrp1, Sfrp2 and Frzb were en- 199 riched in HH6-sub-1 (Figure S3C). Sp8 was identified in HH6-sub-2 (caudal neural), in vivo we found Sp8 expression 200 commenced from HH7 in the neural plate border, partially overlapping with Tfap2A (Figure 4B). The homeobox tran- 201 scription factor, Dlx6, was present in HH7-sub-1, (neural plate, Figure 4A) and was expressed predominantly in the 202 anterior neural plate border or pre-placodal region (Figure 4B). This pattern continued with higher levels of both Sp8 203 and Dlx6 at HH8- (Figure 4C). Sp8 expression was restricted to the dorsal neural tube, whereas Dlx6 spread more 204 laterally (Figure 4C’). At HH9, Sp8 was detected predominantly in the anterior neural tube but was also found in prem- 205 igratory neural crest cells in the mid-brain region (Figure 4D, 4D’) but largely absent from the hindbrain region, though 206 some expression was seen in the trunk premigratory neural crest (Figure 4D). Dlx6 was also present in premigratory 207 neural crest cells at HH9 (Figure 4D’), as well as in the lateral non-neural ectoderm (Figure 4D).

208 Sp5 was enriched in HH6-sub-2 (Figure 4E) and HH7-sub-2 (Figure S3E). Sp5 was detected in the anterior neural 209 plate border where it overlapped with Pax7 and Msx1, and in the posterior primitive streak/early mesoderm at HH7 210 (Figure 4F/F’). Later, (HH8-HH9) Sp5 transcripts were restricted to the neural tube (Figure 4G/H) including premi- 211 gratory neural crest cells (Figure 4G’/H’). At HH9 we also saw the onset of expression in the developing otic placodes 212 (Figure 4H).

213 At HH6, HH6-Cl8 and HH6-Cl10 harboured Znf703 and Gbx2, both of which were enriched in HH6-sub-2 (Fig- 214 ure 4I). Znf703 has recently been demonstrated as RAR responsive factor important for neural crest development in 215 Xenopus (Hong and Saint-Jeannet, 2017, Janesick et al., 2019). Consistent with this, we found Znf703 expression in 216 the posterior neural plate extending into the neural plate border from HH6 (Figure 4J/K). At HH8, Znf703 transcripts 217 also populated the posterior neural folds (Figure 4L) and partially colocalized with Msx1 in premigratory neural crest 218 cells (Figure 4L’). We found Gbx2 broadly expressed across the epiblast from HH6 (Figure 4J/K), becoming more

7 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

219 enriched in the neural folds by HH8 (Figure 4L). While both Znf703 and Gbx2 partially localized with Msx1, Znf703 220 expression continued medially into the neural plate whereas Gbx2 transcripts extended laterally into the non-neural 221 ectoderm (Figure 4L’).

222 Neural clusters expressed numerous transcription factors including Sox21 which was which was present in HH7- 223 sub-0 and HH7-sub-3 (Figure S3E). FezF2 together with Otx2 and Sox2 were also enriched in neural subclusters 224 HH7-sub-3 (Figure S3C/E). FezF2 has previously been shown to regulate Xenopus neurogenesis by inhibiting Lhx2/9 225 mediated Wnt signaling (Zhang et al., 2014) Accordingly, in vivo, FezF2 was expressed in the anterior neural folds 226 at HH7 where it overlapped with Otx2, and in the neural tube at later stages (Figure S4F). Lmo1 was also found in 227 HH7-sub-0 (Figure S3E) and was expressed across the neural plate at HH6 through HH8, where it was also seen in the 228 neural folds (Figure S4G).

A Dlx6 B HH7 B’ C HH8- C’

B’ Merge Ci Merge Dlx6

1.6 1.2 Sp8 0.8 0.4 HH6-subcluster 0.0 Merge Dlx6 Dlx6 Merge Sp8 Dlx6 Tfap2a Tfap2a Sp8 D HH9 D’ Merge D’

Sp8 Dlx6

1.6 1.2 0.8 Sp8 0.4 HH6-subcluster 0.0 Tfap2a Sp8 Tfap2a Merge Sp8 Dlx6 Tfap2a Tfap2a E Sp5 F HH7 F’ G HH8 G’ Merge F’ G’ Merge Pax7

2 Sp5 1

HH6-subcluster 0 Merge Pax7 Pax7 Merge Sp5 Pax7 Msx1 Msx1 Msx1 H HH9 H’ Merge H’

Sp5 Pax7

op Sp5 HH6-subcluster 0 Msx1 Sp5 Msx1 Merge Sp5 Pax7 Msx1 Msx1 I Znf703 J HH6 J’ K HH7 K’ Merge

Merge Gbx2 K’ J’ 1.0 Znf703 0.5

HH6-subcluster 0.0 Merge Gbx2 Gbx2 Merge Znf703 Gbx2 Msx1 Msx1 Gbx2 L HH8 L’ Merge

Znf703 Gbx2 L’

2.0 1.5 Znf703 1.0 0.5 HH6-subcluster 0.0 Msx1 Znf703 Msx1 Merge Znf703 Gbx2 Msx1 Msx1

Figure 4: HCR validation of novel genes identified from extracted ectoderm clusters. (A) Feature plots of Dlx6 and Sp8 in HH6 subclusters. (B) Whole mount HCR shows co-expression of Dlx6 and Sp8 with neural plate border marker Tfap2A at HH7. (B’) Transverse cryosection of (B) (dashed line). Dashed boxed region shown at high magnification in right panel. (C-D) Whole mount HCR shows co-expression of Dlx6 and Sp8 with neural plate border marker Tfap2A at HH8- (C) and HH9 (D). (C’-D’) Transverse cryosections of (C) and (D) respectively.(E) Feature plots of Sp5 and Msx1 in HH6 subclusters. (F) Whole mount HCR shows co-expression of Sp5 with neural plate border markers Msx1 and Pax7 at HH7. (F’) Transverse cryosection of (E) (dashed line). Dashed boxed region shown at high magnification in right panel. (G-H) Whole mount HCR shows co-expression of Sp5 with neural plate border markers Msx1 and Pax7 at HH8 (G) and HH9 (H). (G’-H’) Transverse cryosections of (G) and (H) respectively. (I) Feature plots of Znf703 and Gbx2 in HH6 subclusters. (J) Whole mount HCR shows co-expression of Znf703 and Gbx2 with neural plate border marker Msx1 at HH6. (J’) Transverse cryosection of (J) (dashed line). Dashed boxed region shown at high magnification in right panel. (K-L) Whole mount HCR shows co-expression of Znf703 and Gbx2 with neural plate border marker Msx1 at HH7 (K) and HH8 (L). (K’-L’) Transverse cryosections of (K) and (L) respectively. Op; otic placode.

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229 scVelo analysis shows developmental trajectories across the epiblast

230 The precise time at which the neural plate border segregates into the neural crest, placode and neural lineages has 231 not been previously ascertained. To address this question, we used scVelo (Bergen et al., 2020) to resolve gene-speciﬁc 232 transcriptional dynamics of these embryonic populations across time. This method is derived from RNA velocity (La 233 Manno et al., 2018), which measures the ratio of spliced and unspliced transcripts and uses this information to predict 234 future cell states, incorporating a critical notion of latent time that allows reconstruction of the temporal sequence of 235 transcriptional steps. By generalising the RNA velocities through dynamic modelling, this method is ideally suited to 236 our analysis, as it reﬂects transitions in progressing, non-stationary embryonic

237 Our whole embryo data demonstrated segregation of the germ layers at HH4 (Figure S5A) with all ectoderm cells 238 (HH4-Cl0) aligning along the same trajectory. Epiblast stem cells (HH4-Cl2) were contributing to all other clusters 239 (Figure S5A). At HH5, the majority of ectoderm cells (HH5-Cl4) still followed the same trajectory (Figure S5B); 240 however, a portion of cells marked by Tfap2A and Dlx5 were more transcriptionally stable (Figure S5C) indicating 241 these are non-neural ectoderm cells whereas the differentiating population was marked by neural factors including 242 Zeb2 and Cdh11 (Figure S5C).

