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

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.

HH5-Cl4 cells, co-expression of neural and non-neural ectoderm factors here (Figure S1E/F)   Neural plate border Notochord progenitors As the chick embryo undergoes gastrulation, ectodermal cells that will become neural, neural plate border, placode or epidermis remain in the upper layer of the embryo (epiblast), while mesoendodermal cells ingress and internalise at 112 the primitive streak and Hensen's node. To further resolve the transcriptional complexity of the developing neural plate 113 border, we extracted and subclustered the ectodermal clusters at stages HH5, HH6 and HH7. These are designated 114 HH5-Cl4, HH6-Cl8/10 and HH7-Cl3/9 ( Figure 1E, H, K).

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In summary, analysis of our single cell HH5 and HH6 transcriptomes did not identify a distinct cell cluster marked of gene expression. 167 We identified several new genes in the ectoderm, many of which persisted into neural or non-neural tissues. At

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Ing5 was also detected in the neural plate border and in the neural folds ( Figure 3C). By HH8, Astl expression was 175 prominent in the neural folds, most strongly in the posterior hindbrain ( Figure 3D, 3D'). Ing5 was detected along the 176 neural tube at HH8 ( Figure 3D, 3D'); however, by HH10 Ing5 transcripts were no longer detectable. Astl expression 177 was also broadly decreased at HH10, where activity was restricted to a small region of the neural tube in the hindbrain 178 and the emerging otic placodes ( Figure S4A). Consistent with these observations Astl and Ing5 were detected in HH5-

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We identified a number of transcription factors in HH6-Cl8 (neural) and HH6-Cl10 (non-neural ectoderm). The 187 latter was characterised by Tfap2A enrichment and also harboured Grhl3 and Irf6 ( Figure 3E). In situ analysis showed

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Grhl3 was specifically expressed in the neural plate border, most strongly in the posterior region at HH6 and HH8-

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( Figure S4E, Figure 3F). Irf6 was expressed in the neural plate border as well as the surrounding non-neural ectoderm at 190 HH6 and HH8-( Figure S4E, Figure 3F). Furthermore, we observed significant cellular co-expression of all three factors 191 in the neural plate border ( Figure 3F'). At HH8+, Irf6 was restricted to the dorsal neural tube including premigratory 192 neural crest cells, Grhl3 was also detected here at lower levels but was present in the emerging otic placode ( Figure   193 3G). By HH9+ Grhl3 levels were broadly diminished but remained in the developing otic placode. Irf6 was maintained 194 in the neural tube during neural crest delamination and also enriched in the otic placode ( Figure 3H). In the ectodermal subclusters, we identified a number of novel factors including Wnt pathway genes. In HH6-  Figure S3C). Sp8 was identified in HH6-sub-2 (caudal neural), in vivo we found Sp8 expression 199 commenced from HH7 in the neural plate border, partially overlapping with Tfap2A ( Figure 4B). The homeobox tran-200 scription factor, Dlx6, was present in HH7-sub-1, (neural plate, Figure 4A) and was expressed predominantly in the 201 anterior neural plate border or pre-placodal region ( Figure 4B). This pattern continued with higher levels of both Sp8 202 and Dlx6 at HH8-( Figure 4C). Sp8 expression was restricted to the dorsal neural tube, whereas Dlx6 spread more 203 laterally ( Figure 4C'). At HH9, Sp8 was detected predominantly in the anterior neural tube but was also found in prem-204 igratory neural crest cells in the mid-brain region ( Figure 4D, 4D') but largely absent from the hindbrain region, though 205 some expression was seen in the trunk premigratory neural crest ( Figure 4D). Dlx6 was also present in premigratory 206 neural crest cells at HH9 ( Figure 4D'), as well as in the lateral non-neural ectoderm ( Figure 4D).

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Neural clusters expressed numerous transcription factors including Sox21 which was which was present in HH7-222 sub-0 and HH7-sub-3 ( Figure S3E). FezF2 together with Otx2 and Sox2 were also enriched in neural subclusters 223 HH7-sub-3 ( Figure S3C/E). FezF2 has previously been shown to regulate Xenopus neurogenesis by inhibiting Lhx2/9 224 mediated Wnt signaling (Zhang et al., 2014) Accordingly, in vivo, FezF2 was expressed in the anterior neural folds 225 at HH7 where it overlapped with Otx2, and in the neural tube at later stages ( Figure S4F). Lmo1 was also found in 226 HH7-sub-0 ( Figure S3E) and was expressed across the neural plate at HH6 through HH8, where it was also seen in the 227 neural folds ( Figure S4G). Znf703 Pax7, Tfap2A, ( Figure 1I), Bmp4, and Msx1 ( Figure 5B). Whilst these cells appeared to contribute to both neural and 249 non-neural programmes, the low degree of splicing kinetics as compared to other subpopulations indicated they were 250 relatively transcriptionally stable at this stage ( Figure 5A).

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

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Here we also included 10X scRNA-seq data obtained from premigratory neural crest cells (HH8+) from our previous

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Combining these datasets revealed that the non-neural ectoderm from both HH6 and HH7 grouped together, as 288 did the neural plate clusters from the same stages ( Figure 6A). This suggests successful inter-sample incorporation 289 into a single reference with no resultant batch bias ( Figure S5F). Ectoderm cells from HH5 were spread amongst the 290 neural plate and the non-neural ectoderm clusters from HH6 and HH7. The neural crest clusters were more discrete,  scVelo showed that non-neural ectoderm cells from HH6/HH7 were split between two trajectories ( Figure 6A).

