NR6A1 is essential for neural crest cell specification, formation and survival

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
Neural crest cells (NCC) are a migratory progenitor cell population unique to vertebrates that contribute to nearly every organ system throughout the body. Disruptions in NCC development can result in congenital disorders (neurocristopathies). Yet, our understanding of the cellular mechanisms and signals that govern mammalian NCC formation remains poor. Here, we discovered nuclear receptor superfamily 6 group member 1 (NR6A1/GCNF/RTR) is a novel, critical regulator of mammalian NCC specification, formation and survival. Nr6a1 expression in mouse embryos spatiotemporally overlaps with early NCC development. NR6A1 loss-of-function perturbs anterior cranial NCC formation and survival, with complete agenesis of migratory NCC caudal to the first pharyngeal arch. Using targeted molecular and genomic approaches, we demonstrate that these phenotypes are associated with perturbation of NCC specification and epithelial-mesenchymal transition, and with persistent expression of pluripotency-associated factors. Supporting these observations, in vivo overexpression of Oct4 in gastrulating mouse embryos disrupts NCC specification and formation. Conditional temporal deletion revealed that Nr6a1 is required during mid-late gastrulation, demonstrating that the initiation of murine NCC specification likely occurs during gastrulation-earlier than previously thought, but in close alignment with the established timeline of NCC development in other vertebrate model organisms. These findings also reveal that the gold standard transgenic mouse line, Wnt1-Cre, is unsuitable for studying genetic function during NCC specification and formation. In summary, NR6A1 is essential for mammalian NCC development and may function during gastrulation as a bimodal switch modulating pluripotency-associated factors in the neuroepithelium, while concomitantly activating NCC specifiers and regulators of EMT.

Introduction
Neural crest cells (NCC) are a heterogeneous migratory cell population. With varying degrees of potential, NCC contribute to nearly every tissue and organ system throughout the body and are therefore essential for proper development. During early embryogenesis, NCC are specified at the neural plate border, undergo an epithelial-mesenchymal transition (EMT), delaminate from the neuroepithelium and then migrate throughout the embryo. Depending on their axial level, NCC differentiate into cartilage, bone and connective tissues in the head and neck, neurons and glia of the peripheral nervous system, smooth muscle and cardiomyocytes in the heart, endocrine cells in many organs, and pigment cells in the skin, among many others1. Imbued with considerable inherent plasticity, NCC have served as a conduit for intraspecies and interspecies variation, and the evolution of anatomical novelties - particularly in the head and face2–5. However, perturbations in NCC development are responsible for a diverse class of congenital anomalies known as neurocristopathies6–8. Uncovering the mechanisms by which NCC regulate vertebrate development and evolution, and devising therapeutic approaches for preventing congenital neurocristopathies, requires a comprehensive understanding of the signals, switches and gene regulatory networks that govern each stage of NCC development.

Decades of research have elucidated many of the tissue interactions and signals that drive NCC specification and formation. NCC are induced at the neural plate border through contact-mediated interactions between the neural ectoderm, surface ectoderm and paraxial mesoderm9–18. Signals from these surrounding tissues include BMP, FGF and WNT which influence the expression of neural plate border genes such as, Tfap2a, Zic1, Msx1 and Pax3/711,12,16,17,19–30. These inductive signals together with the neural plate border factors upregulate NCC-specifier genes Foxd3, Sox9, and Sox10 and EMT genes Snai1/2, Zeb2 and Twist1 to facilitate NCC specification, formation and delamination17,18,23,31–46. However, many of these factors and their functions, which were uncovered in non-mammalian model organisms, appear dispensable in mouse47–49. For example, BMP4 and BMP7 function as critical inductive signals that regulate NCC specification in chicken, but when BMP4 and BMP7 are knocked out in mouse, either individually or in combination, NCC still form16,50–55. Similarly, BMP2 is also not required for NCC induction in the mouse, although it is important for NCC migration56. Neural plate border genes Pax3 and Pax7 are also expendable for NCC formation in mouse as is evident from single and double knockouts57–59. Finally, NCC still form, delaminate and migrate in mouse embryos even when EMT master regulators Snai1, Snai2, Zeb2 or Twist1 are knocked out60–63. These discrepancies between mammalian and non-mammalian species are surprising given the fundamental importance of NCC in vertebrate development, but may be attributable to developmental heterochronies, and differences in species-specific experimental approaches48. Thus, the gene regulatory network underpinning mammalian NCC formation has been difficult to define and remains poorly understood.

Previously, we uncovered nuclear receptor subfamily 6 group A member 1 (Nr6a1) as transcriptionally downregulated in a mouse model of the craniofacial neurocristopathy, Treacher Collins syndrome64, suggesting this nuclear receptor plays an unknown role in NCC development. While the greater nuclear receptor superfamily consists of 70 receptors organized into 7 subclasses, NR6A1 is in a subclass on its own. Nuclear receptors like NR6A1 contain an N-terminal domain, a DNA-binding domain and a ligand binding domain65–67. However, the ligand that interacts with NR6A1 is unknown and therefore NR6A1 is considered an orphan nuclear receptor. First isolated from testes and heart tissue, NR6A1 was originally named germ cell nuclear factor (GCNF) and retinoid receptor-related testis-specific receptor (RTR)68,69. Dynamically expressed during folliculogenesis and spermatogenesis, as well as in the embryo throughout pre- and post-implantation, Nr6a1 is critical for mouse embryo development and survival70–73. NR6A1 loss-of-function in mouse results in an open neural tube, axial truncation, cardiac defects, and failure of chorioallantoic fusion, which leads to embryonic lethality by embryonic day (E) 10.573.

In this work we investigate the role of Nr6a1 in NCC development—determining this orphan nuclear receptor is a novel pivotal regulator of murine NCC specification, formation and survival. We demonstrate that Nr6a1 is spatiotemporally expressed in the neuroepithelium and newly emigrating NCC indicating that Nr6a1 regulates NCC formation and survival. Indeed, through loss-of-function analyses in mice we show that Nr6a1 null embryos exhibit a deficiency in anterior cranial NCC, and complete agenesis of migrating NCC caudal to the first pharyngeal arch. These phenotypes are associated with downregulation of NCC specifiers (Foxd3, Sox9, Sox10) and EMT master regulators (Snail1, Zeb2), in concert with expansion of the neural stem cell marker Sox2, increased proliferation, and persistent expression of pluripotency-associated factors (Oct4 and Nanog) in the neuroepithelium. Using mouse embryonic stem cells (mESC), and human induced pluripotent stem cells (hiPSC) differentiated into NCC, as well as chromatin immunoprecipitation and multiomic approaches, we demonstrate that NR6A1 modulates the chromatin landscape and directly binds to putative DR0 motifs in the promoter regions of NCC, EMT and pluripotency-associated factors to regulate their expression. Furthermore, global temporal and conditional spatiotemporal deletion reveal that Nr6a1 is specifically required during mid-late gastrulation, suggesting that NCC specification in mouse embryos commences earlier than previously recognized. Consistent with this model, in vivo overexpression of Oct4 in gastrulating mouse embryos disrupts NCC specification and formation. Altogether, our work has uncovered NR6A1 as a novel regulator of mammalian NCC specification and formation. NR6A1 may therefore function as a bimodal switch that modulates the repression of pluripotency factors associated with neural stem cell maintenance and proliferation, while being required to activate a gene regulatory network of NCC specifiers and regulators of EMT.

Results
Spatiotemporal distribution of Nr6a1 mirrors NCC development
Treacher Collins syndrome is a neurocristopathy caused primarily by variants in the TCOF1 gene74–76. Characterized by downward slanting of the palpebral fissures, hypoplasia of the zygomatic complex, micrognathia and cleft palate74,75, previous studies of mouse and zebrafish models of Treacher Collins syndrome determined these characteristic craniofacial anomalies to be caused by a deficiency in NCC. Tcof1+/− haploinsufficiency results in diminished rRNA synthesis and ribosome biogenesis, as well as DNA damage, leading to increased p53-dependent apoptosis, which compromises the formation, proliferation and survival of NCC64,76–80. Transcriptomic analyses of E8.5 Tcof1+/− and control littermate embryos revealed numerous gene expression changes in association with the molecular and cellular pathogenesis of Treacher Collins syndrome64. We hypothesized that downregulated genes in this mouse neurocristopathy model might also be important for normal NCC development.

Interestingly, Nr6a1, which has been shown to regulate the transition of primitive neural stem cells to definitive neural stem cells81, was decreased two-fold in Tcof1+/− embryos compared to controls64. We therefore posited that Nr6a1 may also play a critical role in NCC development. Nr6a1 expression can be induced in embryonic stem cells at E4.5 and P19 cells when treated with retinoic acid and its expression pattern inversely correlates with Oct482,83. Furthermore, previous studies reported strong expression of Nr6a1 throughout the embryo at E7.5 before being restricted to the neural ectoderm and posterior of the embryo (E8.0-8.5)73.

To better characterize the spatiotemporal expression of Nr6a1 during NCC development, we performed a detailed temporal analysis by in situ hybridization on CD1 mouse embryos from E6.5 to 9.5 (Fig. 1). Nr6a1 expression is absent at E6.5, consistent with the known high levels of Oct4 present in the embryo at this stage (Fig. 1A). Nr6a1 expression commences between E6.5 and E7.5 and is observed broadly throughout the embryo (Fig. 1B). At E8.0, Nr6a1 is expressed in the neural plate and in the trunk progenitors required for axial elongation (Fig. 1C,D). Nr6a1 expression then becomes more pronounced along the entire neuraxis from E8.5-8.75 (Fig. 1E,F). Transverse histological sections of stained E8.5-E8.75 embryos showed Nr6a1 is dynamically expressed in the dorsal most region of the neuroepithelium, along the basal edge and in cells directly outside the neuroepithelium consistent with a pattern of newly delaminated NCC (Fig. 1E, arrows). At E9.5, Nr6a1 expression is maintained throughout the neuraxis, but with an absence in the posterior growth zone (Fig. 1F), consistent with the trunk to tail transition as we have previously reported84. Furthermore, we also previously showed that Nr6a1 expression becomes restricted to the somites at E10.5 before being downregulated by E12.584. Our expression analyses, therefore, indicated that Nr6a1 is expressed at the right time and in the right location to potentially function in the specification, formation and early migration of NCC.

NCC formation, delamination and survival require Nr6a1
Nr6a1 null mutant mice are embryonic lethal by E10.5 due to cardiac defects and a failure of chorioallantoic fusion73. Nr6a1−/− embryos also exhibit significant defects in axial elongation and tail bud formation73,84,85. To determine if Nr6a1 is required for NCC development, we evaluated the expression of NCC-specifier genes Foxd3, Sox9 and Sox10 in E8.75 Nr6a1−/− embryos compared to stage-matched control littermates, prior to embryonic lethality, using in situ hybridization chain reaction (HCR). Foxd3 is one of the earliest markers of NCC specification and it maintains the potency of NCC86 while also regulating cadherin turnover to facilitate delamination by EMT39. Compared to wild type littermate controls, Foxd3 expression was significantly reduced in the Nr6a1 null embryos with only a minor cluster of cells expressing Foxd3 in the head (Fig. 2A,B, paired ratio t-test p=0.0339).

Sox9 is also required for NCC formation and delamination and is expressed in pre-migratory and early migratory NCC, as well as later during their chondrogenic differentiation42,87,88. We could detect only a few cells expressing Sox9 in the craniofacial mesenchyme, with no labeled cells in the frontonasal prominences in Nr6a1 null embryos (Fig. 2A,B; paired ratio t-test p=0.0190). Similarly, Sox10, a marker of migrating NCC40,43, was significantly reduced in Nr6a1−/− embryos with a complete absence of labeled cells in the first pharyngeal arch (Fig. 2A,B; paired ratio t-test p=0.0300).

We next evaluated whether the expression of NCC EMT master regulators Snai1, Zeb2 and Twist1 was also disrupted in Nr6a1−/− embryos. Snai1 is typically expressed in neuroepithelial cells during NCC formation, and its expression persists in delaminating and migrating NCC31,32,36,89. However, Snai1 expression was reduced in Nr6a1−/− embryos, with an absence of Snai1 labeled cells in the first pharyngeal arch compared to the control stage-matched littermate (Fig. 2A,B; paired ratio t-test p=0.0129). Zeb2 is also expressed in the neuroepithelium and migratory NCC37,63. Unfortunately, we were unable to detect Zeb2 by HCR or SABER-FISH, and therefore, instead performed traditional in situ hybridization to investigate the dynamics of Zeb2 expression. Although Zeb2 was expressed in the neural plate in Nr6a1−/− embryos, it was absent from the NCC derived facial and pharyngeal mesenchyme (Fig. 2C). Twist1, like Snai1 and Zeb2, is considered a master regulator of EMT. Interestingly, Nr6a1−/− embryos exhibit Twist1 expressing cells in the frontonasal prominences and pharyngeal arches (Fig. 2A,B), but previous studies have shown that Twist1 is not required for NCC formation and instead regulates NCC migration and differentiation during mouse embryo development60,90,91. Altogether, our gene expression analysis suggested Nr6a1 is required for the expression of NCC-specifier and EMT genes during development.

Gene expression is not necessarily an indicator of lineage, but the perturbations in NCC-specifier and EMT gene expression implied that Nr6a1 null embryos might be deficient in NCC formation and delamination. However, the expression of Twist1 suggested there were some mesenchymal cells present in the frontonasal prominences and pharyngeal arches. Therefore, to more conclusively assess the presence or absence of NCC in Nr6a1 null embryos, we performed lineage tracing by crossing the Wnt1-Cre transgene92 into the background of Nr6a1−/− embryos. Wnt1-Cre is expressed in the dorsal neuroepithelium encompassing the territory in which neural crest cells are formed. In agreement with this idea, E9.0-9.5 Nr6a1−/−;Wnt1-Cre;R26R-LacZ embryos completely lacked migrating NCC caudal to the first pharyngeal arch (Fig. 2D), but the presence of LacZ-positive cells within the frontonasal region and first pharyngeal arch in Nr6a1−/− embryos suggests some cells are still capable of delaminating and migrating from the midbrain and anterior hindbrain neuroepithelium (Fig. 2D). We observed identical cranial and trunk patterns using an eYFP reporter instead of LacZ (Fig. 2E). E9.0-9.5 Nr6a1−/−;Wnt1-Cre;R26R-eYFP embryos exhibited fewer migrating cranial NCC and complete absence of migrating NCC caudal to the first pharyngeal arch. Transverse histological sections of E9.0-9.5 Nr6a1−/−;Wnt1-Cre;R26R-eYFP embryos further illustrate the diminishment of eYFP-labeled putative NCC within the first pharyngeal arch and their complete absence from the second pharyngeal arch compared to controls (Fig. 2E). This is consistent with the expression of NCC-specifier and EMT genes as visualized by HCR (Fig. 2A), which had predicted Nr6a1−/− mutant embryos would have very few NCC in the head.