243 A clear split in the ectoderm populations was ﬁrst observed at HH6 with neural plate (HH6-Cl8) and non-neural ec- 244 toderm (HH6-Cl10) initiating separate trajectories (Figure 5A). Sox2+ cells contributed to the proliferating multipotent 245 population. The remainder of HH6-Cl8 cells joined the posterior cell clusters with a discreet subset of cells engaged 246 in a separate trajectory (Figure 5A). The Sox2+ subpopulation was speciﬁcally enriched for anterior neural markers 247 Otx2 and Six3, and pluripotency factor FoxD3 (Figure 5B). In addition to the neural markers that characterised the rest 248 of HH6-Cl8 (Sox3, Hes5, Sfrp2, Frzb) (Figure S2B/C), these cells also expressed neural plate border genes including; 249 Pax7, Tfap2A, (Figure 1I), Bmp4, and Msx1 (Figure 5B). Whilst these cells appeared to contribute to both neural and 250 non-neural programmes, the low degree of splicing kinetics as compared to other subpopulations indicated they were 251 relatively transcriptionally stable at this stage (Figure 5A).

252 Interestingly, at HH7 we observed a convergence of trajectories between the neural plate (HH7-Cl3) and non-neural 253 ectoderm (HH7-Cl9), likely representing the maturing neural plate border (Figure 5C). Within this population of cells, 254 there was an overlap of neural plate border, ectodermal and placode gene expression (Pax7, Bmp4, Tfap2A, Msx1, Dlx5 255 and Six3; Figure 5D) suggesting that they are not yet lineage-restricted but, rather, remain an intermingled heteroge- 256 neous progenitor population. Expression of early neural crest genes (Draxin, Tfap2B) also emerged in these converging 257 cells (Figure 5D). Importantly, Pax7 was exclusively found within this region (Figure 5D). Meanwhile, other cells from 258 HH7-Cl3 and HH7-Cl9 were linking to the neural plate/tube trajectories (HH7-Cl4). We observed a portion of neural 259 plate (HH7-Cl3) cells joining the posterior mesoderm cluster (HH7-Cl0), likely containing neuromesodermal precur- 260 sors (NMPs) as suggested by the enrichment in Tbxt and Sox2 expression (Figure S5D).

261 Neural plate border lineages emerge progressively from the ectoderm over time

262 To further resolve the ectodermal trajectories, the extracted ectoderm clusters were modelled using scVelo. At HH5, 263 each population (HH5-sub-0, caudal epiblast; HH5-sub-1, neural plate; HH5-sub-2, non-neural ectoderm) initiated a 264 separate trajectory (Figure S5E). However, cells located centrally contributed to all 3 subclusters (Figure S5E) and 265 expressed neural plate border genes including Pax7 (Figure 2B).

266 At HH6 the trajectories were more distinct (Figure 5E). Caudal epiblast cells (HH6-sub-2) contributed to both 267 neural plate and non-neural ectoderm populations. Within the neural plate cluster (HH6-sub-1) some cells were directed 268 towards the non-neural ectoderm and others followed a separate trajectory. Likewise, the non-neural ectoderm cluster 269 was subdivided between 2 parallel pathways with some mutual cross-over. Neural plate border cells found across all 3 270 clusters and at their interfaces (as depicted by Pax7; Figure 2D) largely joined the non-neural ectoderm clusters (Figure 271 5E).

272 HH7 trajectory map was more complex (Figure 5F). Here Pax7+ neural plate border cells (HH7-sub-1) projected 273 toward all neural plate border derivatives, neural crest and placodes (HH7-sub-1) and neural tube (HH7-sub-0; Fig- 274 ure 5F). Some neural plate border cells appeared to be derived from the neural plate (HH7-sub-0/3), these cells ex- 275 pressed neural crest progenitor genes (Tfap2B, Draxin, Snai2,) (Figure 2F, Figure S3F). Pax7+ cells heterogeneously 276 co-expressed other established neural plate border genes Tfap2A, Msx1 and Bmp4 (Figure 5G). This demonstrates that 277 Pax7+ cells are capable of giving rise to multiple lineages and highlights heterogeneous combinations of factors at

9 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

278 the neural plate border reﬂect multipotency. The remainder of HH7-sub-1 cells (Pax7-) were engaged in a separate 279 trajectory (Figure 5F); these cells were enriched for Dlx5 (Figure 2F), Six1, Pax6 and Gata3 (Figure S3F), suggesting 280 a placodal fate. Overall, at HH7 scVelo analysis showed the neural plate border was beginning to segregate according 281 to progenitor populations.

Cardiac Sox2 Pax7 Bmp4 A Anterior B Six3 C D mesoderm HH6 Posterior HH7 LPM LPM Cardiovascular progenitors Hensens LPM Paraxial Paraxial Neuro- mesoderm Node Sox2 mesoderm Tfap2A Otx2 ectoderm Msx1

Neural plate/tube

Endoderm Sox2 Dlx5 Posterior FoxD3 Six3 LPM 0 Pax7+ 1 2 Head Neural 0 Primitive 3 mesenchyme Non-neural 1 streak plate 4 2 5 Msx1 ectoderm 3 Draxin Proliferating 6 Bmp4 Hensens 4 Tfap2B 7 Endoderm Node 5 cells 8 6 9 7 Sox2+ 10 HH78 Non-neural Ectoderm 11 9 10

E HH6-Cl8+Cl10 F HH7-Cl3+Cl9 G Caudal Pax7 Pax7 Tfap2A Msx1 epiblast Neural Plate Border NMP’s Pax7+ Tbxt+ Sox2+ Caudal Neural

Pax7 Pax7 Bmp4 Tfap2B Mid-Neural Non-neural Rostral ectoderm Neural 0 1 0 Notochord 2 1 3 Neural plate 2 progenitors 4

Figure 5: scVelo infers developmental trajectories across early epiblast stages (A) UMAP embedding of clusters from whole epiblast data at HH6 showing predicted trajectories. (B) Colocalisation of selected genes across HH6 clusters (C) UMAP embedding of clusters from whole epiblast data at HH7 showing predicted trajectories. (D) Colocalisation of selected genes across HH7 clusters. (E) UMAP embedding of extracted ectoderm subclusters from HH6 showing predicted trajectories. (F) UMAP embedding of extracted ectoderm subclusters from HH7 showing predicted trajectories. (G) Colocalisation of selected genes across HH7 subclusters.

282 Inferring developmental trajectories from presumptive ectoderm to premigratory neural crest cells

283 To assess the progression of ectodermal progenitors across time-points from epiblast to onset of neural crest em- 284 igration, we ﬁrst integrated the ectoderm clusters from each stage namely; HH5-Cl4, HH6-Cl3+9 and HH7-Cl8+10. 285 Here we also included 10X scRNA-seq data obtained from premigratory neural crest cells (HH8+) from our previous 286 work (Williams et al., 2019), such that we could trace trajectories from the neural plate border to the bona ﬁde neural 287 crest.

