Mouse embryos were dissected in phosphate-buffered saline, pH 7.4 (PBS), fixed in 3.7% formaldehyde overnight at 4°C, and washed in PBS containing 0.1% Triton (PT). Embryos were stored at −30°C in methanol and were rehydrated at the time of use in PT. Embryos were cryopreserved using a sucrose gradient of 10%, 20% then 30% w/v sucrose in PT, followed by 1:1 30% sucrose:OCT Compound (Fisher Scientific 23730571) and 100% OCT. Embryos were flash-frozen in OCT for cryosectioning using dry ice and 100% ethanol bath and were stored at −80°C until sectioning. Cryo-sectioning was performed at 10 um and slides were stored at −80°C until staining. Sections on slides were washed with PT and blocking was performed at room temperature for 1 h in 5% Gibco normal goat serum (16210064) and 1% bovine serum albumin (Fisher Scientific BP1600100). Primary antibodies were diluted in blocking solution and applied to tissue overnight at 4°C [Sox9 (EMD Millipore AB5535; 1:1,000) Pax3 (DSHB PAX3; 1:100)]. Secondary antibodies (AlexaFluor) diluted in blocking buffer (1:500) were applied for 1.5 h at room temperature. Sections were mounted with Fluoromount G (Fisher Scientific OB10001). Images of the cross-sections were taken on Zeiss LSM780 or LSM 980. Wholemount embryos were imaged on Leica M165FC dissecting microscope with a Leica DFC 3000G camera or Zeiss LSM780.

Wnt1-Cre2 (Lewis et al., 2013) or Sox10-Cre (Stine et al., 2009) mice were crossed with ROSA26^Tomato/+ mice (Luche et al., 2007) to lineage trace neural crest cells at E9.5. Mouse embryos were decapitated anterior to the otic placode. The cranial region was enzymatically dissociated with papain at room temperature combined with gentle pipetting until a single-cell suspension was achieved. An equal volume of FBS was used to quench the enzyme. The single-cell suspension was filtered, spun at 300 g for 5 min, and resuspended in PBS with 1% BSA. Samples were filtered and fluorescent cells were sorted on a BD FACSAria III instrument with a 70-μm nozzle into a 1.5 ml Eppendorf tube that contained Trizol-LS (Thermo Fisher Scientific, 10296028).

Total RNA was extracted from cells using RNeasy Micro Kit (QIAGEN 74004). For mRNA sequencing cDNA synthesis was performed using SMART-Seq Ultra Low Input RNA Kit for Sequencing (Takara 634889) from approximately 500 pg of total RNA. cDNA was validated using the High Sensitivity NGS Fragment Analysis Kit (Agilent formerly AATI DNF-474-0500) on a 12-Capillary Fragment Analyzer. Quantification was determined using the Quant-iT dsDNA Assay Kit, high sensitivity (Thermo Fisher Q33120), and 100 pg of cDNA was tagmented and ligated using the Nextera XT DNA Library Kit (Illumina FC-131-1024) at 12 volumes to produce sequencing libraries. The resulting libraries were validated using the High Sensitivity NGS Fragment Analysis Kit on a 12-Capillary Fragment Analyzer and quantified using the Quant-iT dsDNA Assay Kit, high sensitivity. Equal concentrations of libraries were pooled, denatured, diluted, and subjected to paired-end sequencing using the Mid Output v2.5 kit (Illumina FC-404-2001) on a NextSeq550 following the manufacturer’s instructions.

Sequencing files from each flow cell lane were downloaded and the resulting FASTQ files were merged. Quality control was performed using fastQC (v0.10.1). Reads were mapped to the mouse genome mm10 assembly using STAR (v2.5.0a). In R (v3.5.2), gene count matrices were built with Bioconductor packages Rsamtools (v2.0.0) and GenomicFeatures (v1.32.2). mRNA-sequencing datasets were annotated with UCSC transcripts downloaded from Illumina iGenomes in GTF file format. We determined reads per million (RPM) using GenomicAlignments (v1.16.0). Principal component analysis (PCA) was performed using an rlog transformed gene expression matrix of global gene expression >1 for each region. DESeq2 (v1.20.0) was used for differential gene expression analysis and read count normalization. Expression heat maps were generated using ComplexHeatmap (v2.0.0). Biological Process GO analyses were determined using Enrichr and visualized using ggplot.