Given the expression of Twist1 in Nr6a1 null embryos, these eYFP-positive and LacZ-positive cells are likely to be mesenchymal (Fig. 2A). However, the downregulated expression of NCC-specifier and other EMT genes, and reduced number of labeled cells, raised questions about their long-term potential. TUNEL staining of E8.75 Nr6a1−/−;Wnt1-Cre;R26R-eYFP revealed visibly higher levels of cell death in Nr6a1 mutants compared to Wnt1-Cre;R26R-eYFP littermate controls (Supplemental Fig. 1). Given the Nr6a1 null embryos do not survive beyond E10.5, we wanted to confirm the cell death was specific to the NCC. We therefore counted the number of TUNEL-positive cranial NCC and non-NCC in E8.75 Nr6a1−/−;Wnt1-Cre;R26R-eYFP and control littermates well before embryonic lethality. Quantifying the percentage of cells in each population undergoing apoptosis confirmed a higher percentage of NCC undergoing apoptosis in the Nr6a1−/−;Wnt1-Cre;R26R-eYFP compared to controls (Supplemental Fig. 1B; one-way ANOVA, p=0.0105; unpaired parametric t-test p=0.0017). Furthermore, no statistical difference was observed in the level of apoptosis in non-NCC tissue in Nr6a1−/−;Wnt1-Cre;R26R-eYFP embryos compared to control littermates, thus revealing the cell death evident in Nr6a1−/− mutant embryos was NCC-specific. Altogether, these results indicate Nr6a1 is required for murine NCC formation and survival.

The neural stem cell to NCC transition requires Nr6a1
Nr6a1 is predominantly expressed in the neural ectoderm during early NCC development and is required for neural tube closure73. This raised the question of whether the deficiency in NCC formation and migration in Nr6a1 null embryos was a secondary consequence of abnormal specification and patterning of the neural ectoderm. Sox2 is a pan-neural marker of progenitor cells in the neural ectoderm and is sufficient to maintain their proliferation and stemness properties93,94. Through in situ hybridization, we observed that Sox2 is expressed in the open neural plate in Nr6a1 null embryos (Fig. 3A), indicating that pan-neural specification of the neural plate occurs in Nr6a1 mutants. Wnt1 and Pax3 are also expressed within the neural ectoderm, but specifically in dorsal domains that help establish dorsoventral patterning11,12. Their spatiotemporal patterns of expression remain unchanged in Nr6a1 null embryos compared to controls indicating that proper dorsal pattering is also established in the neural plate of Nr6a1 mutants.

Immunostaining of transverse sections confirmed SOX2 and PAX3 were expressed in the neural ectoderm of Nr6a1−/− embryos but also illustrated the increased thickness of the neural plate compared to controls (Fig. 3B). SOX2 is a key regulator of proliferating neural progenitor cells throughout the entire anterior-posterior axis of the central nervous system from neural plate stages of embryogenesis through to adulthood95. PAX3 also functions to maintain the neuroepithelium in a proliferative, undifferentiated state, allowing neurulation to proceed96. We therefore hypothesized that the thickened neuroepithelium in the Nr6a1 mutants was due to increased proliferation. Indeed, phospho-histone H3 (pHH3) immunostaining of transverse histological sections confirmed that Nr6a1−/− embryos exhibit considerably higher proliferation than littermate controls (Fig. 3B).

Additionally, we also observed that the expression of SOX2 persisted in the dorsolateral border region of Nr6a1−/− embryos compared to controls (Fig. 3B). Downregulation of Sox2 in the dorsolateral border of the neural ectoderm is essential for neural stem cells to differentiate into NCC and occurs as part of a SoxB (Sox1, Sox2) to SoxE (Sox9, Sox10) switch93,97. Furthermore, although the dorsal domain of PAX3 in the neural ectoderm appeared unchanged in Nr6a1 mutants, PAX3 expression was not observed outside of the neuroepithelium, consistent with the deficiency of migratory NCC in Nr6a1 mutants compared to littermate controls (Fig. 3B).

Thus, NR6A1 loss-of-function does not result in gross mispatterning of the pan-neural or dorsal character of the neural ectoderm. Collectively, these results indicate the neural ectoderm forms and is appropriately patterned in Nr6a1 null embryos. However, the persistence of SOX2 expression in the dorsolateral border region together with elevated proliferation, suggests that the neural ectoderm in the Nr6a1 null mutants is maintained in a proliferative stem cell state which may restrict neural stem cells from differentiating into NCC.

To better understand how NR6A1 regulates NCC formation and survival, we initially performed microarray based transcriptomic analyses of E9.0 wildtype and Nr6a1 null mutant embryos. Consistent with our HCR results (Fig. 2A), the expression of NCC-specifier genes Foxd3, Sox9, and Sox10 was downregulated in Nr6a1 null embryos, together with EMT master regulators Snai1, Zeb2 and Twist1 (Table 1). Although the downregulation of these factors in the microarray did not reach statistical significance, this was likely due to the entire embryo being sampled, of which the NCC are but a relatively small portion at this stage of development. Therefore, we also performed bulk RNA-sequencing on only the anterior portion of the embryos, and again we observed considerable downregulation of NCC-specifier and EMT master regulator gene expression in Nr6a1−/− embryos compared to littermate controls (Table 1). In contrast, we found that pluripotency associated factors such as Oct4 and Nanog, were significantly upregulated in Nr6a1−/− embryos compared to littermate controls through each of the transcriptomic approaches (Table 1).

OCT4 and NANOG, together with SOX2, play essential coordinated roles in preserving the pluripotency and self-renewal capacity of ESCs and adult stem cells98–100. Interestingly, previous studies have shown that Nr6a1 directly represses Oct4 and Nanog during peri-implantation development82,83,101. To test this model in the context of NCC development, we performed a time course analysis of OCT4 expression using the Oct4-EGFP reporter mouse line102. Oct4-EGFP is strongly expressed and clearly observed in the epiblast at E7.5 but is then downregulated by E8.5 (Fig. 4A). We then crossed Oct4-EGFP into the background of Nr6a1 null embryos and observed persistent ectopic Oct4-EGFP expression throughout the neural ectoderm in E8.5 Nr6a1−/−;Oct4-EGFP embryos compared to control littermates (Fig. 4B-C). Although GFP is an indirect read out of OCT4 activity, persistent ectopic Oct4 expression has previously been reported in Nr6a1−/− embryos83. Collectively, these results indicate that OCT4 is typically downregulated in the neural ectoderm in association with NCC formation.

In further support of this idea, the expression of SOX10 did not align with the regions of persistent ectopic Oct4-EGFP activity, suggesting that Oct4 was downregulated in the few NCC that do form in Nr6a1−/− embryos (Fig. 4B-C). The region of persistent ectopic Oct4 expression notably aligns with the location where NCC formation is perturbed along the neuraxis. The persistent expression of pluripotency associated genes such as Sox2, Oct4 and Nanog, therefore suggest that the neural ectoderm in Nr6a1 null mutants is maintained in a proliferative stem cell state which restricts neural stem cells from differentiating into NCC in the head, and especially caudal to the first pharyngeal arch. Thus, Nr6a1 may be required for the transition of neural ectoderm or neural stem cells to NCC by repressing pluripotency and stem cell maintenance genes while concomitantly being required for the activation and expression of NCC specification and EMT genes.

NR6A1 modulates stem cell maintenance and NCC specification
NR6A1 is an orphan member of the nuclear receptor superfamily and shares the highest homology with COUP-TF, which functions as both a transcriptional repressor and activator68,101. NR6A1 binds to a double repeat of the consensus sequence AGGTCA with zero spacing (DR0) motif, to transcriptionally regulate target genes such as Oct4 and Nanog68,85,103. The downregulation of NCC specification and EMT genes in Nr6a1−/− embryos suggests NR6A1 could function by activating these genes during development in parallel with modulating the repression of pluripotency-associated genes. We therefore searched for this putative binding site or motif in NCC-specification and EMT genes to determine the potential for NR6A1 to directly regulate these genes. Sox9, Sox10, Snai1 and Zeb2 each possess DR0 binding sites in their promoter regions, which is consistent with downregulation of their expression in Nr6a1 null embryos.

To further understand how NR6A1 regulates NCC-specifier and EMT genes within the neural ectoderm during NCC development, we performed single cell multiomic analyses of E8.75 Nr6a1 null mutant and control littermate embryos. Combining single cell RNA-sequencing (scRNA-seq) with single nucleus transposase-accessible chromatin sequencing (snATAC-seq) allows for the assessment of differential gene expression and chromatin accessibility between cell populations in the Nr6a1 null and control littermate embryos. The scRNA-seq results from the controls was used to annotate the cell populations (Supplemental Fig. 2A-B). The neural plate border cells were identified based on high differential expression of Wnt1, Pax3/7 and Zic1104–106 (Supplemental Fig. 2A-B). NCC were annotated based on high differential expression of the NCC-specifier genes Foxd3, Sox9, Sox10, Crabp1, Snai1 and Zeb247,106–108 (Supplemental Fig. 2A-B). The neural ectoderm was identified by high differential expression of Sox2, Pax2, Otx2 and Sox3104,106,109 (Supplemental Fig. 2A-B). Finally, neural progenitors were annotated based on high expression of Olig2, Sox2 and Pax694,106,110,111 (Supplemental Fig. 2A-B). The same transcriptional signatures were used to identify the same specific cell populations within the Nr6a1−/− embryos.

Comparison of the Nr6a1−/− embryo scRNA-seq dataset to the control littermate scRNA-seq dataset revealed the NCC population was decreased by greater than 50% in the null, comprising 4% of the total cell population in the controls but only 1.4% in the Nr6a1−/− embryos (Supplemental Fig. 3A-C). The greater than 50% loss of NCC aligned with the results obtained from Nr6a1−/−;Wnt1-Cre;R26R-eYFP lineage tracing (Fig. 2E). Furthermore, the few cells that clustered as NCC in the mutant embryos exhibited a significant decrease in Sox10 expression, while in contrast the neural plate border (NPB) cells exhibited increased Oct4 and Nanog expression compared to control littermates, consistent with our previous microarray, bulk RNA-seq, HCR, immunostaining and transgenic reporter mouse analyses (Table 1).

Next, we evaluated changes in chromatin accessibility between Nr6a1−/− mutant embryos and control littermates (Supplemental Fig. 4). It is well established that NR6A1 directly binds to and represses Oct4. Therefore, we first evaluated the chromatin accessibility of Oct4 in the NCC and ectodermal populations in Nr6a1−/− mutant embryos compared to control littermates (Fig. 5A). NCC, neural ectoderm and neural plate border cells from Nr6a1−/− embryos exhibited increased chromatin accessibility compared to controls, consistent with NR6A1 loss-of-function preventing the repression of Oct4 (Fig. 5A). Furthermore, peaks illustrating the increased accessibility of open chromatin upstream of Oct4, aligned with the locations of putative DR0 binding sites, demonstrating mechanistically how NR6A1 represses Oct4 expression (Fig. 5A).

We next evaluated chromatin accessibility around Sox10, which is significantly decreased in Nr6a1−/− embryos (Fig. 5B). If NR6A1 regulates the transcription of Sox10, then we would expect Sox10 to be less accessible or no longer accessible in Nr6a1 null embryos. Indeed, Nr6a1−/− embryo NCC exhibited decreased chromatin accessibility at Sox10 compared to controls, consistent with NR6A1 being required for access and transcription of Sox10 (Fig. 5B). The DR0 sites overlapped with the Sox10 peaks as well, supporting the idea that NR6A1 binds to and regulates the transcription of Sox10 (Fig. 5B).

snATAC-seq demonstrated that NR6A1 can modulate the accessibility of pluripotency-associated and NCC-specifier genes like Oct4 and Sox10 respectively within NCC (Fig. 5). To validate NR6A1’s ability to directly bind to the DR0 binding sites we performed electrophoretic mobility shift assays (EMSA) and competitive inhibition assays. EMSA revealed the presence of shifted Zeb2 and Sox10 bands following the addition of NR6A1, indicating NR6A1 can directly bind to the DR0 sites in their promoter regions (Fig. 6A). For the competitive inhibition assay, wildtype and mutated DR0 site DNA were introduced in increasing amounts (Fig. 6B). We observed diminished binding in the presence of increasing wildtype DNA compared to mutated DR0 site DNA, supporting the ability of NR6A1 to selectively bind to the DR0 sites of Zeb2 and Sox10 (Fig. 6B).

To confirm NR6A1 binding occurs in vivo and to evaluate all of our NCC and EMT genes of interest, we performed a targeted ChIP assay using mESC that were differentiated into NCC. NR6A1 exhibited statistically significant binding of the DR0 sites in Zeb2, Snai1, and Sox10 (Fig. 6C). We also confirmed that NR6A1 can bind Nanog DR0 sites, which had previously been shown82, further supporting our results (Fig. 6C).

In conclusion, NR6A1 can modulate chromatin accessibility and bind to the DR0 sites found in the promoter regions of pluripotency-associated factors and NCC-specifier and EMT genes. NR6A1 transcriptionally represses pluripotency associated factors, but at the same time, downregulation of NCC-specifier and EMT genes in Nr6a1 null embryos indicates NR6A1 may function as a transcriptional regulator of NCC-specifier and EMT genes. Collectively these results indicate that NR6A1 governs the transition of neural stem cells to NCC by modulating the repression of stem cell maintenance and proliferation in the neural ectoderm, while concomitantly regulating the expression of NCC-specifier and EMT genes within the neural plate border and newly emigrating NCC.

NCC specification begins at mid-late gastrulation
Nr6a1 null embryos are embryonic lethal by E10.5 due to the failure of chorioallantoic fusion73. This early lethality prevents the assessment of a role for Nr6a1 in later NCC differentiation at all axial levels. However, given the important role of Nr6a1 in NCC formation, we posited that Nr6a1 should therefore be essential for craniofacial, cardiac, peripheral nervous system, and gastrointestinal development, as each of these tissues and organs depend heavily on NCC for their proper development. To bypass the early embryonic lethality of Nr6a1 null embryos, we conditionally deleted Nr6a1 from NCC, using Wnt1-Cre. Surprisingly, no phenotypic abnormalities were found in Nr6a1fl/fl;Wnt1-Cre embryos compared to controls (Supplemental Fig. 5). E18.5 Nr6a1fl/fl;Wnt1-Cre embryos appeared morphologically normal, and the NCC-derived craniofacial skeleton (evaluated by alizarin red and alcian blue staining), peripheral nervous system and enteric nervous system (evaluated by TUJ1 immunostaining) were indistinguishable from controls. Furthermore, the NCC-specific Nr6a1 knockouts were produced in Mendelian ratios and survived until adulthood with full reproductive capabilities. This indicated that NCC development was unaffected (Supplemental Fig. 5), which is in stark contrast to all of our other data clearly showing that Nr6a1 is required for NCC specification, formation and survival.