288 Combining these datasets revealed that the non-neural ectoderm from both HH6 and HH7 grouped together, as 289 did the neural plate clusters from the same stages (Figure 6A). This suggests successful inter-sample incorporation 290 into a single reference with no resultant batch bias (Figure S5F). Ectoderm cells from HH5 were spread amongst the 291 neural plate and the non-neural ectoderm clusters from HH6 and HH7. The neural crest clusters were more discrete, 292 with the bona ﬁde neural crest cluster (FoxD3, Tfap2B, Sox10) clearly segregated from neural (Sox3, Pax6, Cdh2) and 293 mesenchymal (Pitx1, Twist1, Sema5B) neural crest clusters as previously described (Williams et al., 2019). The HH8+ 294 dataset also included a neuroepithelial-like cluster, characterised by Cdh1, Epcam and FoxG1 expression. Interestingly 295 these cells grouped with the non-neural ectoderm clusters from HH6 and HH7, including neural plate border cells 296 (Pax7, Tfap2A; Figure 6A/B), suggesting early neural crest cells retain properties of their predecessors.

297 scVelo showed that non-neural ectoderm cells from HH6/HH7 were split between two trajectories (Figure 6A). 298 Placodal markers (Dlx5/6, Six3 and Pax6) deﬁned one distinct trajectory (Figure S5E), whereas the other trajectory

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299 was less well defined but could be distinguished by posterior epiblast markers (HoxB1, Cdx4) (Figure S5H). The 300 neuroepithelial-like cells from HH8+ were emerging between these trajectories where they were joined by other cells 301 from HH6/HH7 non-neural ectoderm clusters. These cells were enriched for Pax7 and other neural plate border markers 302 Tfap2A, Msx1, Bmp4 and (Figure 6B). Early neural crest genes including Tfap2B and Draxin were also emerging here 303 (Figure 6B). While these cells had less defined trajectories, they were generally directed towards the neural-neural crest 304 lineage (Figure 6A). Neural plate border and neural crest markers also extended into a heterogeneous region of cells, 305 where a significant portion of the neural-neural crest and neural plate cells were highly integrated, suggesting some cells 306 from these populations shared transcriptional signatures. This region was further defined by Zfhx4 and Zic1 (Figure 307 6B) which are expressed in the neural plate border, neural plate and premigratory neural crest cells (Khudyakov and 308 Bronner-Fraser, 2009, Williams et al., 2019). Some neural-neural crest cells within this region appeared to be directly 309 derived from the neural plate.

310 Since we did not see evidence of neural crest lineages until HH7, we assessed the developmental trajectories 311 from HH7 ectoderm clusters directly to bona fide neural crest cells (HH8). To this end, we extracted the ectoderm 312 clusters (HH7-Cl3+Cl9) and combined these cells with the premigratory neural crest data set from HH8+ embryos 313 and used scVelo to plot the trajectories across these cells/stages. Analysis of the pooled dataset yielded clearly defined 314 developmental trajectories splitting from the HH7 ectoderm clusters, whereby the non-neural ectoderm cells (HH7-Cl9) 315 gave rise to the neuroepithelial-like cells from the HH8+ dataset, and the neural plate (HH7-Cl3) cells were contributing 316 directly to the neural-neural crest (Figure 6C). Non-neural ectoderm cells were also initiating a separate trajectory, 317 likely representing placodal lineages as indicated by Six1, Dlx5 (Figure 6D). While Tfap2A was expressed across the 318 non-neural ectoderm cluster, Pax7 was found in just a subset of non-neural ectoderm cells and in neuroepithelial-like 319 cells, where Tfap2A was also found. Both factors were also enriched in bona fide neural crest, though Pax7 was also 320 detected in the neural-neural crest (Figure 6D). Other neural plate border markers (Bmp4, Msx1) were found in the 321 non-neural ectoderm and some neuroepithelial-like cells, as well as extending into the interface of neural plate and 322 neural-neural crest populations (Figure 6D).

323 Taken together, combining our 10X data-sets from all stages reinforced the notion that the neural plate border terri- 324 tory is not speciﬁed to a neural crest fate until HH7 since scVelo analysis on the entire dataset (including HH5 and HH6 325 data) generated ambiguous and non-contiguous developmental trajectories (Figure 6A). While neural crest trajectories 326 were not clearly deﬁned until HH7, placodal trajectories could be discerned from HH6. Latent time analysis cor- 327 roborated these observations, indicating the progression of transcriptional maturity and corresponding differentiation 328 status across the HH7 non-neural ectoderm cells into the neuroepithelial-like cells and, similarly, from the neural plate 329 cells into the neural-neural crest and canonical neural crest populations (Figure 6E). Furthermore, the data showed a 330 sub-population of premigratory neural crest cells shared transcriptional signatures with their progenitor cells.

331 Analysis of dynamic transcriptional trajectories during lineage restriction reveals key lineage speciﬁc drivers

332 We next sought to determine key genes driving the observed developmental trajectories in the context of neural 333 crest specification from the neural plate border. Dynamical modelling of transcriptional states within the HH7/8 com- 334 bined data-set revealed a number of highly ranked dynamic genes including Pax7 and Tfap2B. Pax7 splicing increased 335 progressively from the non-neural ectoderm cells to the neuroepithelial-like cells and lower splicing was observed in 336 neural crest cells where the transcripts stabilized such that increased expression was observed (Figure 6F). Splicing of 337 Pax7 transcripts was also increased in the putative placodal lineage from the non-neural ectoderm (Figure 6F). Tfap2B 338 splicing was not evident in the HH7 data, but was increased in the neuroepithelial-like cells and stabilized in neural 339 crest populations (Figure 6F). Tfap2A showed more complex velocities, whereby high levels of spliced transcripts were 340 detected in non-neural ectoderm cells from HH7 likely driving these cells towards the placode trajectory (Figure 6F). 341 We also observed a progressive increase in Tfap2A splicing across the neuroepithelial-like cells to the bona fide neural 342 crest. This suggested that by HH8, Tfap2A transcripts are stable in the neuroepithelial-like population but upregulated 343 in the bona fide neural crest, consistent with the observed expression dynamics (Figure 6F). Another neural plate border 344 gene, Bmp4, was dynamically regulated in non-neural ectoderm but downregulated in the neural plate (Figure S5I).

345 We also identiﬁed a number of more novel factors putatively involved in ectoderm lineage progression. Otx2 was 346 driving neural plate cells towards the neural-neural crest cluster, consistent with previous ﬁndings from functional 347 perturbation studies (Williams et al., 2019). However, Otx2 also seemed to be driving non-neural ectoderm cells 348 towards the neuroepithelial-like population (Figure 6F). Lmo1 was also driving the neural plate cells towards the neural- 349 neural crest; however, unlike Otx2, Lmo1 expression was not maintained in the neural crest populations (Figure S5I).

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350 Bri3 was identiﬁed as a highly ranked dynamic gene and was upregulated in neural plate cells and some non-neural 351 ectoderm cells. Down-regulation of Bri3 in neural crest cells suggested an early role in ectoderm lineage trajectories for 352 this novel factor (Figure 6F). Nav2 and Sox11 were both highly expressed in the neural plate and downregulated in the 353 non-neural ectoderm. Sox11 was also expressed in the mesenchymal neural crest cluster where it was highly spliced, 354 potentially representing a dual segregated role for this factor in both neural plate and mesenchymal crest lineages 355 (Figure S5I). Nav2 velocities increased progressively from the non-neural ectoderm to the neural plate; consistently, a 356 subset of non-neural ectoderm cells joined a trajectory with the neural plate cells (Figure S5I) where Nav2 splicing was 357 highest (Figure S5I). We found Pitx1 and Dlx6 to be dynamically active across the merged HH7/8 data-set, potentially 358 driving non-neural ectoderm to the neuroepithelial-like population (Figure 6F, Figure S5I).

