Background

Cranial neural crest cells (NCCs) form the face and the anterior part of the head (Jiang et al., 2002; Chai and Maxson, 2006; Mishina and Snider, 2014). Cranial NCCs are one of the subpopulations of NCCs that show unique characteristicis by differentiating into bones and cartilage, while other populations of NCCs do not have this differentiation potential (Stemple and Anderson, 1992; Jiang et al., 2000; Ishii et al., 2012). Cranial NCCs delaminate from the neural tube and migrate into branchial arches (BAs) (Santagati and Rijli, 2003; Kaucka et al., 2016; Yang et al., 2021). Subsequently, they give rise to neural tissues, including peripheral nerve systems but also connective tissues, such as bones, cartilage, and adipose tissues (Santagati and Rijli, 2003). Several signaling pathways are known to regulate differentiation, proliferation, migration, and cell specification for cranial NCCs, which have been reported to develop craniofacial anomalies (CFA), such as cleft palate and craniosynostosis (Teng et al., 2008; Komatsu et al., 2013; He and Soriano, 2013). CFA are a diverse group of pathologic conditions caused by environmental and genetic reasons. Therefore, a deeper understanding of the molecular and cellular mechanisms underpinning cranial NCCs function may result in improved identification, prevention, and treatment of CFA in human patients.

In the 1980s, a method to generate chick-quail chimeras was developed, and cell lineage tracing experiments of neural crest cells during embryonic development became popular (Couly and Dourin, 1985). After establishment of Cre-loxP technologies, transgenic mouse lines expressing Cre recombinase driven by promoters of NCC marker genes, such as Wnt1 and Protein zero (P0) (Wnt1-Cre and P0-Cre lines), facilitated cell lineage tracing during neural crest migration and differentiation (Danielian et al., 1998; Yamauchi et al., 1999, Chen et al., 2017). Using the Cre-loxP technologies, genes of interest are specifically mutated in NCCs to introduce either gain-of-function (GOF) or loss-of-function (LOF) mutations, which significantly deepen our understanding of the mechanisms of craniofacial and trunk development mediated by NCCs. To further gain insight into mechanisms which control proliferation, cell fate specification, and differentiation of cranial NCCs, it is important to establish an in vitro culture system to maintain the character of cranial NCCs.

Bone morphogenetic proteins (BMPs) are a group of growth factors that bind to receptors on the plasma membrane that transduce a signal mediated by SMAD proteins. The levels of BMP-SMAD signaling are important to determine a range of cell behaviors including fate specification; therefore, developing an understanding of the fine-tuning mechanisms of BMP signaling in cranial NCCs is critical. We and others have reported that mutant mice with GOF or LOF of BMP type1A receptor (Bmpr1a) in NCCs display deformities in the craniofacial region (Komatsu et al., 2013; Li et al., 2013; Gu et al., 2014). In addition to that, we have recently reported that when ACVR1 (caAcvr1), another BMP type 1 receptor, is constitutively activated in NCCs of transgenic mice (caAcvr1; P0-Cre mice, hereafter), these formed ectopic cartilage in their face (Yang et al., 2021). Thus, we have proposed that enhanced BMP signaling pushes cranial NCCs to the chondrogenic fate at early embryonic stages.

We established the isolation and culture method for cranial NCCs from the first branchial arch (BA1) at an early embryonic stage, day 10.5 embryos (Figures 1, 2). Isolated cranial NCCs from caAcvr1; P0-Cre mice showed greater chondrogenic differentiation capacity than cranial NCCs from control mice (Figure 3), suggesting that the isolated cranial NCCs from BA1 maintain the differentiation potential observed in vivo. This isolation method is an important tool to analyze molecular and cellular mechanisms of craniofacial development.