Recently, however, it was proposed that Wnt1-Cre excision occurs too late during neurulation to properly assess gene function during NCC specification and formation in mouse embryos48. Wnt1-Cre facilitates Cre excision of floxed alleles at approximately E8.5. This is important because it suggests that Nr6a1 may not be required for NCC development after E8.5. Furthermore, it also implies that Nr6a1 may instead be needed earlier in development to elicit its role in NCC specification and formation.

To test this idea, and establish when Nr6a1 is specifically required, we globally deleted Nr6a1 in a temporally specific manner using the tamoxifen inducible Cre line, ERT2-Cre112. Consistent with the Wnt1-Cre knockout, no morphological differences were observed when Nr6a1 was temporally excised with ERT2-Cre at E8.5. We then excised Nr6a1 via tamoxifen administration at progressively earlier half-day time points. Global excision at E7.75 or E7.25 resulted in no observable phenotypic differences in NCC gene expression in conditional Nr6a1 mutants compared to controls (Fig. 7). However, we discovered that global excision of Nr6a1 by E6.75 resulted in a phenotype matching the original Nr6a1 null embryos as evidenced by the downregulated expression of NCC-specifier and EMT genes (Fig. 7). This is consistent with our observations that Nr6a1 expression begins between E6.5 and E7.5 (Fig. 1). Thus, not only is Nr6a1 functionally required during mid-gastrulation but our data indicates that NCC specification commences during mid-gastrulation in mouse embryogenesis, which is considerably earlier than previously appreciated.

NCC formation requires stem cell maintenance downregulation
Interestingly, it was recently proposed that NCC retain or re-activate pluripotency-associated genes to facilitate their characteristic capacity to generate a wide variety of cell and tissue derivatives113–118. However, our analyses of Nr6a1−/− mutant embryos indicate that the expression of pluripotency-associated factors including Oct4, Nanog and Sox2, persists in Nr6a1−/− mutant embryos, coinciding with deficient NCC generation. This suggests that retention of pluripotency-associated factors may actually restrict the generation of NCC from neural stem cells. Consistent with this idea, our analyses of wildtype Oct4-EGFP mouse embryos revealed that Oct4-EGFP expression in E7.5 mouse embryos is downregulated by E8.5, coinciding with the formation of NCC (Fig. 4A). In further support of these findings, our multiomic analyses revealed a similar absence of Oct4 and Nanog expression in E8.75 wildtype mouse embryos (Supplemental Fig. 2B).

To better substantiate whether Oct4 and other pluripotency and stem cell maintenance factors are downregulated in concert with NCC formation, we analyzed our scRNA-seq data of cranial tissues from pre-migratory and migratory NCC stage mouse embryos76,106,119. Sox2, Sox1 and Nr6a1 are highly expressed in the neural ectoderm, in contrast to Oct4 and Nanog (Fig. 8A, Supplemental Fig. 6). Furthermore, the expression of each of these genes is downregulated in the progenitor NCC population (Fig. 8A, Supplemental Fig. 6). In contrast, NCC-specification (Foxd3, Sox9, Sox10) and EMT (Snai1, Zeb2) genes are highly enriched in progenitor NCC as compared to the neural ectoderm, distinguishing these two populations (Fig. 8A, Supplemental Fig. 6). Other groups have reported a similar absence of pluripotency-associated factor expression during NCC development aligning with our data120. Therefore, to expand and further substantiate our assessment of the dynamics of Oct4, Nanog and Sox2 expression during development, we also analyzed a publicly available scRNA-seq dataset that covered gastrulation to neurulation stages in mouse embryos121. Complementary to our data, Oct4, Nanog and Sox2 were absent from the progenitor NCC cluster, and a definitive boundary was evident in the pseudotime trajectory separating the primitive neuroectoderm from progenitor NCC (Supplemental Fig. 7). Nr6a1 expression in this dataset aligned with our in situ hybridization data confirming that Nr6a1 is expressed in the epiblast during gastrulation, before becoming enriched in the neural ectoderm during neurulation (Supplemental Fig. 7).

To further evaluate pluripotency and stem cell maintenance factor expression during NCC formation, we measured by RT-qPCR, the dynamics of Oct4, Nanog, Sox2, Sox9 and Sox10 expression during mESC and hiPSC derivation of NCC (Supplemental Fig. 8A and C). Each approach revealed the absence of pluripotency-associated factor expression in the forming NCC populations, and this was strikingly exemplified by their immediate downregulation in hiPSC upon provision of NCC differentiation media (Supplemental Fig. 8A and C). Interestingly, Nr6a1 was also specifically elevated (days 1 and 2) in hiPSC during the switch between pluripotent stem cell maintenance, and NCC specification (Supplemental Fig. 8C). Immunostaining further substantiated the downregulation in expression of pluripotency-associated factors and upregulation of NCC-specifier gene expression (Supplemental Fig. 8B and D).

To conclusively show that downregulation of pluripotency and stem cell maintenance regulators is required for NCC formation, we overexpressed Oct4 in mouse embryos from E6.5 to E8.5 (during NCC specification and formation) using a doxycycline inducible system122 (Fig. 8B). The temporal initiation of Oct4 overexpression was designed to correlate with the temporal requirement for Nr6a1. Embryos overexpressing Oct4 presented with neural tube closure defects and craniofacial abnormalities, mimicking the Nr6a1 null phenotype. Moreover, the expression of NCC-specifier (Foxd3, Sox9, Sox10) and EMT (Zeb2, Snai1) genes in these Oct4 overexpressing embryos was downregulated, similar to that in Nr6a1 null mutant embryos (Fig. 8B). Downregulation of pluripotency and stem cell maintenance factor expression is therefore required for proper NCC formation.

Altogether, our data suggest that NR6A1 is a key regulator of the transition of neural stem cells to NCC by modulating the repression of pluripotency-associated genes while promoting the expression of NCC-specification and EMT genes during mid-late gastrulation.

Methods
Animal husbandry and embryo collection
All mice were housed in the Laboratory Animal Services Facility at the Stowers Institute for Medical Research under a 14-hour light/10-hour dark/light cycle, and experiments were performed according to IACUC animal welfare guidelines and approved protocol (#2022-014). Embryos were obtained by timed mating, with the morning of the vaginal plug defined as embryonic day (E) 0.5 and genotyping of yolk sac tissue was performed as previously described73,138. For all experiments, both male and female embryos were included as there are no sex specific differences in NCC development. At least 5 embryos were used in each control and mutant group across two litters for each experiment, unless otherwise specified. The use of embryos from multiple litters ensures the findings were not litter specific.

For temporal excision of Nr6a1, pregnant dams were orally gavaged with 5mg of Tamoxifen and 1mg of progesterone dissolved in 100µL of corn oil. Treatment was performed one day prior to the desired stage of allele excision as it takes at least 24h for complete recombination to occur.

Oct4/rtTA mice were housed and maintained in the animal facility at the CBM (Madrid, Spain) in accordance with national and European legislation. Procedures were approved by the CBM Animal Welfare Committee and by the Area of Animal Protection of the Regional Government of Madrid (ref. PROEX196/14).

To overexpress Oct4, CD1 females were mated with Oct4/rtTA (Col1a1-tetO-Oct4;R26R-M2rtTA) transgenic males (Jax stock #006911). Transgene expression was induced from E6.5 until the desired stage by replacing the drinking water of pregnant females with a 7.5% sucrose solution containing 0.5mg/mL doxycycline. Embryos from non-treated mice of the same genotype were used as controls.

In situ hybridization
Following dissection, embryos were fixed in 4% paraformaldehyde for 2 hours at room temperature. Embryos were then dehydrated through an ascending series from 0.1% Tween in DEPC treated PBS (PBSTw) to 100% methanol, before being stored at −20°C overnight and until further use. In situ hybridization was then performed with anti-sense digoxigenin-labeled mRNA riboprobes as previously described139. Briefly, embryos were gradually rehydrated from 100% methanol into PBSTw then bleached for 5 min in 6% hydrogen peroxide in PBSTw at room temperature. Two washes in PBSTw were then performed at room temperature followed by a 5 min digestion in 10µg/mL proteinase K in PBSTw without rocking at room temperature. After three washes, each for 5 min in PBSTw without rocking at room temperature, the embryos were post-fixed in 4% PFA and 0.2% glutaraldehyde in PBSTw for 20 min at room temperature with rocking. Two additional washes in PBSTw were performed before gradually equilibrating the embryos in hybridization solution. The embryos were then left rocking at 68°C in hybridization solution with 2µg/µL of probe overnight. The next day, the embryos were repeatedly washed in PBSTw before being placed in a blocking solution of 10% lamb serum and 0.1% Roche blocking reagent in TBST for 90 min at room temperature. The embryos were then equilibrated in blocking solution with Roche Anti-Digoxigenin AP Fab fragments (1:2000) overnight at 4°C. One day of TBST washes was performed followed by color development the next day. The embryos were washed in NTMT 3-times before developing in NTMT with 125µg/mL BCIP and 250µg/mL NBT at room temperature, protected from light, overnight. The embryos were then washed in PBSTw and re-fixed in 4% PFA to stop the reaction. Anti-sense and sense probes were run at the same time to ensure the signal was specific and not background. Embryos of the same stage were placed in the same tube to confirm lack of staining in E6.5 was real. Experiments were performed twice and a minimum of 5 embryos of each stage were evaluated each time.

Hybridized chain reaction (HCR)
HCR probes were commercially available and purchased from Molecular Instruments (V3.0) and performed according to the manufacturer’s protocol140 with the following modifications. E8.75 embryos were bleached in 6% hydrogen peroxide in PBSTw for 5 min at room temperature with rocking. The embryos were digested in 10µg/mL proteinase K in PBSTw for 5 min at room temperature without rocking. 8µL of probe was then added to 500µL of Hybridization Buffer before adding 10µL of each Hairpin. The embryos were mounted in 0.8% low melting point agarose for imaging and cleared with CE3D141.

Immunohistochemistry
For section immunohistochemistry, Nr6a1 litters were collected at E9.0 and fixed overnight in 1% PFA at 4°C. Embryos were processed through a sucrose gradient, mounted in Tissue Tek OCT (VWR, West Chester, PA) and sectioned at 10 microns. Sections were rinsed 3 times with PBS 0.1% Trition X-100 (PBT) for 5 min and blocked in 10% goat serum (Invitrogen, Carlsbad, CA) in PBT for 1 hour at room temperature. Slides were incubated in primary antibody diluted in blocking solution at 4°C overnight. The monoclonal antibodies SOX2 (R&D Systems, Minneapolis, MN) and pHH3 (Upstate/Millipore, Billerica, MA) were both used at 1:500. The antibody to PAX3 was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA 52242). TUNEL staining (Roche Life Sciences) was performed according to the manufacturer’s instructions. Slides were rinsed 3 times for 10 min in PBT at room temperature on a shaker and incubated in the appropriate Alexa secondary antibody (Molecular probes/Invitrogen, Carlsbad, CA) at 1:250 for 2 h at 4°C. Sections were counterstained with a 1:1000 dilution of 2mg/mL DAPI (Sigma, St. Louis, MO) in PBS for 5 min, followed by rinses in PBS before mounting slides with VectaShield (Vector Laboratories, Newark, CA). All images were collected using a Zeiss Axioplan microscope and processed using Illustrator (Adobe, San Jose, CA).

Immunostaining of whole embryos and cultured cells was performed as previously described142,143. For whole mount immunostaining a SOX10 antibody (1:500; Abcam, ab155279) was used together with GFP (1:1000; Abcam, ab13970). The WTC-11 human induced pluripotent stem cell line was obtained from the Coriell Institute (#GM25256). hiPSCs were plated in an Ibidi 8-well plate (Ibidi #80826) and maintained as pluripotent stem cells or differentiated into NCC. Cells were fixed in 4% PFA (Alfa Aesar via VWR Cat. No. AA43368-9M) for 15 min at room temperature and then permeabilized in PBS + 0.3% Tx-100 (Millipore Sigma, #TX1568, PBST) for 20 min at room temperature. Cells were blocked using PBST + 0.5% BSA (Millipore Sigma, #2910) for 1–2 h at room temperature and incubated in primary antibody in blocking buffer overnight at 4°C. The following primary antibodies were used on cells: Oct4 (R&D Systems, #MAB1759 1:50), Nanog (R&D Systems, #1997 1:40), Sox10 (R&D Systems, #2864 1:100), and Tfap2a (Invitrogen, #3B5 1:50). The cells were washed 3 times for 5 min with PBST and 0.5% BSA (Millipore Sigma, #2910) before incubating in secondary antibody and blocking solution. The following secondary antibodies were used at a 1:500 dilution: Donkey anti-Rabbit IgG (H+L) Alexa Fluor™ 546 (Thermo Fisher #A10040), Donkey anti-Goat IgG (H+L) Alexa Fluor™ 488 (Thermo Fisher, #A11055), and Donkey anti-Mouse IgG (H+L) Alexa Fluor™ 546 (Thermo Fisher, #10036) at room temperature for 1-2 hours. The cells were then washed 3 times for 5 min with PBST and 0.5% BSA, mounted in Vectashield in DAPI (Vector Laboratories, #H-1200-10) and imaged. All images were individually acquired with the same settings, and brightness and contrast were adjusted the same throughout.

Microarray
E9.0 wildtype embryos and Nr6a1−/− embryos were isolated and total RNA was extracted from whole embryos using TRIzol reagent. Concentration and quality of RNA were determined by spectrophotometer and Agilent bioanalyzer analysis (Agilent Technologies, Inc., Palo Alto, CA). For array analysis, labeled mRNA targets were prepared with 150ng total RNA using MessageAmp II IRNA Amplification Kit (Applied Biosystems / Ambion, Austin, TX) according to the manufacturer’s specifications. Array analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays processed with the GeneChip Fluidics Station 450 and scanned with a GeneChip Scanner 3000 7G using standard protocols. Resulting CEL files were analyzed using RMA and limma in the R statistical environment84. The microarray data are publicly available in the Gene Expression Omnibus using accession number GSE166458.