359 This analysis provides insight into potential candidates driving lineage speciﬁc circuits underlying the progressive 360 segregation of the early ectoderm into neural and non-neural ectoderm and concomitantly initiating the emergence of 361 early neural crest and placode progenitors.

A B Tfap2A C HH7 Neural plate D Six1 HH6 Non-neural HH8+ Neural NC HH7 Non-neural Placodal lineage Pax7 Dlx5 Dlx5/6 HH8+ Neuroepithelial-like Six1/3 Pax6 HH8+ Bona ﬁde NC Cdx2/4 Bmp4 Tfap2A HoxB1 Msx1 Pax7

HH8+ Mesenchymal NC HH8+ Neural NC HH7 Non-neural Tfap2B Bmp4 Draxin ectoderm Msx1 HH8+ Mesenchymal NC

Zfhx4 E + HH8 Bona ﬁde NC HH5 Ectoderm Zic1 HH8+ Neuroepithelial-like HH6 Neural plate HH7 Neural plate

Pax7 Tfap2B Tfap2A 2 Otx2 Bri3 Pitx1 0.16 0.50 4 4 12

1 0.25 2 0.08 unspliced

2 unspliced 6 unspliced unspliced unspliced unspliced

0 0 0 0 0.00 0.00 0 2 0 3 6 0 1 2 0 2 4 0 1 2 0.0 0.3 0.6 spliced spliced spliced spliced spliced spliced

Pax7 Tfap2B Tfap2A Otx2 Bri3 Pitx1

Velocity Velocity Velocity Velocity Velocity Velocity

Pax7 Tfap2B Tfap2A Otx2 Bri3 Pitx1

Expression Expression Expression Expression Expression Expression

Figure 6: Developmental trajectory analysis from the neural plate border to premigratory neural crest cell. (A UMAP embedding of ectoderm clusters combined with premigratory neural crest data showing predicted trajectories. (B) Colocalisation of selected genes across combined data set. (C) UMAP embedding of HH7 ectoderm clusters combined with premigratory neural crest data showing predicted trajectories. (D) Colocalisation of selected genes across HH7/premigratory neural crest combined data set. (E) Latent time data from HH7/premigratory neural crest combined dataset. (F) Phase portraits (top row), velocity plots (middle row) and expression plots (bottom row) of selected genes across the HH7/ premigratory neural crest combined data sets. NC; neural crest.

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362 Discussion

363 Here we use single-cell RNA-sequencing of chick embryos from late gastrula through early neurula to characterise 364 the development of the neural plate border and its derivatives, the neural crest and cranial placode precursors. Our data 365 show that the neural plate border, as defined by co-expression of Tfap2A and Pax7 first emerges at HH5, but is not fully 366 transcriptionally defined until HH7. Previous work has pointed to the presence of a pre-border region at blastula stages 367 harbouring neural crest progenitors demarcated by Pax7 expression (Basch et al., 2006; Prasad et al., 2020). However, 368 this was observed in explanted cultures. In contrast, we do not detect significant Pax7 expression until HH5. This 369 suggests that cells within the explants may not have been fully specified at the time of explantation but cell interactions 370 coupled with autonomous programmeming enabled the cells to continue their specification programme.

371 We identiﬁed several genes in our dataset that were not previously known to function in the neural plate border. 372 For example, Irf6 has a well-established role in craniofacial development (Fakhouri et al., 2017, Ingraham et al., 2006, 373 Wang et al., 2003) but has yet to be explored at earlier stages of development. Grhl3 has been shown to work in 374 a module with Tfap2A and Irf6 during neurulation (Kousa et al., 2019). In addition, Wnt signaling pathway genes 375 like Sp5 and Sp8 were prominent. Wnt signaling has a well-established role in neural plate border speciﬁcation; for 376 example, Wnt signals are required to activate the earliest neural crest genes Tfap2A, Gbx2 and Msx1. Sp8 is known 377 to play a role in processing Wnt signals during limb development (Haro et al., 2014, Kawakami et al., 2004), but has 378 also been described in neural patterning and craniofacial development (Sahara et al., 2007, Zembrzycki et al., 2007), 379 whereby loss of Sp8 in mice caused severe craniofacial defects due to increased apoptosis and decreased proliferation 380 of neural crest cells (Kasberg et al., 2013). However, an early role for Sp8 has not been explored. Sp5 has recently been 381 implicated in the neural crest gene regulatory network, where it was found to negatively regulate Axud1 to help maintain 382 neural crest cells in a na¨ıve state (Azambuja and Simoes-Costa, 2021). Therefore, Sp5 and Sp8 may represent hitherto 383 unknown components of Wnt mediated induction of neural plate border lineages. Gbx2 was recently reported as the 384 earliest Wnt induced neural crest induction factor in Xenopus. Perturbation of Gbx2 inhibited neural crest development 385 while the placodal population was expanded (Li et al., 2009). Interestingly, our analysis reveals distinct heterogeneity 386 of Wnt signaling factors, indicating differential cellular responses to Wnt signaling within the developing neural plate 387 border, which may contribute to driving different lineage trajectories.

388 We used scVelo analysis of transcriptional and splicing dynamics across clustered and aligned single-cell transcrip- 389 tomes to observe the dynamics of developmental trajectories during ectoderm lineage segregation. This enabled us to 390 follow the transcriptional velocity of individual genes and resolve their dynamics at the single-cell level. Significantly 391 this provides a high-resolution temporal dimension to our understanding of the changing ontology of the neural plate 392 border and its derivatives. The results suggest that the ectoderm is initiating the division of trajectories between neural 393 and non-neural progenitors at HH5, but the neural plate border is not yet transcriptionally distinct. At HH6 and HH7 394 the neural plate border population is emerging at the interface of these tissues, from which multiple lineages manifest 395 by HH7. This analysis also shows neural-neural crest cells are the first to surface from the neural plate border and 396 neural plate. From these, the bona fide neural crest emerges followed by mesenchymal neural crest. Furthermore, 397 this analysis shows a subset of premigratory neural crest cells share signatures with their precursors from neural plate 398 border cells from HH6 and HH7, demonstrating that some early neural crest cells are not yet restricted to a particular 399 lineage.

400 Taken together our data show that cells of the emerging neural plate border are not characterised by unique tran- 401 scriptional signatures, but share features with cells of the surrounding ectoderm. This highlights the heterogeneity of 402 the neural plate border whereby Pax7+ cells are integrated with other ectoderm populations. While Pax7 is the predom- 403 inant factor in the neural plate border and has been suggested to label neural crest precursors as early as HH5 (Basch 404 et al., 2006), the complexity and co-expression signatures are what endow neural plate border cells with their unique 405 multipotency compared to neural plate and ectoderm cells. Interestingly, we note that neural plate border signatures 406 are not apparent until HH7, later than previously suggested. By contrast, placodal trajectories were discernible from 407 HH6. Moreover, we ﬁnd Pax7+ cells give rise to all neural plate border lineages but lose multipotency signatures at the 408 onset of lineage speciﬁc trajectories. Our analysis provides important insights into genes underlying the progressive 409 segregation of the emergence of early neural crest and placode progenitors at the neural plate border. By revealing 410 lineage trajectories over developmental time, this resolves the timing of neural plate border lineage segregation whilst 411 also informing on dynamics of multipotency programmemes.