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Placodal markers (Dlx5/6, Six3 and Pax6) defined one distinct trajectory ( Figure S5E), whereas the other trajectory 298 was less well defined but could be distinguished by posterior epiblast markers (HoxB1, Cdx4) ( Figure S5H). The 299 neuroepithelial-like cells from HH8+ were emerging between these trajectories where they were joined by other cells 300 from HH6/HH7 non-neural ectoderm clusters. These cells were enriched for Pax7 and other neural plate border markers 301 Tfap2A, Msx1, Bmp4 and ( Figure 6B). Early neural crest genes including Tfap2B and Draxin were also emerging here 302 ( Figure 6B). While these cells had less defined trajectories, they were generally directed towards the neural-neural crest 303 lineage ( Figure 6A). Neural plate border and neural crest markers also extended into a heterogeneous region of cells, 304 where a significant portion of the neural-neural crest and neural plate cells were highly integrated, suggesting some cells  Since we did not see evidence of neural crest lineages until HH7, we assessed the developmental trajectories 310 from HH7 ectoderm clusters directly to bona fide neural crest cells (HH8). To this end, we extracted the ectoderm 311 clusters (HH7-Cl3+Cl9) and combined these cells with the premigratory neural crest data set from HH8+ embryos 312 and used scVelo to plot the trajectories across these cells/stages. Analysis of the pooled dataset yielded clearly defined 313 developmental trajectories splitting from the HH7 ectoderm clusters, whereby the non-neural ectoderm cells (HH7-Cl9) 314 gave rise to the neuroepithelial-like cells from the HH8+ dataset, and the neural plate (HH7-Cl3) cells were contributing 315 directly to the neural-neural crest ( Figure 6C). Non-neural ectoderm cells were also initiating a separate trajectory, 316 likely representing placodal lineages as indicated by Six1, Dlx5 ( Figure 6D). While Tfap2A was expressed across the 317 non-neural ectoderm cluster, Pax7 was found in just a subset of non-neural ectoderm cells and in neuroepithelial-like 318 cells, where Tfap2A was also found. Both factors were also enriched in bona fide neural crest, though Pax7 was also 319 detected in the neural-neural crest ( Figure 6D). Other neural plate border markers (Bmp4, Msx1) were found in the 320 non-neural ectoderm and some neuroepithelial-like cells, as well as extending into the interface of neural plate and 321 neural-neural crest populations ( Figure 6D).

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

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Analysis of dynamic transcriptional trajectories during lineage restriction reveals key lineage specific drivers 331 We next sought to determine key genes driving the observed developmental trajectories in the context of neural 332 crest specification from the neural plate border. Dynamical modelling of transcriptional states within the HH7/8 com-333 bined data-set revealed a number of highly ranked dynamic genes including Pax7 and Tfap2B. Pax7 splicing increased 334 progressively from the non-neural ectoderm cells to the neuroepithelial-like cells and lower splicing was observed in 335 neural crest cells where the transcripts stabilized such that increased expression was observed ( Figure 6F). Splicing of 336 Pax7 transcripts was also increased in the putative placodal lineage from the non-neural ectoderm ( Figure 6F). Tfap2B 337 splicing was not evident in the HH7 data, but was increased in the neuroepithelial-like cells and stabilized in neural 338 crest populations ( Figure 6F). Tfap2A showed more complex velocities, whereby high levels of spliced transcripts were 339 detected in non-neural ectoderm cells from HH7 likely driving these cells towards the placode trajectory ( Figure 6F). 340 We also observed a progressive increase in Tfap2A splicing across the neuroepithelial-like cells to the bona fide neural 341 crest. This suggested that by HH8, Tfap2A transcripts are stable in the neuroepithelial-like population but upregulated 342 in the bona fide neural crest, consistent with the observed expression dynamics ( Figure 6F). Another neural plate border 343 gene, Bmp4, was dynamically regulated in non-neural ectoderm but downregulated in the neural plate ( Figure S5I). 344 We also identified a number of more novel factors putatively involved in ectoderm lineage progression. Otx2 was 345 driving neural plate cells towards the neural-neural crest cluster, consistent with previous findings from functional 346 perturbation studies (Williams et al., 2019). However, Otx2 also seemed to be driving non-neural ectoderm cells 347 towards the neuroepithelial-like population ( Figure 6F). Lmo1 was also driving the neural plate cells towards the neural-348 neural crest; however, unlike Otx2, Lmo1 expression was not maintained in the neural crest populations ( Figure S5I).
Bri3 was identified as a highly ranked dynamic gene and was upregulated in neural plate cells and some non-neural ectoderm cells. Down-regulation of Bri3 in neural crest cells suggested an early role in ectoderm lineage trajectories for 351 this novel factor ( Figure 6F). Nav2 and Sox11 were both highly expressed in the neural plate and downregulated in the 352 non-neural ectoderm. Sox11 was also expressed in the mesenchymal neural crest cluster where it was highly spliced, 353 potentially representing a dual segregated role for this factor in both neural plate and mesenchymal crest lineages 354 ( Figure S5I). Nav2 velocities increased progressively from the non-neural ectoderm to the neural plate; consistently, a 355 subset of non-neural ectoderm cells joined a trajectory with the neural plate cells ( Figure S5I) where Nav2 splicing was 356 highest ( Figure S5I). We found Pitx1 and Dlx6 to be dynamically active across the merged HH7/8 data-set, potentially 357 driving non-neural ectoderm to the neuroepithelial-like population ( Figure 6F, Figure S5I).

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

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