Bulk RNA-sequencing
E8.75 Nr6a1−/− and wildtype control littermates of between 9-11 somites were isolated and bisected into anterior (rostral to and including the first branchial arch) and posterior (caudal to and including the second branchial arch) samples. Total RNA was extracted from whole embryos using an RNA micro Kit. Concentration and quality of RNA were determined by bioanalyzer analysis. RNA-seq was run using single-end 50bp sequencing reads with Illumina HiSeq 4000. FASTQ files were aligned with STAR, and EdgeR was used for differential expression analysis between mutant and wildtype84. The RNA-seq data are publicly available in the Gene Expression Omnibus using accession number GSE180427.

Single-cell RNA sequencing
Single-cell RNA sequencing of E8.5 Wnt1-Cre;R26R-eYFP and Mef2c-F10N-LacZ embryos, and subsequent bioinformatic analyses were performed as previously described76,106,119 and the dataset is publicly available at the Gene Expression Omnibus using accession number GSE168351. Briefly, 10X Genomics Chromium Next GEM Single Cell 3’ Library + Gel Bead Kit (v3.1) was used on dissociated cells to generate libraries. Sequencing was performed with an Illumina NovaSeq 6000 and 10X Genomic cell ranger was implemented to process the data. Normalization and downstream analysis all utilized Seurat (v3) in R. Clustering was performed using the shared nearest neighbor method first at a resolution of 0.05, then, within the NCC cluster at a resolution of 0.26. Finally, the pre-migratory NCC were then clustered at a resolution of 2.0 to produce the final 15 distinct NCC clusters. For single cell RNA sequencing gastrulation to neurulation time courses, the data was obtained and images generated from the interactive server: https://marionilab.cruk.cam.ac.uk/MouseGastrulation2018/ described in ref. 121.

Multiomic single-cell RNA-sequencing and single-nucleotide ATAC-sequencing
Nr6a1−/− and wildtype control littermates were collected at E8.75. 3-4 embryos were used per genotype across two litters. Nuclei were isolated with the 10X Genomics Nuclei Isolation kit. Following isolation, the nuclei quality was assessed via bright field imaging and standard QC analysis. A total of 3200-3500 nuclei were captured per sample. Libraries were prepared and pooled such that the pooled gene expression and ATAC libraries could then be run on individual flow cells, resulting in 20,000 reads per nuclei for scRNA-seq and 25,000 reads per nuclei for snATAC-seq. Paired end sequencing was performed on an Illumina NextSeq 2000. Raw data was processed with 10X Cell Ranger and analysis was performed in R (v4.4.1) with Seurat (v5)144. Data was filtered using standard QC methods: ATAC reads greater than 50,000 and less than 500 were filtered out along with RNA reads greater than 25,000 and less than 1000. Cells with percent RNA mitochondria reads greater than 10% were filtered out. Finally, cells in which transcription start site enrichment was less than 1 or nucleosome signal greater than 2 were also filtered out. The final dataset analyzed following filtering and SCT normalization145 included 2987 mutant cells and 2728 wildtype cells. Wildtype and mutant cells were integrated together with Seurat’s CCA Integration146. Clustering was performed on the integrated dataset at a resolution of 0.5 using the find nearest neighbor method. Seurat FindMarkers was used to compare gene expression across clusters to aid in cell type annotation and to find differences between genotypes. Annotations from the scRNA-seq clustering were used to annotate the snATAC-seq. The dataset is publicly available at the Gene Expression Omnibus using accession number GSE287235.

Electrophoretic mobility shift assay (EMSA) and competition binding assay
We produced NR6A1 protein in vitro using the TNT translation system (Promega). Oligo DNA 5’–hydroxyl end labeled with (γ-32P) ATP using T4 PNK system was used as a probe. EMSA experiments were performed at 30°C for 30 min in 10 µL of EMSA buffer (10 mM HEPES, pH 7.5; 50 mM KCl; 1 mM EDTA, 5 mM MgCl2; 2 mM DTT; 10% Glycerol; Poly [dI-dC] 1 µg). For competition assays, unlabeled wildtype or mutated competitor DNA was added to the reaction at 5x, 10x or 50x concentrations relative to labelled probe. The total reactions were directly loaded onto a 5% acrylamide gel. Electrophoresis was carried out at 4°C for 100 min at 300 V. The WT sequence for the competition assay was AGGTCA and the Mutant sequence was CTTGAC.

Mouse stem cell to primitive neural crest cell differentiation
KH2 cells were maintained on MEFs in KOSR media. Prior to initiating differentiation, the cells were adapted to feeder free conditions for 1 passage. For differentiation, 12,500 cells/cm2 were plated on Matrigel (Sigma-Adlrich) coated plates. Base differentiation media consisted of DMEM/F12 with Glutamax: Neurobasal 1:1 media containing 1X Embryomax, 2-mercaptoethanol, 0.5X N2, 0.5X, B27, and 1X NEAA. bFGF (10 ng/mL) (StemCell Technologies) and Heparin solution (0.0002%) (StemCell Technologies) were added to the media for the entirety of differentiation. After the first 4 days of differentiation, Bmp2 (10 ng/mL) was added to the culture until the cells were harvested.

Human induced pluripotent stem cell to neural crest cell differentiation
hiPSCs were obtained from the Allen Institute (GM25256). All experiments were performed according to the regulations of approved IBC registration HPCL-014) and protocol (IBC2003-24). Undifferentiated hiPSCs were maintained and passaged on plates coated with hESC-qualified Matrigel (Corning #354277) in mTeSR1 (StemCell Technologies #85850) media supplemented with 1% penicillin/streptomycin (Thermo Fisher, #15070063). Cells were passaged approximately every 3–5 days (70–85% confluency) using Accutase (Gibco, #11105–01) to detach cells. Cells were then plated in mTeSR1 + 1% P/S and 10 μM Rock Inhibitor (Y-27632, StemCell Technologies, #72308). For NCC differentiation, hiPSCs were detached with Accutase (Gibco, #11105–01) and resuspended in STEMdiff™ Neural Crest Differentiation media (StemCell Technologies, #08610). Cells were plated at 7000 cells/cm2 in STEMdiff™ Neural Crest Differentiation media supplemented with 1% penicillin/streptomycin (Thermo Fisher, #15070063) and 10 μM Rock Inhibitor (Y-27632, StemCell Technologies, #72308) for the first 24 h. Media was changed every day. On day 8 of differentiation, cells were harvested for RNA extraction or fixed for immunostaining.

Chromatin immunoprecipitation
ChIP experiments were performed as previously described147, using mouse KH2 stem cells that were differentiated into to NCC over 5 days. These experiments were repeated 3 times for rigor, reproducibility and statistical analysis. Antibodies used were: NR6A1 (Proteintech Ca. No. 12712-1-AP) and IgG control (Proteintech Ca. No. 30000-0-AP). Snai1 promoter sequence: CAGCGCCCAAAGGTCAGCAGCTCGGGGATG; Snai2 promoter sequence: AAGCCAAGTCGCCGTAGGTCACCTAGCGGAA; Sox9 Exon 3 sequence: GTTCCGGCCACCCACGGCCAGGTCACCTACAC; Sox9 3’ UTR sequence: GCTGTTCCCCGTGGAGGTCAGAGGGGAGAGGTA; Sox10 Exon 2 sequence: GTGAACTGGGCAAGGTCAAGAAGGAACAGCA; Zeb2 Intron 2 sequence: CTTGGCTCCAGCAGTGAGTCAAGCCACAGCC; Zeb2 Intron 2 sequence 2: AGAGGTCATGTGAACCTCAGAGTCAGGCCCTCG.

Quantitative PCR
RNA was extracted from WTC-11 hiPSC or differentiated NCC using the Qiagen miRNeasy Micro Kit (Qiagen, #217084) with on-column DNase treatment (Qiagen, #79254). The Superscript III Kit (Invitrogen, 18080051) was used to synthesize cDNA for quantitative RT-PCR (qPCR) using random hexamer primers. qPCR was performed on ABI7000 (Thermo QuantStudio 7) using Perfecta Sybr Green (Quantbio #95072-250). Primers are listed in supplemental Table 1. No template controls were run as negative controls. The ΔΔCt method was used to calculate fold change. Student’s t-test and ANOVA were used for statistical analysis of n=18 samples at each timepoint for each gene, and significance was determined based on p<0.05.

Imaging and image analysis
Brightfield images of in situ hybridization embryos were captured as previously described106. In brief, images were taken on a Leica MZ16 microscope using a Nikon DS-Ri1 camera. Manual Z-stacks were acquired and then images were merged using Helicon Focus. Fluorescent images were taken on upright Zeiss LSM-700, inverted Zeiss LSM-800 equipped with a GaAsP detector and a Nikon CSU-W1 inverted spinning disk equipped with a sCMOS camera. Image analysis was performed using Fiji.

HCR imaging and quantification
All embryos were imaged using the same settings on a Nikon CSU-W1 inverted spinning disk with a 20x long working distance objective. Z-stacks of each embryo were max-projected in Fiji and the same contrast and brightness were used to generate the fluorescent patterns in Fig. 2A. To ensure equivalent tissue was analyzed during quantification, the same number of z-slices (2.5µm per slice) were used to make a sum projection. A region of interest was drawn around the head of the embryo ending at the caudal edge of the first pharyngeal arch. The measure feature was used in Fiji to acquire the area, integrated density and mean of the signal for each channel/probe. An additional box region of interest was drawn next to the tissue in the empty space of the image to measure the mean intensity of the background for each channel/probe. The total corrected fluorescent signal was then calculated by taking the integrated density measurement and subtracting the area of the head that was measured, multiplied by the background. There can be differences in the number of cells in the head that are captured in the z-stack as well as distance from the objective that can affect signal. To account for these differences, the total corrected fluorescent signal of each probe was then divided by the total corrected fluorescent DAPI signal such that the final value graphed was the relative expression of each probe/gene in the cranial region of each embryo. Variation in development can occur across and within litters during early craniofacial development. Therefore, we chose to stage match our embryos to the closest developmental stage control littermate, and by evaluating the total corrected fluorescence for each probe over the total corrected DAPI fluorescence, and matching to the developmentally closest control littermate, we ensured the most precise quantitative comparisons. Finally, as the values are a ratio of total corrected fluorescence for each gene normalized to DAPI, a ratio paired t-test was performed to assess the statistical significance of the differences in relative fluorescent expression of each gene between control and Nr6a1 null embryos. Graphing and statistical analyses were performed in GraphPad Prism 10.

Quantification of cell death
Imaris was used to count the number of NCC and non-NCC labeled with TUNEL in a z-stack of each embryo. The spot count tool was used on a region of interest confined to the first pharyngeal arch and more rostral region of the head in the DAPI channel to count the total number of cells in the head. A surface was then generated of the same region using DAPI, followed by a mask of the surface using the TUNEL channel. The cells that were within the TUNEL masked region were then counted with the spot tool to identify all the cells in the head that were undergoing cell death. Next, a surface was generated using the GFP channel to capture all of the NCC. The GFP signal in the Wnt1-Cre;R26R-mTmG line is localized to the cell membrane, therefore, to count the cells that were GFP positive and thus NCC, we generated a mask of the GFP surface with DAPI. The DAPI-stained nuclei located in the GFP-positive region were then counted to determine the total number of NCC in the head. To determine the total number of NCC undergoing cell death, another mask was generated with the GFP surface using the TUNEL channel. Cells were once again counted using the spot tool. Please note that a cell is ~10µm in diameter, therefore we chose a spot size of 8µm for all spot counting steps assuming that the nuclei are slightly smaller than the overall size of a cell. To determine the number of non-NCC present in the measured cranial regions, we subtracted the total number of NCC from the total number of cells in the head. The total number of NCC with TUNEL staining was subtracted from the total number of cells in the head with TUNEL staining to find the total number of non-NCC undergoing cell death. We then calculated the percentage of TUNEL+ cells in each population and graphed the values with GraphPad Prism 10. We chose to evaluate the overall percentage of cell populations undergoing cell death as opposed to total cell numbers to account for any population size differences that might be found due to variation in developmental stages in Wnt1-Cre;R26R-mTmG embryo littermates. An un-paired parametric t-test was performed using GraphPad Prism to determine statistical significance.

Oct4-EGFP expression quantification
The average maximum intensity of GFP in the SOX10-positive NCC and neural ectoderm was measured using Fiji. A maximum projection was generated from the z-stack acquired of each embryo. A box was then drawn around 3 different NCC and 3 different neural ectoderm cells keeping the same size area each time. The measure feature was then used to obtain the mean intensity within each boxed cell. The three measurements for each cell population were then averaged to determine the average mean intensity for each cell population of each embryo. A total of 3 mutants and 4 controls were analyzed. Values were then graphed using GraphPad Prism 10. A one-way ANOVA was performed across the entire dataset and un-paired parametric t-tests were then performed to compare individual cell populations.

Statistical analysis and reproducibility
All experiments were performed at least three times to confirm results were reproducible, and sample sizes for each experiment are noted. Results were individually plotted with error bars representing standard error of the mean (SEM). Statistical analysis was performed by unpaired two-tailed Student’s t-test unless otherwise stated, and p-values <0.05 were considered significant.