13 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

412 Supplementary results:

413 HH4: Other clusters at HH4 were readily identified as mesoderm (HH4-Cl1) or endoderm (HH4-Cl3) as charac- 414 terised by the expression of Cdx4 and Pitx2 for mesoderm and Sox17, FoxA2 and Cxcr4 for endoderm (Figure 1B, 415 Figure S1B/C. HH4-Cl2 was enriched for genes associated with pluripotency including Sall4, Tgif1, ElavL1 (Lee et 416 al., 2015, Ye and Blelloch, 2014, Zhang et al., 2006) (Figure S1B/C). Mitochondrial genes and cell migration factors 417 (Cxcl12, Itgb1, Tgfbr1) were enriched in HH4-Cl4 suggesting these are highly active migrating cells (Figure 1B, Fig- 418 ure S1C). scVelo analysis revealed that mesoderm (HH4-Cl1) and endoderm cells (HH4-Cl3) were following distinct 419 trajectories as individual populations (Figure S5A). At HH5, mesoderm cells (HH5-Clusters 0-3) were segregating and 420 endoderm cells (HH5-Cl5) were also maintaining their distinct trajectory (Figure S5B). 421 HH6: Mesodermal cells were found in several clusters: anterior lateral plate mesoderm (HH6-Cl2; Pitx2, Alx1); 422 posterior lateral neural plate (HH6-Cl0; Gata2, HoxB5). Cardiac mesoderm markers were enriched in HH6-Cl4 423 (Tcf21 and Gata5) and HH6-Cl11 (Lmo2, Ets1, Kdr). HH6-Cl1 and HH6-Cl3 represented paraxial mesoderm (Msgn1, 424 Mesp1). Endoderm cells formed HH6-Cl6 (Sox17, FoxA2). Hensen’s node and primitive streak markers (Dll1, Fgf8, 425 Noto, Chrd) were identified in HH6-Cl9 and HH6-Cl7 respectively. We also detected a cluster of cells (HH6-Cl5) 426 with high levels of mitochondrial genes (Cox1/3), this cluster was also enriched for factors associated with highly 427 proliferative cells (Pdia3, Igf1r) (Figure S2C). 428 HH7: Aspects of the mesoderm were discernible across several clusters (HH7-Cl0, 1, 2, 6, 7, 10). HH7-Cl5 was 429 defined by endoderm markers and HH7-Cl8 represented Hensen’s node and the primitive streak (Figure S2F).

430 Author contributions

431 Conceptualisation, R.M.W., T.S.S., M.E.B; Methodology, R.M.W. Software, R.M.W., M.L., Validation, R.M.W.; For- 432 mal Analysis, R.M.W., M.L.; Investigation, R.M.W.; Writing R.M.W.; Writing – Review & Editing, all authors; Visu- 433 alisation R.M.W., T.S.S., M.E.B; Supervision, T.S.S., M.E.B; Funding Acquisition, M.E.B.

434 Acknowledgements

435 Fluorescence activated cell sorting was performed at California Institute of Technology Flow Cytometry Facility using 436 BD Biosciences FACSAria IIu Cell Sorter with Patrick Cannon. 10X libraries were prepared in the Thomson lab at 437 California Institute of Technology with assistance from Jeff Park. Illumina sequencing was performed at the Millard 438 and Muriel Jacob at California Institute of Technology with Igor Antoshechkin. HH4 10X library was constructed 439 at MRC Weatherall Institute of Molecular Medicine, University of Oxford with assistance from Kevin Clark (FACS 440 facility), Dr Neil Ashley (single-cell facility) and Tim Rostron (NGS sequencing facility). Confocal microscopy was 441 performed within the Biological Imaging Facility at the Beckman Institute, California Institute of Technology with 442 assistance from Dr Giada Spigolon. We thank members of the Bronner and Sauka-Spengler labs for their support and 443 helpful discussions. This work was funded by NIH R01DE027538 to MEB.

444 Materials and Methods

445 Chick embryos:

446 Fertilized chicken eggs, obtained from Sunstate Ranch Sylmar CA, were incubated at 37°C with approximately 447 40% humidity. Embryos were staged according to (Hamburger and Hamilton, 1951).

448 Preparing embryos for FAC-sorting:

449 Appropriately staged embryos were extracted using the filter paper based ‘easy-culture’ method. Eggs were opened 450 after desired incubation period, albumin was removed and embryos were lifted from the yolk using punctured filter 451 paper, this procedure is described in detail elsewhere (Williams and Sauka-Spengler, 2021b). Embryos were kept in 452 Ringers solution and dissected to remove all extra-embryonic material. Embryos were then dissociated for fluorescence 453 activated cell sorting (FACS) as previously described (Williams and Sauka-Spengler, 2021a). Embryos were processed 454 by FACS using 7-AAD as a live/dead stain such that healthy individual cells were obtained with a reliable cell count.

14 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

455 10X single-cell RNA-seq library preparation and sequencing:

456 Approximately 10,000 cells/stage were collected by FACS into 2ul Hanks buffer then loaded onto the 10X Ge- 457 nomics Chromium platform. Single-cell RNA-seq libraries were generated using the Chromium Single Cell 3’ Library 458 and Gel Bead Kit v3 (10X Genomics, Cat. #1000075) (HH4 data was obtained using v2 chemistry) as per the manu- 459 facturers protocol. Libraries were quantiﬁed using Qubit (Life Tech Qubit high sensitivity DNA kit Cat. #Q32854) and 460 Kapa (Kapa Biosystems, KAPA Library Quantiﬁcation Kit, Cat. #KK4835). scRNA-seq libraries were sequenced on 461 Illumina HiSeq2500 platform in rapid run mode with on-board clustering and sequencing depth of 300 million reads. 462 The run type was: paired end 28(read1)-8(index)-91(read2). HH4 10X scRNA-seq library was sequenced on Illumina 463 NextSeq500 platform using high output v2.5 150-cycle kit in 26 x 8 x 0 x 98 mode.

464 10X single-cell RNA-seq data analysis:

465 Fastq files were generated using cellranger v3.1.0 mkfastq (Zheng et al., 2017). Cellranger was also used for base 466 calling, demultiplexing and mapping to the galgal6 genome assembly. A custom galgal6 genome was constructed 467 using the mkref function whereby selected 3’UTRs were extended according to manually annotation. From HH4 em- 468 bryos 2398 cells were recovered with 144,605 mean reads/cell, 1836 median genes/cell, 6180 median UMI counts/cell 469 and 15,370 total genes. From HH5 embryos 6723 cells were recovered with 44,343 mean reads/cell, 3659 median 470 genes/cell, 14,229 median UMI counts/cell and 18,916 total genes. From HH6 embryos 4553 cells were recovered 471 with 30,466 mean reads/cell, 3396 median genes/cell, 13,309 median UMI counts/cell and 17,828 total genes. From 472 HH7 embryos 6585 cells were recovered with 20,044 mean reads/cell, 2696 median genes/cell, 8821 median UMI 473 counts/cell and 17,873 total genes. Count matrices were generated using the cellranger count function and exported to 474 R-studio for downstream analysis using Seurat-v3 (Stuart et al., 2019). Matrices were filtered to remove barcodes with 475 fewer than 500 genes and more than 3500-5500 genes (Figure S1/S2) and high mitochondrial content (>0.5%). UMI 476 counts were normalised and following principal component analysis linear dimension reduction was conducted (HH4; 477 resolution 0.2, dims 1:20, HH5; resolution 0.4, dims 1:15, HH6; 0.6, dims 1:20, HH7; resolution 0.4, dims 1:20). 478 Clustered cells were visualised on a UMAP plot and differentially expressed genes were identified. Ectoderm clusters 479 were extracted and re-clustered using the ‘subset’ command, analysis was performed as for whole embryo data.