Wildtype mouse embryos were decapitated at the otic placode and the cranial region was enzymatically dissociated with papain at room temperature combined with gentle pipetting until a single-cell suspension was achieved. At which time the dissociation was quenched with an equal volume of FBS. The single-cell suspension was filtered (Falcon 352235), spun at 300 g for 5 min, and resuspended in DMEM with 10% FBS. For single-cell ATAC sequencing, nuclei were isolated by resuspending cell pellet in lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.01% Nonidet P40, 0.01% Digitonin, 1% BSA). Lysis was diluted with wash buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 1% BSA, 0.1% Tween-20) and nuclei were pelleted by spinning at 500 g for 5 min. Nuclei were then washed, pelleted, and resuspended in nuclei buffer (10× Genomics 2000153). Nuclei count and quality were assessed on a hemocytometer using 0.4% Trypan blue to stain nuclei. GEM generation was performed on a 10× Chromium Controller Instrument on Chromium Next GEM Chip G for mRNA (10× Genomics 1000120) and Next GEM Chromium Chip H for ATAC (10× Genomics 1000161). Libraries were subsequently prepped using Chromium Next GEM Single Cell 3’ Library Kit v3.1 for mRNA (10× Genomics 1000158) and Chromium Next GEM Single Cell ATAC Library Kit v1.1 for ATAC (10× Genomics 1000163). Chromium Single Index Kit T Set A (10× Genomics 1000213) was used to index mRNA libraries and i7 Multiplex Kit N Set A (10× Genomics 1000084) was used to index ATAC libraries. Libraries were validated using the High Sensitivity NGS Fragment Analysis Kit on a 12-Capillary Fragment Analyzer and quantified using the Quant-iT dsDNA Assay Kit, high sensitivity. Equal concentrations of libraries were pooled, denatured, diluted, and subjected to paired-end sequencing using the Mid Output v2.5 kit (Illumina FC-404-2001) on a NextSeq550 following the manufacturer’s instructions. At E9.5 16,949, cells were sequenced from a C57BL/6 wildtype mouse embryo (128, 243, 041 reads; an average of 7,566 reads per cell). Single-cell mRNA Raw bcl files were downloaded using Illumina’s BaseSpaceCLI version 0.10.7 and were converted to fastq files using 10× Genomics Cell Ranger mkfastq and were aligned to the mm10 genome using 10× Genomics Cell Ranger count. Data were subsequently log normalized and clustered using five statistically significant principal components in Seurat version 3.1.5 (Butler et al., 2018). Mitochondrial genes and cells with <200 UMI and >2,500 UMIs were filtered out. Cell populations were identified for single-cell mRNA data as in previous studies (Pijuan-Sala et al., 2019) and cell markers are provided in Supplementary Table 1. Single-cell ATAC raw bcl files were downloaded using Illumina’s BaseSpaceCLI version 0.10.7 and were converted to fastq files using 10× Genomics Cell Ranger ATAC mkfastq (version 1.1.0) and were aligned to the mm10 genome using 10× Genomics Cell Ranger ATAC count. Peaks were unified, quantified, and visualized in Seurat and Signac (version 0.2). Single-cell ATAC data was overlayed with cell populations from annotated single-cell mRNA similar to previous studies (Stuart et al., 2019, 2020 preprint and motifs were called within 2 kb of the loci of interest by Homer. Enrichr was used for gene ontology analyses.