Skeletal staining of Wnt1-Cre conditional knockout
E18.5 embryos were anesthetized by immersion in ice cold PBS for at least 60 min until no reflex movements were observed following a pinch test. The embryos were then fixed in 95% ethanol for 1 hour and dehydrated in fresh ethanol overnight at 4°C. The skin and viscera were manually removed, and the embryos were incubated in 70% ethanol/5% acetic acid for 30 min at room temperature, before staining overnight in alizarin red/alcian blue at room temperature as previously described148,149. The embryos were then washed in 70% ethanol/5% acetic acid for 30 min and quickly rinsed in water. Soft tissue digestion was performed in 2% KOH overnight for at least 6 h, followed by further digestion and clearing, initially in 0.25% KOH, and then through an increasing gradient of glycerol in 0.25% KOH before being stored in 50% glycerol.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Zajic and colleagues explore the function of orphan receptor NR6A1 in mouse neural crest specification and development. NR6A1 was originally identified as altered in the TCOF1 mutants, a model for the Treacher Collins syndrome. The authors find that NR6A1 is expressed in ectoderm during gastrulation and neural crest progenitors during neurulation. NR6A1 mutation strongly depletes the NCC cell population as well as the early specification marker expression, while cell death is increased. Further, coarse dorsal-ventral neural patterning seems normal despite an open neural tube, and pluripotency genes Oct4 and Nanog are increased in the mutant epiblast and neuroepithelium. Using a large panel of state-of art techniques, the authors carefully explore the mechanisms of NR6A1 action. They propose that NR6A1 is a switch repressing pluripotency genes while activating the early neural crest ones.\nThis is a remarkable piece of work. The experiments are well designed, and the data are well described. The manuscript is rigorous and well written. However, below are a few major points meant to challenge and improve the hypotheses of the work and enrich the discussion, in the light of some recent other analyses that were not considered yet are fully relevant to interpret the data.\nMajor point 1: There has been a long-standing debate about the apparent discrepancies between different vertebrate animal models used for neural crest studies, many regulations demonstrated in fish, frog and chick being apparently absent in the mouse. A key finding in this work is the demonstration that the \"gold standard\" tool used for several decades in mouse neural crest analyses, wnt1-cre line, is inadequate to study early neural crest specification, and that mouse neural crest specification, as found in other vertebrate models, starts during gastrulation. This alignment of the models in the vertebrate lineage is crucial for the neural crest community to unite behind a common understanding of the NC-gene regulatory network and should be put forward very clearly both in abstract and discussion.\nMajor point 2: In situ hybridizations in Figure 2A are not very clear; especially, several gene's expressions seem lower but not abolished (Foxd3, Snail1) while Twist1 staining looks more like background staining. Stronger staining would be more convincing when the authors state that \"Surprisingly, even though the expression of many NCC-specifier and EMT genes are lost in Nr6a1-/- embryos, the presence of LacZ-positive cells within the frontonasal region and first pharyngeal arch suggests some cells...\" page 7. It rather seems that the patterns observed match the reduced population of cells seen in the GFP-lineage tracing (Figure 2B-C) as well as the Microarray results: \"Although the downregulation of these factors in the microarray did not reach statistical significance, ...\". This observation correlates with the possibility that the genes are expressed albeit at a lower level.\nMajor point 3: Although major domains (Pax3 / Sox2) form in the open neural tube of mutants, there is a possibility that the entire neural plate is now entirely biased towards neural fate or alternatively that dorsal cells are frozen in a neural border state. Sox2 maintenance in the neural folds (by DNMT3A, Hu et al., 2012) was previously associated with prolonged expression of neural border specifiers (Zic1): Are Zic1 or proneural gene expression expanded dorsally in NR6A1 mutants?\nMajor point 4: Increased expression of Nanog and Oct4 in NR6A1 mutants:\nFigure 4: Oct4/rtTA embryos treated with doxycycline (+Dox) as compared to untreated embryos (-Dox). Consistent with the Nr6a1 null embryos, NCC and EMT marker expression is no longer present through the cranial mesenchyme of the embryos, indicating a disruption in NCC formation.\nM&M: \"To overexpress Oct4, CD1 females were mated with Oct4/rtTA (Col1a1-tetO-Oct4;R26R-M2rtTA) transgenic males (Jax stock #006911). Transgene expression was induced from E6.5.\nIn line with the finding that NR6A1 acts from gastrulation stages, the authors here activate exogenous Oct4 expression thoughout gastrulation and recapitulate the NR6A1 mutant NCC phenotype. This is a critical experiment that should focus attention as it was shown that the activation of pluripotency genes (Nanog and its target Oct4) needs to be dynamically regulated during gastrulation: continuous activation from early gastrulation results in neural border and neural crest defects, while Nanog activation at the end of gastrulation is required for proper NC development (Scerbo and Monsoro-Burq, Science Advances 2020). At that stage, pluripotency factors are reprogrammed towards NC target genes by cooperation with the neural border specifiers (Hovland et al., Developmental Cell 2022). This litterature is essential to quote for this point and in discussion.\nIn Col1a1-tetO-Oct4;R26R-M2rtTA mouse model, is there a difference in the neural border and NC phenotypes when activating Oct4 at early (E6.5, as in Figure 4) versus later time point in gastrulation (via tamoxifen administration at progressively later half-day time points)?\nAccording to results of this temporal analysis, Oct4 expression E7.5 and E8.5 may not be relevant for neural crest earliest initiation step but rather for later specification stage (page 14).\nIndeed, in single cell data Nanog/Oct4 expression seem present in neural border/neuroectodermal cells but not specified NCC (page 15, lettering in Figure S6A is very small and fuzzy), as found in other scNC datasets (Kotov et al., PNAS 2024 is more adequate than Briggs et al., 2018 for NC studies). There is a sharp difference in Oct4 levels between neuroectodermal clusters: does this support the possibility of a dose-dependent role of Oct4? Would NR6A1 be \"modulating\" Oct4 \"levels\" during gastrulation rather than being an \"on/off\" switch at the edge of the neural plate when NC progenitors are initiated but not yet specified?\nMajor point 5: Introduction, pages 3,\nThe authors make a commendable job to quote original classic research articles from the 1990s in many instances. However, they should also include some more recent references which provide important mechanistical insights. Below are some key references to include.\n- The first formal demonstration of the interplay between secreted factors BMP, WNT and FGF with neural border specifiers Zic1, Msx1 and Pax3, providing the seed for the neural crest gene regulatory network known today, was done in the xenopus model (Monsoro-Burq et al., Developmental Cell 2005).\n- The temporal and spatial tissue-layer dynamics of BMP and WNT signals controlling the sequential steps neural border and neural crest induction was studied in Alkobtawi et al., Cell Reports 2021.\n- TFAP2a is a major neural border specifier in fish, frog and chick, activated the earliest at the edge of the neural plate and regulating the other ones as shown in De Croze et al., PNAS 2011 and Rothstein and Simoes-Costa, 2020 Genome Research.\nMajor point 6: In discussion : The difference between cranial and caudal NCC should be highlighted, as the phenotype of mutant NR6A1 NCC differs along the body axis: this is also the case for the Pax3/Pax7 mutants : Van Ho et al., Developmental Cell 2011.", "reviewer_2": "The paper by Zajic et al addresses the role of the nuclear receptor gene Nr6a1 in neural crest specification and epithelial to mesenchymal transition. A posited mechanism of this phenotype is the persistent expression of pluripotency associated factors. While the topic of neural crest specification and potency has garnered much interest, the data in this manuscript do not convincingly determine that these processes are novel or specific to the neural crest. Several findings have previously been reported in other cell types and are rather generalisable to progenitor or early tissue (neural ectoderm, germ cell) in regulating proliferation vs. differentiation. The data and previous work on GCNF/Nr6a1 suggest that this factor plays a role in deciding if cells stay in a proliferative state or begin to make fate specific decisions. Furthermore, the data presented appears to affect early neural ectoderm in totality, not presumptive neural crest. Throughout the course of the paper, there are several distinct mechanistic conclusions (hyperproliferation v. cell death v. neural crest specification) drawn, but no definitive support shown in for any. The figures are not constructed in a manner that highlights or emphasize key concepts, making it difficult to support conclusions drawn. Finally, the quality of some data also contributes to questioning some conclusions draw by the authors.\nIntroduction- There is a lack of introduction of the nuclear receptor family and their well-established role in progenitor potency and cell fate decision. (e.g., Nr5a2 in skeletal progenitors).\n-introduction states that some discrepancies across species regarding neural crest specification. how does nr6a1 function in other species?\nFigure1- In general, the in situ gene expression experiments are not of high quality. Specifically in Fig.1 the staging of the in situ is not in alignment with future conclusions (i.e., Nr6a1 is active at E6.75 from Fig. 7) and does not definitively support the conclusion that Nr6a1 is expressed in early neural crest. For that to be proven, authors would need to do double in situ with known markers of crest at various stages. Furthermore, for stages that do not appear to express Nr6a1 there are no positive controls. Scale bars and stage matched embryos are not included in Fig.1 as well as many other figures.\nFigure 2-Again, stage matching and quality of in situ is a concern and detracts from the overall conclusion of the figure. YFP experiments are not done at equivalent stages and there are still neural crest in Nr6a1 mutants.\nSupp. Fig. 1- there are a significant number of TUNEL positive cells that are not labeled as neural crest, yet the conclusion is still that loss of Nr6a1 is required for neural crest survival. While this data is not directly in contradiction to that statement, it also does not fully support that conclusion. More likely it seems that Nr6a1 is a factor expressed in progenitor type cells (whether neural ectoderm or germ cells) that restricts potency and proliferation, and promotes differentiation.\n-Furthermore, more the text refers to YFP labels, but the figure refers to GFP.\n-A more comprehensive panel of various stages of neural crest development would be needed to definitively support this conclusion.\nFigure 3- States that Nr6a1 is required for the transition of neural stem cells to neural crest. If this was the case and this was specific to the crest, you would expect to see the lateral edges where the crest arise to be thickened while the rest of the neuroectoderm is the same thickness as in controls. Instead, the entire neuroectoderm is thickened likely due to hyperproliferation at some point. Thus, this is not specific to neural crest, but a generalizable mechanism for cells to decide to leave a proliferative state and begin fate decisions.\nFigure 4- Again, stage matching is of concern, as the conclusion is there is more Oct4 expression in the Nr6a1-/-, but the Nr6a1-/- embryo looks to be younger, thus that could be an explanation for increased Oct4 expression. There is no discussion of what Figures 4C and D are in the results section for figure 4 making it difficult to understand what the conclusions are. Despite repeatedly referring to Nanog, there are no in vivo experiments testing the expression of Nanog in Nr6a1 mutants. Expression patterns of Foxd3 between Figs 1 and 4 is vastly different, supporting the critique that the quality of the in situs is low.\nSupplemental fig 2- multiome data isn’t fully analyzed and is presented in a manner difficult to interpret. There is no quantification of neural crest on integrated plot and no individual UMAPs to show if/how the neural crest are transcriptionally defined in the Nr6a1 mutants. This presentation/analysis is superficial and does the data a disservice.\nFigure 5- Further represents data that is not specific to the neural crest and is not novel mechanistically, as this has been shown in germ cells.\nFigure 7- Using the tamoxifen approach specifies the temporal window of Nr6a1 (note: this window is not consistent with what is shown in figure 1), however it again does not specifically target neural crest and there is still significant Foxd3 expression.\nDiscussion: Several statements in the discussion are not supported by the data. For example-\n- “NR6A1 is essential for NCC formation”. Data= Foxd3 expression is shown in Nr6a1 knockouts. Thus neural crest formation is occurring at some level.\n-“ novel regulator of the transition of neural stem cells to NCC.” Data= nr6a1 has been shown to play a role in progenitor cell populations previously and there is no reason given to think it would act differently in neural crest progenitors.\n-“ Our work provides some of the first evidence in accord with this idea, since global temporal deletion revealed that Nr6a1 is precisely required by E6.75 to replicate the original Nr6a1 null mutant phenotype.’ Data= null embryo in Figure 2 has low/none Foxd3, but in Figure 7 Foxd3 is still clearly expressed. Thus, the phenotype is not replicated.\n-“ However, the expression of AP2 and Sox10 was not affected and changes in proliferation were not evaluated despite the global loss of Oct4.” Data= This statement was referring to Zalc et al. Zalc reported that AP2 and Sox10 expression was affected in the frontonasal mass, but NOT in the cranial ganglia, which was the data used to support the conclusion that Oct was necessary for ectomesenchymal specification.\n“Our analyses of Nr6a1 null embryos challenge the re-acquisition model and role for Oct4 in NCC specification and formation. We evaluated the expression of Oct4 during normal development using the Oct4-EGFP transgenic mouse line, which revealed that although Oct4 is strongly expressed in the epiblast at E7.5, it is downregulated by E8.5 coinciding with the specification and formation of NCC.” Data=Authors state neural crest are specified much earlier than classical models suggest (i.e., 6.75), yet show Oct4 expression in areas akin to where neural crest would be at e7.5.\n-“ Our scRNA-seq results matched those obtained in Xenopus where even when clustering with a bias towards re-activation of pluripotency, presumptive NCC do not express these factors”. Data= Oct4 is expressed in neural ectoderm to the same extent as Nr6a1 is expressed in neural ectoderm. Thus, to say that Oct4 is not expressed in presumptive neural crest but Nr6a1 is expressed in neural crest seems contradictory.", "reviewer_3": "Zajic et al. have shown that NR6A1 plays an important early role in mouse neural crest cell (NCC) development. The authors show that through downregulating pluripotency genes and upregulating neural crest specification genes this orphan nuclear receptor molecule functions to promote NCC fates. Key data include morphological, expression, and transcriptomic/multiomic analysis of null NR6A mutants; EMSA and targeted ChIP to evaluate interactions of the protein with neural crest specification genes; and analysis of stage-specific conditional mutants. An important finding is that the normal downregulation of the Oct4 pluripotency factor by E8.5 does not occur in NR6A1 mutants, which instead continue to show Oct4 expression in their neural ectoderm. The work is of significance because it uncovers a requirement to down regulate stem cell factors for NCC specification and differentiation to occur. This contradicts an alternative model in the field that states pluripotency is maintained long term in NC progenitors, and has biomedical significance given that the NR6A1 was identified as functioning downstream of Tcof1 (the causative gene for Treacher Collins syndrome). While the function of NR6A1 has been tested, it was not previously considered in the context of the neural crest. A role for this orphan nuclear receptor in neural crest development is thus a novel finding.\nA primary strength of the study lies in the combination of direct in vivo genetics, transcriptomic approaches, and biochemistry. The methodology and its description are sound. However, there are several problems with this study in its current form. The biggest hole is that while the timing of NR6A1 action is shown convincingly to be during gastrulation, all the phenotypic analysis provided is carried out at much later stages. As a consequence, we don’t know how direct these outcomes are. A related issue is that we lack information on the spatial requirement for this molecule. The authors appropriately attempted to use the Wnt-Cre system to ablate NR6A1 function specifically in NCCs, but this approach instead revealed that by the stage Wnt-Cre acts (E8.5) the molecule is no longer needed. They then switched to using a tamoxifen inducible Cre-system to show that NR6A1 functions much earlier, during gastrulation, at E6.75. They do not show us where the gene is expressed at its stage of action, and it is quite possible that it functions non cell autonomously rather than directly. This point should at minimum be acknowledged and discussed.