Cranial neural crest shortage leads to extensive craniofacial anomalies in mice mutant for the NR2F1/2 nuclear receptors

The NR2Fs are a family of highly conserved nuclear receptors. Heterozygous variants in Nr2f1&2 cause human disease. While primary patient phenotypes occur in separate tissues, both populations share mild craniofacial malformations, suggesting that expression and function may overlap in the developing face. While strong human in vitro and zebrafish in vivo evidence supports this hypothesis, it has not been assessed in vivo in mammals. Here we show overlapping expression of mouse Nr2f1&2 in the cranial neural crest from mid- to post-migratory stages. Conditional ablation of Nr2f1&2 in the cranial crest causes major reduction of all facial skeletal elements except the mandible. This originates before the 16 somite stage as a loss of the trailing stream in an apparently apoptosis-independent manner. While the mechanism is not clear, we note downregulation of several migratory crest genes at the 12 somite stage. Additionally, we publish a comparison of Wnt1-Cre and Pax3Cre activation while investigating the preservation of the mandible in mutants. Neither appear to activate before specification and likely cannot cause total protein loss before delamination of the cranial crest is complete. Bulk RNAseq was performed on individual heads cut caudal to the first pharyngeal arch of control and Wnt1-Cre;Nr2f1fl/fl;Nr2f2fl/fl mouse embryos at the 8 somite (1 somite) and 12 somite (1 somite) stages. Total of 12 samples, 3 replicates each genotype and stage.NR2Fs是一类高度保守的核受体家族。Nr2f1与Nr2f2的杂合变异可导致人类疾病。尽管患者的原发表型累及不同组织，但两类变异均会引发轻度颅面畸形，提示二者在发育中的面部组织内可能存在表达与功能的重叠。尽管已有的人类体外研究与斑马鱼体内研究均为该假说提供了有力支持，但该假说尚未在哺乳动物体内得到验证。本研究证实，小鼠Nr2f1与Nr2f2在颅神经嵴（cranial neural crest）的迁移中期至迁移后期均存在重叠表达。在颅神经嵴中条件性敲除Nr2f1与Nr2f2，会导致除下颌骨外的所有面部骨骼结构显著减少。该表型出现于16体节期之前，表现为神经嵴尾侧细胞流的丢失，且该过程似乎不依赖细胞凋亡。尽管具体调控机制尚未明确，但我们观察到在12体节期，多个迁移性神经嵴相关基因的表达出现下调。此外，在探究突变体下颌骨得以保留的机制时，我们对比了Wnt1-Cre与Pax3Cre的激活模式。二者均未在颅神经嵴特化阶段前被激活，且大概率无法在颅神经嵴完成分层前造成总蛋白的完全缺失。本研究对8体节（1体节）和12体节（1体节）期的对照组小鼠以及Wnt1-Cre;Nr2f1fl/fl;Nr2f2fl/fl基因工程小鼠胚胎的第一咽弓尾侧切割的单个头部组织进行了批量RNA测序（bulk RNAseq）。共获得12个样本，每种基因型与每个发育阶段均设置3个生物学重复。