RNAScope was performed using the Multiplex Fluorescent Reagent Kit v2 (ACD Bio 323100). Embryos were dissected, fixed, cryopreserved, and cryosectioned as above. Slides were stored at −80°C until use and were brought to room temperature. Tissue sections were re-fixed for 15 min at room temperature and subsequently dehydrated in 50% ethanol, followed by 70% and 100% ethanol. Hydrogen peroxide was applied to tissue sections for 10 min at room temperature and antigen retrieval was performed for 5 min at >99°C in a Black and Decker HS800 steamer. Sections were subsequently washed in distilled water, dehydrated in 100% ethanol, dried at room temperature, and subsequently treated with RNAScope Protease III for 30 min at 40°C. Mm-Wnt1-C2 RNAScope 2.5 LS Probe (ACD Bio 4011098-C2) was diluted 1:10 with Mm-Sox10 RNAScope 2.5 LS Probe (ACD Bio 435938) and the probe mixture was hybridized at 40°C for 2 h. Signal was amplified with RNAScope Multiplex FL v2 AMP1-3 for 30 min each at 40°C. Sox10 signal was developed by incubating tissue sections for 15 min at 40°C in RNAScope Multiplex FL v2 hP-C1 and Opal 570 [Perkin Elmer FP1488A (diluted 1:2,000 in TSA buffer)] was applied for 30 min at 40°C. HRP blocker was applied for 15 min at 40°C and Wnt1 signal was subsequently developed by incubating tissue sections for 15 min at 40°C in RNAScope Multiplex FL v2 hP-C2 and Opal 690 [Perkin Elmer FP1497A (diluted 1:500 in TSA buffer)] was applied for 30 min at 40°C. Images were taken on a Zeiss LSM 980.

All research and animal care procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee and housed in the Association for Assessment and Accreditation of Laboratory Animal Care-approved animal facility at Baylor College of Medicine. All strains were maintained on the C57BL/6 background. Adult genotyping was performed by lysing 1–2 mm ear clippings in 75 μl 25 mM NaOH 0.2 mM EDTA at 98°C for 1 h and neutralized with 75 μl 40 mM Tris-Cl, pH 5.5. Embryo genotyping was performed by digesting yolk sac tissue overnight in lysis buffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 mM NaCl, 0.1% SDS, and 5 mg/ml proteinase K]. Cell debris was removed and an equal amount of isopropanol was used to precipitate DNA at −30°C for 1 h. DNA was pelleted by a 30 min centrifugation, washed with 70% ethanol, and resuspended in water. PCR for all alleles was performed using 40 cycles 95 °C for 20 s and touch-down annealing at 64°C, 62°C, 60°C, 58°C, followed by 40 s extension at 72°C. All PCR primers and expected band sizes are in Table 1.

We use multiple approaches to shed light on the extent of labeling from common neural crest Cre-drivers and demonstrate the importance of carefully profiling recombination. We find using IF of transverse serial cross-sections that Wnt1-Cre2 ubiquitously labels the neural tube near the mid/hindbrain boundary at E9.5 resulting in a neuronal-like gene signature from cells isolated using FACS as compared to cells harvested with Sox10-Cre. Previously, Wnt1-Cre2 was profiled in E8.5 and E9.5 wholemount embryos and transverse section through the E9.5 pharyngeal arch to show that labeling of the neural crest with Wnt1-Cre2 was similar to that of the original Wnt1-Cre2 (Lewis et al., 2013). Our results expand on the understanding of labeling by Wnt1-Cre2 by analyzing serial cross-sections along the anterior/posterior axis at E9.5. While Wnt1-Cre2 corrects for the ectopic activation of Wnt signaling and associated phenotypes, Wnt1-Cre2 robustly labels cells of the neural tube, which may be a limitation for studies that require neural crest specificity. The labeling of the neuroepithelium by Wnt1-Cre2 and the endogenous expression of Wnt1 in the neuroepithelium at E9.5 does allow for the continual labeling of the premigratory neural crest. However, the labeling of premigratory neural crest by Wnt1-Cre2 may not accurately target the process of neural crest formation as previous studies have found that recombination by Wnt1-Cre2 may occur too late to allow for early neural crest studies (Brault et al., 2001; Hari et al., 2002; Jia et al., 2007; Büchmann-Møller et al., 2009). Our work profiling the recombination of Wnt1-Cre2 and Sox10-Cre focuses on E9.5 neural crest populations. Future studies may be aimed at profiling the recombination of Wnt1-Cre2 during neural crest formation. It will be important to determine how Wnt1-Cre2 labels cells at the earliest stages of neural crest development. For example, it remains to be determined whether premigratory cells possess an ectomesenchymal vs. neural fate bias and whether both fates are comparably captured by Wnt1-Cre2 in mice. Furthermore, it has been shown that Wnt1 expression declines in neural crest as they delaminate from the neural tube (Zervas et al., 2004; Kléber et al., 2005; Rabadán et al., 2016; Bhattacharya et al., 2018; Hutchins and Bronner, 2018). We similarly found robust Wnt1 expression in neuroepithelial cells compared to neural crest and little co-expression of Wnt1 and Sox10 in migratory neural crest at E9.5. This may reflect the downregulation of Wnt1 after the neural crest migrates away from the neural tube or the possibility that Wnt1 and Sox10 label different neural crest cell populations or derivatives.