\nAnother significant concern is that quantification is completely lacking for several of the in vivo analyses. This needs to be corrected. For example, in Fig. 2A, the NR6A1 mutants show some morphological – or perhaps staging – variation. This should be commented on, especially as there appears to be Foxd3 expression present in the anterior of the mutant shown. It would be helpful to understand the extent of variation between specimens. Related to this, the authors need to take care to discriminate between situations when expression patterns are missing, versus the cells or tissues themselves are absent. For example, Zeb2 is stated to be absent from NCC-derived facial and pharyngeal mesenchyme, but these tissues appear to be missing (at least in part).\nFinally, the analysis provided focuses exclusively on cranial neural crest, although this point is never made explicit. Is Nr6a1 also required for trunk neural crest fates? It seems that trunk somitic mesoderm may be missing or aberrant in Fig. 2B. Proper analysis of trunk phenotypes is needed."}, "author_response": "We sincerely thank the reviewers for their thorough and insightful critiques of our paper, and their recognition of the importance of our work. The reviewers provided a lot of comments and suggestions, which were extremely helpful. We have therefore revised our manuscript accordingly as described below, to address each and all of the reviewers’ concerns. Changes to the text have been tracked or highlighted throughout the manuscript as requested in the author guidelines. We think that these revisions have considerably strengthened the paper, and we trust the reviewers will now find the work satisfactorily addresses their concerns and is suitable for publication.\nReviewer #1\nMajor point 1: There has been a long-standing debate about the apparent discrepancies between different vertebrate animal models used for neural crest studies, many regulations demonstrated in fish, frog and chick being apparently absent in the mouse. A key finding in this work is the demonstration that the \"gold standard\" tool used for several decades in mouse neural crest analyses, wnt1-cre line, is inadequate to study early neural crest specification, and that mouse neural crest specification, as found in other vertebrate models, starts during gastrulation. This alignment of the models in the vertebrate lineage is crucial for the neural crest community to unite behind a common understanding of the NC-gene regulatory network and should be put forward very clearly both in abstract and discussion.\nWe appreciate the reviewer’s enthusiasm about these findings as we also think it they are foundational for improving our evolutionary comprehension of NCC development across species. Accordingly, and as the review suggested, we have added the text below to the abstract and discussion to better emphasize these points:\n“Conditional deletion revealed that Nr6a1 is required during mid-late gastrulation, demonstrating that the initiation of murine NCC specification occurs during gastrulation, which is earlier than previously thought, but in close alignment with the established timeline of NCC development in other vertebrate model organisms, such as chick. Moreover, these findings also reveal that the gold standard transgenic mouse line, Wnt1-Cre, is inappropriate for studying genetic function during early NCC specification and formation”.\n“Many of the genes identified in chicken, frog and fish NCC development have been suggested to be dispensable in mouse 46,47,48. However, our findings that NCC specification in mouse may occur earlier than previously through, and at a stage parallel to these other vertebrates, indicate that perhaps some of these genes need to be re-evaluated at earlier developmental stages for roles in early NCC development. Revisiting the role of these specification factors during early mouse development could demonstrate that the gene regulatory networks are more unified or conserved across vertebrates than currently thought. Evaluating the role of NR6A1 in NCC development in other model organisms would also provide further evidence for functional conservation across vertebrates. It has already been established that there is high Nr6a1 sequence conservation between zebrafish and mouse (98.5%), human (98.5%) and frog (97%) species (Braat et al Goos 1999) and comparable Nr6a1 expression in oocytes and the neural tube (Thisse and Thisse 2008; Adams et al Bronner-Fraser 2008). Consistent with this data, conserved Nr6a1 function has recently been observed in mouse, human and zebrafish with respect to axial elongation (Chang et al 2022) and the etiology and pathogenesis of oculo vertebral renal syndrome (Neelathi et al Brooks 2025)”.\nMajor point 2: In situ hybridizations in Figure 2A are not very clear; especially, several gene's expressions seem lower but not abolished (Foxd3, Snail1) while Twist1 staining looks more like background staining. Stronger staining would be more convincing when the authors state that \"Surprisingly, even though the expression of many NCC-specifier and EMT genes are lost in Nr6a1-/- embryos, the presence of LacZ-positive cells within the frontonasal region and first pharyngeal arch suggests some cells...\" page 7. It rather seems that the patterns observed match the reduced population of cells seen in the GFP-lineage tracing (Figure 2B-C) as well as the Microarray results: \"Although the downregulation of these factors in the microarray did not reach statistical significance, ...\". This observation correlates with the possibility that the genes are expressed albeit at a lower level.\nWe appreciate and understand the reviewer’s concern, and therefore we performed in situ hybridization chain reaction (HCR) and quantified the expression based on overall fluorescence in the head of the Nr6a1 mutant and control stage-matched embryos (Figure 2A and 2B). We hope the reviewer can appreciate the clarity of the HCR over the previous in situ data and agrees that few cells express these genes, plus the levels of expression are considerably lower, although not completely abolished. We also further validated these observations in the multiomic data sets presented later in the manuscript which highlights the consistency of our results across multiple experimental platforms and approaches. We have updated the text accordingly to describe this new data.\nMajor point 3: Although major domains (Pax3 / Sox2) form in the open neural tube of mutants, there is a possibility that the entire neural plate is now entirely biased towards neural fate or alternatively that dorsal cells are frozen in a neural border state. Sox2 maintenance in the neural folds (by DNMT3A, Hu et al., 2012) was previously associated with prolonged expression of neural border specifiers (Zic1): Are Zic1 or proneural gene expression expanded dorsally in NR6A1 mutants?\nWe thank the reviewer for this interesting and insightful question about whether the mutants now exhibit a bias towards a neural fate. Analyses of our multiome data and comparisons of gene expression in the control and Nr6a1 mutant samples, did not reveal an upregulation or expansion in the proneural gene expression domains (see Zic1 below), beyond the persistence of Sox2 in the dorsolateral most domain. The neural plate border domain/cell number appears to be larger, perhaps consistent with the open neural plate and increased dorsolateral neural plate thickness, however, the neural progenitor population was deemed to be reduced (Supplemental Figure 3).\nMajor point 4: Increased expression of Nanog and Oct4 in NR6A1 mutants:\nFigure 4: Oct4/rtTA embryos treated with doxycycline (+Dox) as compared to untreated embryos (-Dox). Consistent with the Nr6a1 null embryos, NCC and EMT marker expression is no longer present through the cranial mesenchyme of the embryos, indicating a disruption in NCC formation.\nM&M: \"To overexpress Oct4, CD1 females were mated with Oct4/rtTA (Col1a1-tetO-Oct4;R26R-M2rtTA) transgenic males (Jax stock #006911). Transgene expression was induced from E6.5.\nIn line with the finding that NR6A1 acts from gastrulation stages, the authors here activate exogenous Oct4 expression throughout gastrulation and recapitulate the NR6A1 mutant NCC phenotype. This is a critical experiment that should focus attention as it was shown that the activation of pluripotency genes (Nanog and its target Oct4) needs to be dynamically regulated during gastrulation: continuous activation from early gastrulation results in neural border and neural crest defects, while Nanog activation at the end of gastrulation is required for proper NC development (Scerbo and Monsoro-Burq, Science Advances 2020). At that stage, pluripotency factors are reprogrammed towards NC target genes by cooperation with the neural border specifiers (Hovland et al., Developmental Cell 2022). This literature is essential to quote for this point and in discussion.\nIn Col1a1-tetO-Oct4;R26R-M2rtTA mouse model, is there a difference in the neural border and NC phenotypes when activating Oct4 at early (E6.5, as in Figure 4) versus later time point in gastrulation (via tamoxifen administration at progressively later half-day time points)?\nWe timed the overexpression of Oct4 to coincide with the key temporal required for Nr6a1, so have not performed a time course experiment in the way that the reviewer suggested. It is an interesting idea but one that addresses a more specific and later developmental role for Oct4, which is tangential to the focus of our questions here on Nr6a1. It’s important to note that Nr6a1 is not the only factor that regulates Oct4, and also that there is persistence of Sox2 as well as Nanog, in addition to down regulation of neural crest cell specifier and EMT genes as part of the pathogenic mechanism underlying the Nr6a1 knockout phenotype. Nonetheless, we think that further exploration of the specific role of Oct4 in neural crest cell development is warranted for the field but that it also requires more cell and tissue specific tools, and that work is beyond the scope and focus of the current paper. We have added more discussion concerning the different results that have been obtained from overexpressing Nanog and Oc4 and cited the work suggested by the reviewer as part of that discussion.\nAccording to results of this temporal analysis, Oct4 expression E7.5 and E8.5 may not be relevant for neural crest earliest initiation step but rather for later specification stage (page 14). Indeed, in single cell data Nanog/Oct4 expression seem present in neural border/neuroectodermal cells but not specified NCC (page 15, lettering in Figure S6A is very small and fuzzy), as found in other scNC datasets (Kotov et al., PNAS 2024 is more adequate than Briggs et al., 2018 for NC studies). There is a sharp difference in Oct4 levels between neuroectodermal clusters: does this support the possibility of a dose-dependent role of Oct4? Would NR6A1 be \"modulating\" Oct4 \"levels\" during gastrulation rather than being an \"on/off\" switch at the edge of the neural plate when NC progenitors are initiated but not yet specified?\nThe reviewer raises a very important point with which we agree. As noted above, Nr6a1 is not the only factor that regulates Oct4, and indeed, even though there is strong evidence for direct binding in ChIP assays, Nr6a1 could modulate the repression and levels of Oct4 rather than act as an absolute on/off switch. Accordingly, we have modified the text in the manuscript to include this more nuanced possibility for how Nr6a1 regulates Oct4.\nWe thank the reviewer for pointing out the difficulties with visualizing Figure S6A and we have replaced the original with an improved figure with bigger text.\nMajor point 5: Introduction, pages 3,\nThe authors make a commendable job to quote original classic research articles from the 1990s in many instances. However, they should also include some more recent references which provide important mechanistical insights. Below are some key references to include.\n- The first formal demonstration of the interplay between secreted factors BMP, WNT and FGF with neural border specifiers Zic1, Msx1 and Pax3, providing the seed for the neural crest gene regulatory network known today, was done in the xenopus model (Monsoro-Burq et al., Developmental Cell 2005).\n- The temporal and spatial tissue-layer dynamics of BMP and WNT signals controlling the sequential steps neural border and neural crest induction was studied in Alkobtawi et al., Cell Reports 2021.\n- TFAP2a is a major neural border specifier in fish, frog and chick, activated the earliest at the edge of the neural plate and regulating the other ones as shown in De Croze et al., PNAS 2011 and Rothstein and Simoes-Costa, 2020 Genome Research.\nWe appreciate the reviewer’s recognition of our efforts to cite the original research publications which has laid the foundations for our current understanding of neural crest cell biology. We also recognize the need to include more recent key literature and thank the reviewer for pointing out some missing references with respect to signaling pathways and neural plate border genes, as well as requesting the addition of citations related to TFAP2A. Accordingly we have added these references as well as additional critical publications on TFAP2A (references 17, 22-28).\nMajor point 6: In discussion: The difference between cranial and caudal NCC should be highlighted, as the phenotype of mutant NR6A1 NCC differs along the body axis: this is also the case for the Pax3/Pax7 mutants: Van Ho et al., Developmental Cell 2011.\nWe thank the reviewer for this comment and for reminding us about the cranial-trunk neural crest cell differences in Pax3/Pax7double mutants. We have cited the Van Ho et al work in addition to other publications such as Zalc et al 2015 but more importantly we have expanded upon our discussion about the Nr6a1 mutant phenotype, cranial-trunk differences, the potential for functional redundancy, and conservation between mouse, chick, frog, fish and human species.\nReviewer #2\nIntroduction- There is a lack of introduction of the nuclear receptor family and their well-established role in progenitor potency and cell fate decision. (e.g., Nr5a2 in skeletal progenitors).\nWe respect the reviewer’s request to provide more information on the background of nuclear receptor’s and Nr6a1 and accordingly we have added more detail about the nuclear receptor family to the introduction, and we specifically note the role of Nr5a2 in pluripotency maintenance and neural crest cell progenitor differentiation in the discussion about the role of orphan nuclear receptors in pluripotency and neural crest cell progenitor differentiation.\nFor example, the following paragraphs were added:\n“We discovered nuclear receptor subfamily 6 group A member 1 (NR6A1) as a novel pivotal regulator of murine NCC specification, formation and survival. While the greater nuclear receptor superfamily consists of 70 receptors organized into 7 subclasses, NR6A1 is in a subclass on its own. Nuclear receptors like NR6A1 contain an N-terminal domain, a DNA-binding domain and a ligand binding domain 63. However, the ligand that interacts with NR6A1 is unknown and therefore NR6A1 is considered an orphan nuclear receptor”.\n“One explanation for the cranial-trunk disparity could be the presence and function of redundant orphan nuclear receptors in NCC development. Interestingly Nr5a2, which maintains Oct4 expression, keeps NCC progenitors in an undifferentiated state, while priming them for later differentiation (Chen et al 2023). This contrasts with Nr6a1’s repression of Oct4 activity and promotion of NCC specification but illustrates the importance of investigating the roles of other orphan nuclear receptors in NCC development”.\n-introduction states that some discrepancies across species regarding neural crest specification. how does nr6a1 function in other species?\nAn evolutionary assessment of Nr6a1 function in neural crest cell development has not yet been performed in other species, which this is one reason why this work is significant, because it represents the first description of a role for Nr6a1 in neural crest cell development in general. We do however note that evaluating the role of NR6A1 in neural crest cell development in other model organisms would provide evidence for functional conservation across vertebrates. Furthermore, we discuss the high Nr6a1 sequence conservation between zebrafish and mouse (98.5%), human (98.5%) and frog (97%) species (Braat et al Goos 1999) and comparable Nr6a1 expression in oocytes and the neural tube across species (Thisse and Thisse 2008; Adams et al Bronner-Fraser 2008). We also discuss, consistent with this philosophy, the conserved function of Nr6a1 that we recently described in mouse,89 with human and zebrafish with respect to axial elongation and the etiology and pathogenesis of oculo vertebral renal syndrome (Neelathi et al 2025). We trust our novel work on Nr6a1 in neural crest cell specification in mice will inspire others utilizing different model organisms to evaluate the role of Nr6a1 in their systems.\nFigure1- In general, the in situ gene expression experiments are not of high quality. Specifically in Fig.1 the staging of the in situ is not in alignment with future conclusions (i.e., Nr6a1 is active at E6.75 from Fig. 7) and does not definitively support the conclusion that Nr6a1 is expressed in early neural crest. For that to be proven, authors would need to do double in situ with known markers of crest at various stages. Furthermore, for stages that do not appear to express Nr6a1 there are no positive controls. Scale bars and stage matched embryos are not included in Fig.1 as well as many other figures.\nWe appreciate the reviewer’s suggestion of aligning the traditional in situ hybridization expression stages with our understanding of Nr6a1 activity based on our functional tests. We have therefore repeated the temporal analysis of Nr6a1 expression with a focus on E6.5-9.5, covering the key stages of gastrulation, neurulation and the specification and formation of neural crest cells. No Nr6a1 expression was detected in the embryo at E6.5, but by E7.5 Nr6a1 was broadly expressed throughout the embryo. Shortly thereafter, Nr6a1 expression becomes refined and restricted to the neuroepithelium by E8.0-8.5, consistent with a specific role in NCC development and a requirement for excision between E6.5 and E7.5 during NCC specification.