480 scVelo analysis:

481 The analysis was performed using the scVelo dynamical modelling pipeline (Bergen et al., 2020) in python. scVelo 482 analysis on combined datasets from different stages: HH5, HH6, HH7 and HH8 datasets were integrated using Seurat 483 v3 by finding inter-sample anchors followed by integration. UMI counts and mitochondrial transcript percentage were 484 regressed out during integrated object scaling, 30 npcs were used for dimension reduction to plot a single reference 485 UMAP. For downstream scVelo analysis HH5, HH6, HH7 and HH8 ectodermal/pro-neural clusters or HH7 and HH8 486 ectodermal/pro-neural clusters were subtracted from the integrated dataset, followed by rescaling and PCA reduction 487 using 10 or 9 dimensions respectively to plot UMAPs. Loom files containing spliced /unspliced transcript expression 488 matrices were generated for each individual stage using velocyto.py pipeline (La Manno et al., 2018). Loom cell names 489 were renamed to match Seurat object cell names and only Seurat-filtered cells were selected for trajectory analysis. 490 Loom files from individual stage samples were merged using loompy.combine function. Seurat generated UMAP 491 coordinates, clusters and cluster or stage colours were added to the filtered loom cells.

492 Hybridisation chain reaction:

493 Fluorescent in situ hybridsation chain reaction was performed using the v3 protocol (Choi et al., 2018). Briefly, 494 embryos were fixed in 4% paraformaldehyde (PFA) for 1 hour at room temperature, dehydrated in a methanol series 495 and stored at -20°C at least overnight. Following rehydration embryos were treated with Proteinase-K (20mg/mL) 496 for 1-2.5 minutes depending on stage (1 min HH4-6, 2.5 mins for older embryos) at room temperature and post-fixed 497 with 4% PFA for 20 min at room temperature. Embryos were washed in PBST for 2x 5 min on ice, then 50% PBST 498 / 50% 5X SSCT (5X sodium chloride sodium citrate, 0.1% Tween-20) for 5 min on ice and 5X SSCT alone on ice 499 for 5 min. Embryos were then pre-hybridized in hybridisation buffer for 5 min on ice, then for 30 min at 37°C in 500 fresh hybridization buffer. Probes were prepared at 4pmol/mL (in hybridization buffer), pre-hybridization buffer was 501 replaced with probe mixture and embryos were incubated overnight at 37°C with gentle nutation. Excess probes were 502 removed with probe wash buffer for 4x 15 min at 37°C. Embryos were pre-amplified in amplification buffer for 5 min 503 at room temperature. Hairpins were prepared by snap-cooling 30pmol (10ml of 3mM stock hairpin) individually at 504 95°C for 90 s and cooled to room temperature for minimum 30 min, protected from light. Cooled hairpins were added

15 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

505 to 500µl ampliﬁcation buffer. Pre-ampliﬁcation buffer was removed from embryos and hairpin solution was added 506 overnight at room temperature, protected from light. Excess hairpins were removed by washing in 5X SSCT 2x 5 507 min, 2x 30 min and 1x 5 min at room temperature. Embryos were mounted on slides and imaged using Zeiss LSM 508 880 Upright confocal microscope. Images were processed using Zeiss Zen software, Z-stacks scans were collected at 509 6µm intervals across approximately 70-100µm, maximum intensity projections of embryo z-stacks are presented. Tile 510 scanning was used (2x3) and stitched using bidirectional stitching mode, with overlap of 10%. For sectioned samples, 511 images were obtained on a Zeiss LSM 880 Upright confocal with 20X and 40X oil immersion objectives, single z-slices 512 are shown.

513 Cryosectioning:

514 Following HCR and whole mount imaging, selected embryos were prepared for cyrosectioning Embryos were 515 placed in a 15% sucrose solution at 4°C overnight then 15% sucrose / 7.5% Gelatin overnight at 37°C. Embryos were 516 transferred to 20% gelatin and incubated at 37°C for 4 hr then mounted in 20% gelatin, snap frozen in liquid nitrogen 517 and stored at -80°C. Sections were taken at 10µm intervals.

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18 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Segregation of neural crest speciﬁc lineage trajectories from a heterogeneous neural plate border territory only emerges at neurulation.

Ruth M Williams1,2, Martyna Lukoseviciute2,3, Tatjana Sauka-Spengler2, and Marianne E Bronner1,∗

Supplemental Material

Ectoderm Mesoderm Epiblast stem cells Endoderm mito A nFeature nCount percent C 0 1 2 3 4 3 MAFA 4000 RNA RNA mt PLPP1 ING5 NAV2 AP1S3 3000 20000 CLDN1 2 CA2 HK1 FABP3 ASTL 2000 AGRP CLEC19A 10000 1 GATA5 CDX4 PITX2 1000 NUCKS1 CRABP−I FIBIN AKAP12 0 0 CIRBP UMAP_1 G3BP1 LARP7 SALL4 B Cxcr4 FoxA2 5.0 5.0 HTRA1 TFDP1 2.5 2.5 SLC2A14

RPS28 UMAP_1 0.0 0.0 SOX17 −2.5 −2.5 APOA1 5 MYL4 4 2.0 −5.0 3 −5.0 1.5 2 1.0 KRT7 1 0.5 RBP4A −7.5 0 −7.5 0.0 −5 0 5 −5 0 5 SHISA2 ElavL1 Tgif1 APOC3 5.0 5.0 CG−16 BSG 2.5 2.5 PDIA3 0.0 0.0 HSPA5 PDIA6 −2.5 −2.5 LAPTM4A 3 CTSV −5.0 2 −5.0 2 1 1 TSPAN3 0 0 DEGS1 −5 0 5 −5 0 5 LGMN Mesoderm Ectoderm Endoderm Mito Node D nFeature nCount percent F 0 1 2 3 4 5 6 7 RNA RNA mt ALDH1A2 6000 40000 MSGN1 PTN DLL1 RDH10 0.75 MEIS1 CLEC19A 30000 RNH1 CDH2 AGRP 4000 NPBWR1 SCGN 0.50 SFRP1 PCSK5 20000 FST CDH11 TWIST1 GCHFR 2000 SHISA2 0.25 SIX1 10000 Wpkci−7 CDX4 MT4L LDHA HSPB9 HSPA2 0.00 NUAK1 0 BAG3 CLDN1 PLPP1 SFRP2 PDGFA E FRZB 10 Nav2 10 MafA EPCAM AP1S3 CTSV 5 5 GJA1 SOX17 KRT7 0 COTL1 0 MYL4 APOA1 3 2.0 EZR −5 2 −5 1.5 1.0 JARID2 1 0.5 VCAN 0 0.0 KRT18 −5 0 5 −5 0 5 ND5 Sox21 Dag1 ND6 10 10 COX3 CYTB COII 5 5 ATP6 COX1 ND3 0 0 SNX3 FAM204A OTUD6B 2 3 −5 −5 2 C7orf50 1 1 ENY2 0 0 UBE2D3 −5 0 5 −5 0 5 FAM133B MEOX1