We identified predicted motifs of transcription factors that may promote the expression of Wnt1 in both the neural tube and neural crest. We identified predicted motifs for Otx2 and Tead2 in the accessible regions of chromatin around the Wnt1 locus and both of these factors are expressed in the neural tube. Otx2 expression parallels that of Wnt1; Otx2 was expressed in more cells of the neural tube than neural crest at E9.5. Furthermore, Otx2 has been suggested to be an early mammalian neural crest transcription factor and have a role in the neural specification (Finkelstein and Perrimon, 1991; LeDouarin et al., 1999; Williams et al., 2019). Otx2 expression may decline in more mature neural crest cells. Tead2 is known to interact with an enhancer element to regulate the expression of Pax3, a factor that is also expressed in both the neural crest and the dorsal neural tube. Similar to the labeling achieved with Wnt1-Cre2, we find Tead2 is expressed in both neural crest cells and cells of the neural tube. We also identified predicted motifs of transcription factors that may promote the expression of Sox10 specifically in the neural crest. We found that accessible regions of chromatin around the Sox10 locus at E9.5 contain motifs for Ets1 and Ebf1. Ets1 is a canonical cranial neural crest specification transcription factor (Barembaum and Bronner, 2013). Ebf1 has been identified as a neural crest migration transcription factor (Simões-Costa et al., 2014). Our multiomics approach enables the identification of accessible motifs which may promote expression of Wnt1 in both migratory neural crest and neural tube, as well as endow specificity of Sox10 to migratory neural crest. However, with our approach, single-cell ATAC data was obtained from one embryo and overlayed with single-cell mRNA data from a second embryo and future studies may benefit from newer technologies where both types of libraries can be constructed from the same cell.

Our findings reveal cellular heterogeneity that should be taken into account when selecting a Cre-driver for labeling the nervous system in mice. The heterogeneity of neural crest has long been a question of interest as these cells must maintain the multipotent differentiation potential to form various derivatives during migration. The degree to which migratory neural crests are a homogeneous cell population that will subsequently diverge vs. heterogeneous populations that exist immediately after delamination from the neural tube remains to be determined. Heterogeneity of neural crest cells was evident via immunofluorescence for canonical neural crest transcription factors SOX9 and PAX3 at most stages of neural crest development. However, we did find considerable SOX9 and PAX3 co-expression during delamination, similar to Wnt1 and Sox10 co-expression, and a recent study which found that neural crest cells are similar during EMT and then subsequently diverge into various lineages (Soldatov et al., 2019). Studies in chick revealed subpopulations of the neural crest that largely do not co-express neural crest transcription factors consistent with our findings (Roellig et al., 2017). Regardless of the transcription factors expressed in each subpopulation of neural crest, our analysis revealed that neural crest subpopulations are similarly metabolically active, consistent with previous studies (Bhattacharya et al., 2020; Keuls et al., 2020). Taken together, our findings uncover unique aspects of cellular diversity amongst early cranial neural crest and identify specific populations of cells targeted by Wnt1-Cre2 and Sox10-Cre during early neural development in mice.