\nThe embryos used in analysis of Nr6a1 expression are CD1 background (wildtype) but this is a temporal analysis for which stage-matched littermates are not needed as we processed all embryos in the litter at the same time. However, in terms of positive controls, all of the embryos underwent color development in the same tube in the same experiment and the E7.5+ stages and above serve as positive controls for E6.5. We have also made sure to add scale bars to these and all other images. We have included additional commentary noting the use of littermate controls in all of our other experiments, which minimizes developmental variation within litters and stage matches the mutants as closely as possible.\nFigure 2-Again, stage matching and quality of in situ is a concern and detracts from the overall conclusion of the figure. YFP experiments are not done at equivalent stages and there are still neural crest in Nr6a1 mutants.\nWe recognize the reviewer’s concern with the traditional in situ hybridization staining, the resolution of which may also have been affected during PDF conversion. Nonetheless, we have therefore performed in situ hybridization chain reaction (HCR) instead to improve the clarity of this figure and quantified the relative expression of each gene. We made sure to use stage-matched littermates in the HCR experiments and quantification, and consequently, this revealed a significant reduction in NCC and EMT gene expression consistent with our original traditional in situ hybridization results. We have also made sure, wherever possible that we explicitly state the use of control stage matched littermates in our experiments, especially for the Cre-recombination experiments, since it is well known in the mouse field that development varies subtlety between litters and particularly with respect to developmental phenotypes like in the Nr6a1 mutant embryos.\nWe have also revised the text to repeatedly note the distinct effects on cranial neural crest cells and trunk neural crest cells and we make it very clear that there is a considerable deficiency in cranial neural crest cells as evidenced from gene expression, lineage tracing and multiome analyses. Furthermore, our lineage tracing analyses, make it readily apparent that there are no migrating neural crest cells caudal to the first pharyngeal arch, which is additional evidence supporting a critical role for Nr6a1 in early NCC development.\nSupp. Fig. 1- there are a significant number of TUNEL positive cells that are not labeled as neural crest, yet the conclusion is still that loss of Nr6a1 is required for neural crest survival. While this data is not directly in contradiction to that statement, it also does not fully support that conclusion. More likely it seems that Nr6a1 is a factor expressed in progenitor type cells (whether neural ectoderm or germ cells) that restricts potency and proliferation, and promotes differentiation.\n-Furthermore, more the text refers to YFP labels, but the figure refers to GFP.\n-A more comprehensive panel of various stages of neural crest development would be needed to definitively support this conclusion.\nWe appreciate et the reviewer’s question about effects on tissues other than neural crest cells, but due to a failure in chorioallantoic fusion, Nr6a1 mutant embryos begin to die around E9.5. Therefore, it’s not possible to perform a detailed later time course of apoptosis. However, we have quantified the percentage of neural crest cells and non-neural crest cells undergoing apoptosis in Nr6a1 mutants and littermate controls and observed a statistically significant increase specifically in neural crest cell death in the Nr6a1 mutants. Thus, Nr6a1 is critical for the survival of NCC (Supplemental Figure 1B). We stage matched littermates for these experiments as best we could despite standard subtle variations within a litter so that the Nr6a1 mutant and control are as appropriately comparable as possible.\nWe note that while Nr6a1 is expressed in the neural crest cell progenitor cells (the neural plate border), the differentiation of the neural plate itself is not affected by Nr6a1 loss-of-function, and thus Nr6a1 is specifically required for the formation of the neural crest cells themselves. This is also supported by our multiomic analyses. Therefore, Nr6a1 is not a universal key for promoting differentiation but functions during spatiotemporally restricted windows of development like during gastrulation or in neurulation by modulating the repression of pluripotency and the activation of gene expression commensurate with the formation a new cell type (ie neural crest cells). Furthermore, Nr6a1 is specifically expressed by the primordial germ cells during their migration, yet these cells are not differentiating. We have therefore illustrated the specificity of Nr6a1 function. Currently, we do not know the specific ligand or ligands that interact with Nr6a1 and therefore we still have an incomplete understanding of what drives or regulates Nr6a1activity, and it could vary at different stages of development. This work provides the motivation and firmly sets the stage for identifying the ligand(s) that bind Nr6a1 to regulate its function during neural crest cell specification.\nWe thank the reviewer for noticing the discrepancy between the YFP and GFP and we have corrected this typographical error.\nFigure 3- States that Nr6a1 is required for the transition of neural stem cells to neural crest. If this was the case and this was specific to the crest, you would expect to see the lateral edges where the crest arise to be thickened while the rest of the neuroectoderm is the same thickness as in controls. Instead, the entire neuroectoderm is thickened likely due to hyperproliferation at some point. Thus, this is not specific to neural crest, but a generalizable mechanism for cells to decide to leave a proliferative state and begin fate decisions.\nThe reviewer is correct that the entire neuroectoderm is thicker in the Nr6a1 mutants, and we show this occurs in association with increased phospho-histone H3 throughout the neuroectoderm (Figure 3). This is due in part to the increased expression of Sox2 (Figure 3), which functions as part of a tripartite complex with Oct4 and Nanog to promote proliferation and stem cell maintenance. However, this does specifically affect the transition of neural stem cells to neural crest cells because that transition occurs in the dorsolateral most region of the neural ectoderm, commonly called the neural plate border. During normal development, Sox2 is downregulated in the neural plate border region, and the transition of neural stem cells to neural crest cells coincides with the SoxB (Sox1/Sox2) to SoxE (Sox9/Sox10) transition in gene expression. However, in Nr6a1 mutant embryos, the activity of Sox2 persists in the neural plate border, which perturbs the transition of neural stem cells to neural crest cells. Caudal to the first arch this results in the complete absence of migratory neural crest cells. In the cranial region, there is a striking deficit of neural crest cells, as well as a decrease in Sox9/Sox10 expression and labeled cells consistent with the model described above. It’s also important to point out that overexpression of Sox2 in the neural ectoderm, (as we (Remboutsika et al 2012) and others have shown (Wakamatsu et al., 2004),a nd cite in the paper), can alone restrict the transition of neural stem cells to neural crest cells. Thus, although there is currently a lot of emphasis in the field with respect to just Oct4 activity, it is remiss to ignore foundation studies that have examined the role of Sox2 in the process and the collective impact of those factors.\nAs an aside, we have previously measured the density of the neuroepithelium across different axial regions and levels to determine if any differences in population size might impact neural crest cell delamination, however, we found little to no difference across the distinct domains of the neuroepithelium [Zajic, et al. 2024 PNAS].\nFigure 4- Again, stage matching is of concern, as the conclusion is there is more Oct4 expression in the Nr6a1-/-, but the Nr6a1-/- embryo looks to be younger, thus that could be an explanation for increased Oct4 expression. There is no discussion of what Figures 4C and D are in the results section for figure 4 making it difficult to understand what the conclusions are. Despite repeatedly referring to Nanog, there are no in vivo experiments testing the expression of Nanog in Nr6a1 mutants. Expression patterns of Foxd3 between Figs 1 and 4 is vastly different, supporting the critique that the quality of the in situs is low.\nWe understand the concern with staging and always use littermates as a control. We have performed additional experiments and revised the figure to include better stage matched controls. We have also quantified the level of expression to determine if the expression of GFP is indeed specifically higher in the neural ectoderm of Nr6a1-/- mutant embryos. Finally, we apologize for the confusion with Figures 4C and D, they were discussed but the discussion had been at the end of the results. We have now split the different components of the figure so that Figures 4C and 4D are they’re own figure (Figure 8) in the appropriate order in which they are discussed. Additionally, the expression of Foxd3 was not shown in Figure 1. However, if the reviewer means the pattern shown in Figure 2A, then the update to Figure 2A using HCR better aligns with the staining shown in what is now Figure 8B.\nPersistence of Nanog was previously noted in vivo in Nr6a1-/- mutants (see Gu, et al 2005 Mol Cell Biol) and we felt there was no need to repeat that data here since it was already published. We did however illustrate the binding of Nr6a1 to Nanog in our ChIP assays that confirmed the importance of the DR0 elements in the promoter regions of pluripotency, neural crest specification and EMT genes and the potential role in transcriptional repression or activation. Instead, we chose to focus primarily on Oct4, since Oct4 mediation of pluripotency, and more recently persistence of, or reactivation of neural crest cell pluripotency, has been the intense focus of the field.\nSupplemental fig 2- multiome data isn’t fully analyzed and is presented in a manner difficult to interpret. There is no quantification of neural crest on integrated plot and no individual UMAPs to show if/how the neural crest are transcriptionally defined in the Nr6a1 mutants. This presentation/analysis is superficial and does the data a disservice.\nWe have analyzed our multiomic data with the help of our bioinformatics core experts and the data shown aligns with the focus of the paper. Furthermore, we stated in the paper that we used the control littermate embryo dataset to identify the cell populations in Nr6a1-/- mutant embryos. To clarify this point, we added the following sentence: “The same transcriptional signatures were used to identify the same populations within the Nr6a1-/- mutant embryos.” We added quantification of the number of neural crest cells in both samples as well as other relevant cell populations (Supplemental Figure 3C). We appreciate the reviewer’s suggestion of adding the quantification as it demonstrated the neural crest cell population is significantly decreased in the null. While we appreciate the reviewer’s encouragement to explore the multiome dataset, a more detailed analysis lies outside the focus and scope of this paper. We have limited space and have already demonstrated with microarray, bulk RNA-seq and scRNA-seq that pluripotency associated genes are upregulated and that neural crest cell specification and EMT genes are down regulated, and that there are changes in the cell cluster proportions. One of the primary purposes of the multiomic analysis was to assess chromatin accessibility specifically within neural crest cells to understand how Nr6a1 loss-of-function affects chromatin accessibility around our genes of interest, demonstrating its potential function as a transcriptional modulator of pluripotency repression and inducer of neural crest cell specification. Analyses beyond this would be exploratory and detract from the overall focus of the paper which is that Nr6a1 appears to regulate neural crest cell specification and formation by acting as a critical transcriptional regulator, while at the same time modulating the repression of neural stem cell pluripotency. Evaluating the regulation of novel genes and delving into other aspects of the multiomic data would require additional functional analysis, which is better suited to a follow up publication\nFigure 5- Further represents data that is not specific to the neural crest and is not novel mechanistically, as this has been shown in germ cells.\nWhile it is true that previous publications have demonstrated a role for Nr6a1 in repressing Oct4 activity, the binding of Nr6a1 to Sox10 and other key neural crest cell specification and EMT genes has not been previously demonstrated or reported. Our data is specific to neural crest cell development, as we investigated the chromatin accessibility of genes in the neural crest population and neural plate domains. The change in chromatin accessibility at Sox10 is only observed in the neural crest cells and is thus cell type specific. Furthermore, while it has been shown that Nr6a1 can act as a repressor, it has yet to be suggested or shown that Nr6a1 might also function as a potential activator of gene expression which is what our data suggests considering Sox10 is supposed to be turned on at this stage of development for NCC formation. This in turn leads to a different model for neural crest cell specification and formation. Instead of standard feed forward gene regulatory networks that feature prominently in reviews to describe the formation of neural crest cells, our data demonstrates that neural crest cell specification and formation involves the suppression of a parental pluripotent stem cell state together with activation of specific neural crest cell specifier and EMT gene networks.\nFigure 7- Using the tamoxifen approach specifies the temporal window of Nr6a1 (note: this window is not consistent with what is shown in figure 1), however it again does not specifically target neural crest and there is still significant Foxd3 expression.\nAs mentioned with respect to the results prior to this section (Supplemental Figure 5), when Nr6a1 is conditionally excised with a neural crest cell specific Cre (ie Wnt1-Cre), it does not result in any gross morphological embryonic phenotype. Furthermore, the mutant offspring are postnatal viable, which is vastly different to the early embryonic lethality observed for complete Nr6a1-/- nulls. This is because Wnt1-Cre, despite being the gold standard in the field, functions too late during development to elicit an effect on neural crest cell specification. Currently there is no Cre line available in the field that specifically targets only the presumptive neural crest cells during their specification during gastrulation. Therefore, we used a tamoxifen inducible deletion approach to define the temporal requirement for Nr6a1 and show that Nr6a1 is required prior to E7.5 for proper neural crest cell specification. This closely aligns with our spatiotemporal analyses of Nr6a1 expression. While there is some Foxd3 expression, as evidenced by the quantification in Figure 2B, it is significantly reduced. It’s important to point out however, the technical differences in the tamoxifen inducible temporal approach compared to a complete null deletion of gene function. In a traditional null, complete genetic loss-of-function manifests in the zygote, whereas a tamoxifen inducible deletion may not be quite 100% efficient. Furthermore, tamoxifen inducible recombination typically takes up to 24 hours, may be variable between tissues, and is also subject to standard developmental variability within a litter and between litters despite regular light-dark cycle in a mouse animal facility. Even with well controlled timed mating, we can never know with exact surety the precise developmental stage of the embryos in utero at the time of tamoxifen gavage. Therefore, a tamoxifen inducible global temporal deletion is designed to mimic a complete null, which in our case it does, but it should not be expected to be an absolute 100% replica.\nDiscussion: Several statements in the discussion are not supported by the data. For example-\n- “NR6A1 is essential for NCC formation”. Data= Foxd3 expression is shown in Nr6a1 knockouts. Thus neural crest formation is occurring at some level.\nWe appreciate the reviewer’s nuance here about whether Nr6a1 is “essential” for neural crest cell formation. As we state in the abstract and other locations in the paper, the loss of Nr6a1 leads to a considerable deficiency in cranial neural crest cells, and a complete absence of migrating neural crest cells caudal to the first arch. We agree that some neural crest cells do form as evidenced by Foxd3 expression. We would be happy to revise this type of statement to “NR6A1 is essential for proper NCC formation” or “NR6A1 is a critical regulator of NCC formation”\n-“ novel regulator of the transition of neural stem cells to NCC.” Data= nr6a1 has been shown to play a role in progenitor cell populations previously and there is no reason given to think it would act differently in neural crest progenitors.\nWe respectfully disagree with the reviewer’s statement here, because the same argument could be made and has been made for the function of Oct4 in different cells at different stages of development. Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in cultured embryonic stem cells, and consequently many assumptions are automatically made about a pluripotency function for Oct4 in other cell types and tissues. This has been the case with respect to a potential role for Oct4 in neural crest cell development. But the mere expression of factors such as Oct4 is not automatically consistent with pluripotency. In fact, work from Rudolf Jaenisch’s lab has dispelled this idea. In a paper that his lab published in 2007, Lengner et al demonstrated that genetic deletion of Oct4 from numerous different stem cell compartments was completely dispensable and elicited no effect, and thus concluded that Oct4 expression is not required for mouse somatic stem cell self-renewal. Therefore, expression alone cannot automatically be equated with function as the function of any gene is context and developmentally dependent.