Supplementary figure S1. Quality control and supporting data for HH4 and HH5 10X data. (A) Violin plots showing RNA features, counts and percentage of mitochondrial reads per cell from HH4 data. Cells were filtered to exclude those with <500 and >3500 features (genes) and >0.5% mitochondrial gene content. (B) Feature plots of selected genes in HH4 clusters. (C) Heatmap depicting top 10 differentially expressed genes across each cluster from HH4. (D) Violin plots showing RNA features, counts and2 percentage of mitochondrial reads per cell from HH5 data. Cells were filtered to exclude those with <500 and >5500 features (genes) and >0.5% mitochondrial gene content. (B) Feature plots of selected genes in HH5 subclusters. (C) Heatmap depicting top 10 differentially expressed genes across each cluster from HH5. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Mesoderm Prolif. Endo. Streak NP NodeNNE CV A C 0 1 2 3 4 5 6 7 8 9 10 11 nFeature nCount percent CDX4 CCDC88C RNA RNA mt GATA2 1.00 MSX1 PTN 5000 BAMBI AGRP Wpkci−7 FIBIN DNAJA1 30000 MSGN1 CFC1 4000 0.75 ALDH1A2 MESP1 HSPB9 HSPA2 PITX2 ALX1 SCGN EXOC6 3000 SFRP1 20000 SIX1 0.50 SHISA2 GCHFR OLFML3 SST NPY 2000 MEOX1 TCF15 C5H11ORF96 RNH1 10000 0.25 RDH10 FST TCF21 GATA5 1000 PRRX1 PODXL PERP2 CSRP1 CD24 JCAD 0 0.00 PENK ND6 COX3 COX1 CYTB ND4 B ATP6 ND2 Sox3 FoxD3 COII ND5 5 5 ND3 SOX17 FOXA2 NDRG1 0 0 APOA1 1.6 OTX2 2.0 1.2 KRT7 1.5 MYL4 0.8 CMTM8 −5 −5 1.0 FN1 0.4 0.5 EVX1 FGF8 0.0 0.0 TBXT −5 0 5 10 −5 0 5 10 CDX2 DLL1 CXCL14 Hes5 Ednrb NKAIN4 WNT8A 5 5 HOXB1 GJA1 CLDN1 SFRP2 0 0 FRZB 2.5 2.0 PLPP1 2.0 PDGFA 1.5 1.5 UCH−L1 −5 1.0 −5 EPCAM 1.0 LMO1 0.5 0.5 CYP26A1 0.0 0.0 CHRD NOTO −5 0 5 10 −5 0 5 10 PTGDS NOV POMC Otx2 Gbx2 SIX3 5 5 TFAP2A ASTL DLX5 CSRP2 DAG1 0 0 RGN 4 3 TAL1 3 FLI1 2 HHEX −5 2 −5 LMO2 1 1 ETS1 TGFBI 0 0 KDR Mesoderm Ectoderm Endoderm Mito Node −5 0 5 10 −5 0 5 10 MT4L SLC35E3 0 1 2 3 4 5 6 7 CA2 G H D nFeature nCount percent F Post. LPM Mesenchyme Para.Ax. Meso Neural plate NP/tube Endo. Meso Meso Node NNE LPM RNA RNA mt 0 1 2 3 4 5 6 7 8 9 10 1.00 MSX1 CDX2 5000 EVX1 CDX4 WNT8A WNT5A SKAP2 TBXT CXCL14 4000 0.75 LMO2 20000 PITX2 ALX1 EXOC6 SIX1 TWIST1 SFRP1 SHISA2 3000 PRRX1 SST 0.50 AKAP12 C5H11ORF96 MEOX1 TCF15 AGRP 2000 10000 FST LBX2 CDH20 NPY 0.25 NEFM RDH10 SFRP2 1000 FRZB CLDN1 SOX21 SOX2 DMD PDGFA LMO1 0 0.00 UCH−L1 0 ND6 COX3 COX1 COII E ATP6 ND3 Otx2 Zeb2 ND4 CYTB 5 ND5 5 ND2 SOX17 0 NDRG1 0 OTX2 MYL4 3 EZR −5 −5 2 APOA1 2 SIX3 1 1 FN1 MSGN1 −10 −10 0 ALDH1A2 −5 0 5 −5 0 5 DLL1 PTN Sox3 Pax6 PVALB RNH1 5 5 MEIS1 CDH2 SUB1 NDUFS4 0 0 chPKCI 2.0 2.5 MRPL17 2.0 CER1 −5 1.5 CHRD −5 1.5 1.0 PTGDS 1.0 NOTO 0.5 0.5 MYO3A −10 0.0 −10 0.0 TIMP3 −5 0 5 −5 0 5 NOV POSTN POMC Zic2 Zfhx4 TFAP2A 5 DLX5 5 PLPP1 NET1 EPCAM 0 0 DAG1 TCF21 2.5 3 FIBIN 2.0 PERP2 −5 1.5 −5 2 ACTA2 1.0 1 GATA5 0.5 KRT7 −10 −10 KRT18 0.0 0 CSRP1 −5 0 5 −5 0 5 PODXL Supplementary figure S2. Quality control and supporting data for HH6 and HH7 10X data. (A) Violin plots showing RNA features, counts and percentage of mitochondrial reads per cell from HH6 data. Cells were filtered to exclude those with <500 and >5000 features (genes) and >0.5% mitochondrial gene content. (B) Feature plots of selected genes in HH6 clusters. (C) Heatmap depicting top 10 differentially expressed genes across each cluster from HH6. (D) Violin plots showing RNA features, counts and percentage of mitochondrial reads per cell from HH7 data. Cells were filtered to exclude those with <500 and >4000 features (genes) and >0.5% mitochondrial gene content. (B) Feature plots of selected genes in HH7 clusters. (C) Heatmap depicting top 10 differentially expressed genes across3 each cluster from HH7. bioRxiv preprint doi: https://doi.org/10.1101/2021.08.08.455573; this version posted August 8, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

0 1 2 0 1 2 0 1 2 3 4 A GJA1 CGATA2 E LMO1 NKX6−2 HOXB1 KRT18 FRZB CDX2 LDHB KRT7 BRINP2 GBX2 CLDN3 SOX21 TBXT CMTM8 CSRP2 MARCKSL1 WNT8A FN1 LFNG DLL1 PBX3 NDRG1 CAPN11 VRTN DLX5 ST6GALNAC2 CACHD1 PODXL FABP3 MID1 PDGFA LETM1 CDX1 TFAP2A CYP26A1 KRT19 MYCN SP5 SCX ZNF703 RGN TFAP2A B3GNT7 DLX5 WNT5A FN1 MEIS1 BASP1 KRT18 PRICKLE1 DAG1 CLDN3 PLPP1 PRTG ID3 KRT19 MSX1 HAPLN1 ASTL GATA2 SLC2A14 FIBIN ID3 CDX4 FABP3 LMO1 ASTL CDH2 OTX2 CSRP2 OTX2 MPZL3 FOXD3 NET1 SHISA2 FRZB UNC5B HESX1 DAG1 SOX2 RGN CYP26A1 CDX2 SIX3 LARP7 HOXB1 SHISA2 WNT8A THOC7 PDGFA SFRP1 YEATS4 CDX4 HESX1 GPC3 ITGA6 SKAP2 OTX1 CYP26A1 GJA1 SLC29A4 MARCKSL1 HES5 WNT5A RASGEF1B SFRP1 MEIS1 EHBP1 CDH11 SP5 TBXT LMO1 RAX HELLS SOX21 PRTG SIX3 DLL1 FGFR2 ZEB2 NCAM1 CAPN11 MSX1 ITGA6 RAX DLX2 MFAP2 SIX3 YEATS4 GRP FST HESX1 WDR1 HSPA2 FEZF2 PAWR SHISA2 HOXB1 SFRP1 PODXL CNTNAP1 GATA2 WNT8A Cbln2 CDX2 STK38L ASTL MYLK KRT18 GJA1 SOX21 B3GNT7 TBXT FST SOX2 CLDN3 TRARG1 LRRN1 KRT19 CDX1 ITGA6 DMD NDRG1 ZNF703 OTX2 TFAP2C PSIP1 CDX4 SHH KRT7 SP5 CHRD SGK1 FOXA2 PDGFA FOXD3 TFAP2A WNT5A NOV FN1 CA2 VRTN RASD1 SPTBN1 GBX2 OLFML3 RGN IGSF21 HES5 VTN FABP3 CTSV CXCL12 SKAP2 APOA1 OLFM1 MAP1B FAM89A LY6E BASP1 MSX1 APLP2 Wpkci−7 DLL1 PTGDS B D F Irf6 Grhl3 Irf6 Grhl3 Draxin Snai2 2 2 2.5 2.5 3 3

