Fig. 2: NCC-specifier and EMT genes are downregulated in the Nr6a1 null embryos resulting in a deficiency of NCC.

A HCR of NCC and EMT marker genes (yellow) in Nr6a1 null and heterozygous stage-matched control embryos at E8.75. Foxd3, Sox9, Sox10, and Snai1 expression appear reduced in the Nr6a1 null embryo compared to controls. Twist1 is still expressed in expressed in migrating NCC. B Quantification and statistical analysis of the relative expression of each gene in Nr6a1 null and stage-matched controls based on the total fluorescence of the gene normalized to the total fluorescence of DAPI in each embryo. The expression of Foxd3, Sox9, Sox10 and Snai1 was down in Nr6a1 null embryos compared to controls consistent with the staining shown in the representative images in (A). Ratio paired t-test (two-tailed) determined the expression of Foxd3, Sox9, Sox10 and Snai1 were statistically significant while Twist1 was not. A total of 4 embryos across two litters was used in this quantification of each condition. C Traditional in situ hybridization of Zeb2 (purple stain) on an E8.75 Nr6a1 null embryo compared to a stage-matched control. Zeb2 is expressed throughout the cranial region and in the dorsal neural plate in the control embryos at E8.75. However, Zeb2 expression is restricted to the neural plate in the Nr6a1 null and is not found in the frontonasal region or arches suggesting its expression is also downregulated. D Beta-galactosidase staining (blue) of Nr6a1^−/−;Wnt1-Cre;R26R-LacZ and littermate Wnt1-Cre;R26R-LacZ embryos at E9.5. Nr6a1 null embryos lack expression of LacZ caudal to the first pharyngeal arch indicating the absence of migrating NCC. E eYFP expression (green) in Nr6a1^−/−;Wnt1-Cre;R26R-eYFP and littermate Wnt1-Cre;R26R-eYFP embryos at E9.0 illustrates the absence of NCC caudal to the first pharyngeal arch and fewer migrating NCC in the cranial region consistent with the LacZ stained embryos. Transverse histological sections through the first pharyngeal arch (PA1) in both embryos shows a reduction in migratory NCC in the null embryos. No NCC are present in the second arch (PA2) in the null embryo. A minimum of 4 embryos were assayed for each in situ hybridization marker and lineage tracing experiment.