\nThe same is true with Nr6a1, which has not been previously shown to have a function in neural crest cell development. Therefore, it cannot be assumed that because Nr6a1 is expressed in some progenitor cell population like primordial germ cells that it will always function the same way. For example, Nr6a1 is expressed in a spatiotemporally manner consistent with neural plate development and yet loss of Nr6a1 does not disturb the overall pan-neural or dorsoventral patterning of the neural plate as evidenced by Figure 3. We have uncovered a novel role for Nr6a1 specifically in neural crest cell development, which has not previously been reported. Further, we show that not only does Nr6a1 modulate the downregulation and impact of pluripotency factors, but it also appears to potentially upregulate neural crest cell specifier and EMT genes, which again is novel. Thus, even the transcriptional targets are novel and have not been previously reported. This then allows us to propose a new model for neural crest cell specification and formation that isn’t just the effect of a feed forward gene regulatory network, but one that requires suppression of a parental pluripotent stem cell state together with activation of specific genes important for neural crest cell development.\n-“ Our work provides some of the first evidence in accord with this idea, since global temporal deletion revealed that Nr6a1 is precisely required by E6.75 to replicate the original Nr6a1 null mutant phenotype.’ Data= null embryo in Figure 2 has low/none Foxd3, but in Figure 7 Foxd3 is still clearly expressed. Thus, the phenotype is not replicated.\nWith our update to Figure 2A, we show there is some expression of Foxd3 in the null by HCR which is consistent with the expression observed in Figure 7. Furthermore, as noted above, we used a tamoxifen inducible deletion approach to define the temporal requirement for Nr6a1 and show that Nr6a1 is required prior to E7.5 for proper neural crest cell specification. This closely aligns with our spatiotemporal analyses of Nr6a1 expression, and while there is some Foxd3 expression, it is significantly reduced as is evident in Figures 2 and 7. Again it is important to point out the technical differences in the tamoxifen inducible temporal approach compared to a complete null deletion of gene function. A tamoxifen inducible global temporal deletion is designed to mimic a complete null, which in our case it does, but it should not be expected to be an absolute 100% replica, due to the not quite 100% perfect recombination, as well as the time tamoxifen takes to elicit recombination with subtle variability between tissues, and in utero developmental stage, even with well controlled timed mating. However, in each case we present the data with the most staining we could obtain.\n-“ However, the expression of AP2 and Sox10 was not affected and changes in proliferation were not evaluated despite the global loss of Oct4.” Data= This statement was referring to Zalc et al. Zalc reported that AP2 and Sox10 expression was affected in the frontonasal mass, but NOT in the cranial ganglia, which was the data used to support the conclusion that Oct was necessary for ectomesenchymal specification.\nPrompted by the reviewers comment we realized we needed to clarify our discussion with reference to the Zalc et al paper, because the authors report in response to Oct4 deletion via ActinCreER that immuno-staining against neural crest markers AP2α (Fig. 4D and F) and Alx4 (Fig. S18D–G) demonstrated that although facial prominences were reduced in mutant embryos compared to controls, similar proportions of the remaining cells expressed AP2α and Alx4. Furthermore, the authors only examined their embryos at E9.5 but reported a 10-fold increase in neural crest cell death and 80% reduction in neural crest cell proliferation. Although it was argued that the there was a differential effect on the frontonasal mass versus the cranial ganglia, the graphs presented in the paper report the same decrease/downward trend even though they did not reach statistical significance for the ganglia by E9.5. But given the formation of the neural crest cell derived frontonasal mass mesenchyme occurs temporally before the neural crest cell contributions to the trigeminal and other facial ganglia it is reasonable to expect that analyses a half to a full day later would have revealed a similar phenotype. Nonetheless, the authors concluded in relation to their data “Oct4 was dispensable for cranial neural crest cell induction and delamination”.\n“Our analyses of Nr6a1 null embryos challenge the re-acquisition model and role for Oct4 in NCC specification and formation. We evaluated the expression of Oct4 during normal development using the Oct4-EGFP transgenic mouse line, which revealed that although Oct4 is strongly expressed in the epiblast at E7.5, it is downregulated by E8.5 coinciding with the specification and formation of NCC.” Data=Authors state neural crest are specified much earlier than classical models suggest (i.e., 6.75), yet show Oct4 expression in areas akin to where neural crest would be at e7.5.\nNeural crest cells are not present at E7.5 and we do not show that they are specified at E6.75. We show that Nr6a1 plays an important role in neural crest cell specification and formation beginning around this time, because we only observe a phenotype using our global tamoxifen inducible model that closely mimics the original Nr6a1 complete null, when Nr6a1 is excised around E6.75. By E7.5 Nr6a1, like Oct4, is highly expressed and modulates the down regulation of Oct4. A similar correlation is observed in our analyses of Nr6a1 and Oct4 activity in human induced pluripotent stem cells differentiated into neural crest cells\nThus, classic markers of neural crest cell specification and formation are not expressed at E7.5, but their expression is initiated by E8.5. However, the process of neural crest cell specification begins during gastrulation and before E7.5 with the expression of Nr6a1 and modulation of the downregulation Oct4 activity which is critical for proper neural crest cell development. It’s important to highlight that specification is not the same as the formation and of NCC with definitive identity.\nAlthough there are nuances and semantics to consider when describing what is actually a neural crest cell, we view their development as a progressive process that begins with the specification of neural stem cells/neural plate border cells as progenitor neural crest cells, which occurs during gastrulation (E6.5-7.5), after which the specified neural crest cells undergo EMT and delamination beginning around E8.25-8.5 to become definitive classic migratory neural crest cells.\n-“ Our scRNA-seq results matched those obtained in Xenopus where even when clustering with a bias towards re-activation of pluripotency, presumptive NCC do not express these factors”. Data= Oct4 is expressed in neural ectoderm to the same extent as Nr6a1 is expressed in neural ectoderm. Thus, to say that Oct4 is not expressed in presumptive neural crest but Nr6a1 is expressed in neural crest seems contradictory.\nIt's important to note that the neural ectoderm is not equivalent to presumptive neural crest cells. Although overexpression of Snai1/2, Sox10 and other factors have been shown to be capable of converting neural ectoderm broadly into migratory neural crest cells, in vivo in embryos, neural crest cells only endogenously arise from the dorsolateral region of the neural ectoderm at the junction with the surface ectoderm, a territory known as the neural plate border. Therefore, the co-expression of Oct4//Nr6a1 in the neural ectoderm is not a readout of expression in presumptive neural crest cells, but it is consistent with the ability of Nr6a1 to ultimately modulate the downregulation of Oct4 expression.\nReviewer #3\nA primary strength of the study lies in the combination of direct in vivo genetics, transcriptomic approaches, and biochemistry. The methodology and its description are sound. However, there are several problems with this study in its current form. The biggest hole is that while the timing of NR6A1 action is shown convincingly to be during gastrulation, all the phenotypic analysis provided is carried out at much later stages. As a consequence, we don’t know how direct these outcomes are. A related issue is that we lack information on the spatial requirement for this molecule. The authors appropriately attempted to use the Wnt-Cre system to ablate NR6A1 function specifically in NCCs, but this approach instead revealed that by the stage Wnt-Cre acts (E8.5) the molecule is no longer needed. They then switched to using a tamoxifen inducible Cre-system to show that NR6A1 functions much earlier, during gastrulation, at E6.75. They do not show us where the gene is expressed at its stage of action, and it is quite possible that it functions non cell autonomously rather than directly. This point should at minimum be acknowledged and discussed.\nWe thank the reviewer for their kind words and recognition of the importance of the work, and also their request to provide additional Nr6a1 expression data, which we have included in Figure 1 We repeated the in situ hybridization analyses of Nr6a1 expression focusing on E6.5-9.5, the key period covering gastrulation, neurulation and the specification and formation of neural crest cells. No Nr6a1 expression was detected in the embryo at E6.5, but by E7.5 Nr6a1 was broadly expressed throughout the embryo. Shortly thereafter, Nr6a1 expression becomes refined and restricted to the neuroepithelium by E8.0-8.5, consistent with a specific role in NCC development and a requirement for excision between E6.5 and E7.5 during NCC specification. While we understand it is interesting that excision is required during gastrulation, and that Nr6a1 is broadly expressed, we respectfully point out that if Nr6a1 loss-of-function resulted in a non-cell autonomous or secondary effect, then we would not expect the neural plate to be correctly patterned in the Nr6a1-/- mutants, (which it was both pan neural and dorsoventrally), while manifesting with a NCC specific phenotype.\nAnother significant concern is that quantification is completely lacking for several of the in vivo analyses. This needs to be corrected. For example, in Fig. 2A, the NR6A1 mutants show some morphological – or perhaps staging – variation. This should be commented on, especially as there appears to be Foxd3 expression present in the anterior of the mutant shown. It would be helpful to understand the extent of variation between specimens. Related to this, the authors need to take care to discriminate between situations when expression patterns are missing, versus the cells or tissues themselves are absent. For example, Zeb2 is stated to be absent from NCC-derived facial and pharyngeal mesenchyme, but these tissues appear to be missing (at least in part).\nWe acknowledge this comment and accordingly we have repeated the experiments in Figure 2A by HCR using stage-matched control littermates. We understand the reviewer’s concern about the need for quantification and therefore we have added quantification of the levels of neural crest cell specifier and EMT gene expression, noted the decrease in labeled cells, and quantified the percentage of cells undergoing cell death (Supplemental Figure 1). We’ve also quantified Oct4-EGFP expression (Figure 4), and the number of NCC in the multiome datasets (Supplemental Figure 3C). We have included additional commentary noting the use of littermate controls in all of our experiments, which minimizes developmental variation within litters and stage matches the mutants as closely as possible. We have updated the expression analyses of Zeb2 in Figure 2C which now better shows the restriction of Zeb2 expression to the neural plate.\nFinally, the analysis provided focuses exclusively on cranial neural crest, although this point is never made explicit. Is Nr6a1 also required for trunk neural crest fates? It seems that trunk somitic mesoderm may be missing or aberrant in Fig. 2B. Proper analysis of trunk phenotypes is needed.\nThis is important and we thank the reviewer for pointing this out. We have revised the text to repeatedly note the distinct effects on cranial neural crest cells and trunk neural crest cells and we make it very clear from our lineage tracing that no migrating neural crest cells are detected caudal to the first arch. We have added considerable discussion on the topic and note somewhat similar cranial-trunk neural crest cell differences in Pax3/Pax7 double mutants but more importantly we have expanded upon our discussion about the Nr6a1 mutant phenotype, cranial-trunk differences, the potential for functional redundancy, and conservation between mouse, chick, frog, fish and human species.\nAs the reviewer alludes to, Nr6a1-/- embryos also exhibit defects in axial elongation and tail bud formation and we have previously studied the role of Nr6a1 in trunk somitic mesoderm development and the trunk to tail transition (Chang et al 2022). Somites do form, but Nr6a1 controls vertebral segment number and Hox gene patterning in the trunk autonomously from other axial regions. We cite this work in the discussion and also in relation to potentially conserved functions across species.\nRESPONSE TO REVIEWERS\nWe sincerely thank the reviewers again for their time and effort and thoughtful comments evaluating our revised manuscript. As noted below we have addressed the remaining minor typographical issues and revised our manuscript accordingly. Changes to the text have been highlighted for convenient referral. We trust the work will now be suitable for publication.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nAs stated in the first review of this article, this work addresses and demonstrates broadly important findings in the field of neural crest biology. With this revision, the authors have addressed adequately my previous comments. I thus support publication without delay of this work.\nThank you for recognizing and appreciating the importance of our work and we are glad to know our revisions satisfactorily addressed your concerns.\nReviewer #2 (Remarks to the Author):\nThe authors have greatly improved the manuscript during revision. While there remain some minor concerns with the in situ (Zeb2), the conclusions are now better supported.\nThank you for acknowledging the revisions we made in response to your original comments, questions, concerns and suggestions. This really helped us to improve our manuscript.\nMinor typographical errors:\n1. flox alleles being referred to differently in text (fx) versus figure (fl)\n2. line 465 'out data'\n3. Figure legend 1 typo\n4. Figure legend 2 typo\nThese typographical errors have all been corrected.\nReviewer #3 (Remarks to the Author):\nThis revised manuscript from Trainor and colleagues is much improved since the previous submission. The authors have added significant additional analysis - in particular adding HCR and quantification in multiple places - as well as clarifying the text. My concerns have been adequately addressed.\nThank you for recognizing and appreciating the importance of our work and we are glad to know we our revisions satisfactorily addressed your concerns."}, {"round": 2, "reviewer_comments": {"reviewer_1": "As stated in the first review of this article, this work addresses and demonstrates broadly important findings in the field of neural crest biology. With this revision, the authors have addressed adequately my previous comments. I thus support publication without delay of this work.", "reviewer_2": "The authors have greatly improved the manuscript during revision. While there remain some minor concerns with the in situ (Zeb2), the conclusions are now better supported.\nMinor typographical errors:\n1. flox alleles being referred to differently in text (fx) versus figure (fl)\n2. line 465 'out data'\n3. Figure legend 1 typo\n4. Figure legend 2 typo", "reviewer_3": "This revised manuscript from Trainor in colleagues is much improved since the previous submission. The authors have added significant additional analysis - in particular adding HCR and quantification in multiple places - as well as clarifying the text. My concerns have been adequately addressed."}, "author_response": "We sincerely thank the reviewers again for their time and effort and thoughtful comments evaluating our revised manuscript. As noted below we have addressed the remaining minor typographical issues and revised our manuscript accordingly. Changes to the text have been highlighted for convenient referral. We trust the work will now be suitable for publication.\n\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nAs stated in the first review of this article, this work addresses and demonstrates\nbroadly important findings in the field of neural crest biology. With this revision, the\nauthors have addressed adequately my previous comments. I thus support publication\nwithout delay of this work.\nThank you for recognizing and appreciating the importance of our work and we are glad\nto know our revisions satisfactorily addressed your concerns.\nReviewer #2 (Remarks to the Author):\nThe authors have greatly improved the manuscript during revision. While there remain\nsome minor concerns with the in situ (Zeb2), the conclusions are now better supported.\nThank you for acknowledging the revisions we made in response to your original\ncomments, questions, concerns and suggestions. This really helped us to improve our\nmanuscript.\nMinor typographical errors:\n1. flox alleles being referred to differently in text (fx) versus figure (fl)\n2. line 465 'out data'\n3. Figure legend 1 typo\n4. Figure legend 2 typo\nThese typographical errors have all been corrected.\nReviewer #3 (Remarks to the Author):\nThis revised manuscript from Trainor and colleagues is much improved since the\nprevious submission. The authors have added significant additional analysis - in\nparticular adding HCR and quantification in multiple places - as well as clarifying the\ntext. My concerns have been adequately addressed.\nThank you for recognizing and appreciating the importance of our work and we are glad\nto know we our revisions satisfactorily addressed your concerns."}]

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