0 0 0.0 0.0 0 0 1.5 2.0 2.0 1.0 1.0 1.0 1.5 1.5 1.0 −2 −2 −2.5 −2.5 −3 1.0 −3 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 −4 −4 −5.0 −5.0 −6 −6 0.0 −5.0 −2.5 0.0 2.5 5.0 −5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −6 −3 0 3 6 −6 −3 0 3 6 FoxD3 Gbx2 FoxD3 Gbx2 Pax6 Six1 2 2 2.5 2.5 3 3

0 2.5 0 2.5 0.0 2.0 0.0 2.0 0 2.5 0 2.0 2.0 2.0 2.0 1.5 1.5 1.5 1.5 1.5 1.5 −2 −2 1.0 1.0 −3 −3 1.0 1.0 1.0 −2.5 −2.5 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.0 0.0 0.0 0.0 0.0 −4 −4 −5.0 −5.0 −6 −6 −5.0 −2.5 0.0 2.5 5.0 −5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −6 −3 0 3 6 −6 −3 0 3 6 Znf703 Gata2 Znf703 Gata2 Irf6 Grhl3 2 2 2.5 2.5 3 3

0 0 2.5 0.0 0.0 0 0 1.6 2.0 2.0 1.6 2.0 1.5 1.0 1.2 1.2 1.5 1.5 −2 1.0 −2 −2.5 −2.5 0.8 0.8 1.0 0.5 1.0 −3 −3 0.5 0.5 0.5 0.4 0.4 0.0 0.0 0.0 0.0 0.0 −4 −4 −5.0 −5.0 0.0 −6 −6 −5.0 −2.5 0.0 2.5 5.0 −5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −6 −3 0 3 6 −6 −3 0 3 6 Gata3 Six1 Gata3 Six1 Znf703 Tbxt 2 2 2.5 2.5 3 3 2.5 2.0 1.25 1.5 2.0 0 0 0.0 0.0 0 0 1.5 1.00 1.5 1.5 1.2 1.0 0.75 1.0 1.0 1.0 0.5 −2 −2 −2.5 −2.5 0.8 −3 0.50 −3 0.0 0.5 0.5 0.25 0.5 0.4 0.00 0.0 0.0 0.0 0.0 −4 −4 −5.0 −5.0 −6 −6 −5.0 −2.5 0.0 2.5 5.0 −5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −6 −3 0 3 6 −6 −3 0 3 6

Supplementary ﬁgure S3. Supporting data for subclustering HH5, HH6 and HH7 ectoderm clusters. (A) Heatmap depicting top 20 differentially expressed genes across HH5 subclusters. (B) Feature plots of selected genes in HH5 subclusters. (C) Heatmap depicting top 20 differentially expressed genes across HH6 subclusters, (D) Feature plots of selected genes in HH6 subclusters. (E) Heatmap depicting top 20 differentially expressed genes across HH7 subclusters. (F) Feature plots of selected genes in HH7 subclusters.

A HH4 HH6 HH10 B HH5 Astl HH6 Astl HH7 Astl

3 3 3 2 2 2 1 1 1 0 0 0

HH5 Ing5 HH6 Ing5 HH7 Ing5

2.5 Tfap2a Tfap2a 2.0 1.5 2.0 1.5 1.0 1.5 1.0 1.0 Astl Astl Tfap2a 0.5 0.5 0.5 Ing5 Ing5 Astl 0.0 0.0 0.0 C HH7 HH8 HH9 DHH4 Nav2 HH5 Nav2 E HH7

3 3 2 2 1 1 0 0 HH6 Nav2 HH7 Nav2

2 2 Grhl3 Nav2 Nav2 Nav2 1 1 Irf6 Sox21 Sox21 Sox21 0 0 Tfap2a F HH7 HH8 HH10 G HH6 HH7 HH8

FezF2 FezF2 FezF2 Lmo1 Lmo1 Lmo1 Otx2 Otx2 Otx2 Sp8 Sp8 Sp8 Tfap2a Tfap2a Tfap2a Tfap2a Tfap2a Tfap2a

Supplementary ﬁgure S4. HCR validation of selected genes. (A) Whole mount HCR shows co-expression of Astl and Ing5 with neural plate border marker Tfap2A at stages HH4, HH6, HH10. (B) Feature plots of Astl and Ing5 across whole epiblast data sets at stages HH5, HH6 and HH7. (C) Whole mount HCR shows co-expression of Nav2 and Sox21 at stages HH7, HH8 and HH9. (D) Feature plots of Nav2 across whole epiblast data sets. (E) Whole mount HCR shows co-expression of Grhl3, Irf6 and Tfap2A at HH7. (F) Whole mount HCR shows co-expression of FezF2 and Otx2 with neural plate border markers Tfap2A at stages HH7, HH8 and HH10. (G) Whole mount HCR shows co-expression of Lmo1 and Sp8 with neural plate border markers Tfap2A at stages HH6, HH7 and HH8.

A B C D Mitochondrial Ectoderm/ Tfap2A Dlx5 Tbxt genes Neural plate Endoderm Endoderm Ectoderm Streak/ early mesoderm 2 3 2 1 1 0

Zeb2 Cdh11 Sox2 Anterior 0 Epiblast 1 Stem cells mesoderm 2 0 3 1 4 3 3 2.5 5 2.0 Mesoderm 2 2 2 1.5 3 6 1.0 Posterior 1 4 Mesoderm 7 1 0.5 mesoderm 0.0

E Neural plate HH5-Cl4 F G Dlx5 Dlx6 H HoxB1

4 3 1.6 2.5 1.2 2.0 2 1.5 0.8 1 1.0 0.4 0.5 0 0.0 0.0 0 Pax6 Six3 Cdx4

Caudal HH5 −4 HH6 2.5 epiblast 3 3 0 HH7 2.0 Non-neural 2 2 1.5 1 1.0 HH8 1 1 ectoderm 2 0.5 −10 −5 0 5 0 0 0.0

I Bmp4 Lmo1 Sox11 4 Nav2 Dlx6 2 0.10 0.5 2

2 1 unspliced 0.05

unspliced 0.25 unspliced unspliced

0.00 0 0 0 0.00 0 1 0 2 4 0 2 0 1 0.0 0.2 0.4 spliced spliced spliced spliced Bmp4 Lmo1 Sox11 Nav2 Dlx6

0.08 0.25 0.15 0.1 0.15

0.00 0.00 0.00 0.0 0.00

Velocity -0.08 Velocity -0.25 Velocity -0.15 Velocity -0.1 Velocity -0.15 Bmp4 Lmo1 Sox11 Nav2 Dlx6

0.16 0.50 3 2 0.8 2 0.08 0.25 1 0.4 1 Expression 0.00 Expression Expression Expression Expression 0.00

Supplementary ﬁgure S5. scVelo analysis at HH4 and HH5 and supporting data for scVelo analysis at HH6 and HH7. (A) UMAP embedding of clusters from whole epiblast data at HH4 showing predicted trajectories. (B) UMAP embedding of clusters from whole epiblast data at HH5 showing predicted trajectories. (C) Feature plots of selected genes across HH5 whole epiblast data. (D) Feature plots of selected genes across HH7 whole epiblast data. (E) UMAP embedding of subclusters from HH5 ectoderm cluster (HH5-Cl4) showing predicted trajectories. (F) UMAP embedding of clusters from all combined data set showing stages. (G) Feature plots of placode markers across all stages combined data set. (H) Feature plots of other selected genes across all stages combined data set. (I) Phase portraits (top row), velocity plots (middle row) and expression plots (bottom row) of selected genes across the HH7/ premigratory NC combined data sets.