Cranial neural crest cells (NCCs) are the origin of the anterior part of the face and the head. Cranial NCCs are multipotent cells giving rise to bones, cartilage, adipose-tissues in the face, and neural cells, melanocytes, and others. The behavior of cranial NCCs (proliferation, cell death, migration, differentiation, and cell fate specification) are well regulated by several signaling pathways; abnormalities in their behavior are often reported as causative reasons for craniofacial anomalies (CFAs), which occur in 1 in 100 newborns in the United States. Understanding the pathological mechanisms of CFAs would facilitate strategies for identifying, preventing, and treating CFAs. Bone morphogenetic protein (BMP) signaling plays a pleiotropic role in many cellular processes during embryonic development. We and others have reported that abnormalities in BMP signaling in cranial NCCs develop CFAs in mice. Abnormal levels of BMP signaling cause miscorrelation with other signaling pathways such as Wnt signaling and FGF signaling, which mutations in the signaling pathways are known to develop CFAs in mice and humans. Recent Genome-Wide Association Studies and exome sequencing demonstrated that some patients with CFAs presented single nucleotide polymorphisms (SNPs), missense mutations, and duplication of genes related to BMP signaling activities, suggesting that defects in abnormal BMP signaling in human embryos develop CFAs. There are still a few cases of BMP-related patients with CFAs. One speculation is that human embryos with mutations in coding regions of BMP-related genes undergo embryonic lethality before developing the craniofacial region as well as mice development; however, no reports are available that show embryonic lethality caused by BMP mutations in humans. In this review, we will summarize the recent advances in the understanding of BMP signaling during craniofacial development in mice and describe how we can translate the knowledge from the transgenic mice to CFAs in humans.

Craniofacial anomalies, especially midline facial defects, are among the most common birth defects in patients and are associated with increased mortality or require lifelong treatment. During mammalian embryogenesis, specific instructions arising at genetic, signaling, and metabolic levels are important for stem cell behaviors and fate determination, but how these functionally relevant mechanisms are coordinated to regulate craniofacial morphogenesis remain unknown. Here, we report that bone morphogenetic protein (BMP) signaling in cranial neural crest cells (CNCCs) is critical for glycolytic lactate production and subsequent epigenetic histone lactylation, thereby dictating craniofacial morphogenesis. Elevated BMP signaling in CNCCs through constitutively activated ACVR1 (ca-ACVR1) suppressed glycolytic activity and blocked lactate production via a p53-dependent process that resulted in severe midline facial defects. By modulating epigenetic remodeling, BMP signaling–dependent lactate generation drove histone lactylation levels to alter essential genes of Pdgfra , thus regulating CNCC behavior in vitro as well as in vivo. These findings define an axis wherein BMP signaling controls a metabolic/epigenetic cascade to direct craniofacial morphogenesis, thus providing a conceptual framework for understanding the interaction between genetic and metabolic cues operative during embryonic development. These findings indicate potential preventive strategies of congenital craniofacial birth defects via modulating metabolic-driven histone lactylation.

The temporomandibular joint (TMJ) is unique in both developmental origin and functional maintenance. The role of bone morphogenic protein (BMP) signaling in endochondral ossification has been widely investigated but not in the context of the TMJ. We employed a histomorphometric analysis approach to understand how augmented BMP signaling in the cranial neural crest affects the postnatal development of the TMJ. Our analysis showed that cartilage length in the mandibular condyle was reduced in Wnt1 Cre;caBmpr1a mice before the weaning stage (P17). However, following weaning, the mandibular condylar cartilage showed recovered length (P28 and P42). Furthermore, the changes in cartilage length coincide with alterations in cell death in the superficial region of the mandibular condyle. These results suggest that BMP signaling influences chondrocyte cell death and TMJ development in a timepoint-specific manner.

X chromosome inactivation (X-inactivation) is generally regarded as a dosage-compensation mechanism restricted to female mammals. Here we show that BMP signaling induces X-inactivation through upregulation of Xist and promotes chondrogenesis in both sexes. Remarkably, augmented BMP signaling induced ectopic X-inactivation: transiently inactivating both X chromosomes in females and one in males in a tissue-specific manner. In cranial neural crest cells, ectopic X-inactivation suppressed X-linked gene Tmsb4x, leading to ectopic cartilage formation. Genetic reduction of Xist or pharmacological restoration of the Tmsb4x product suppressed this phenotype. Moreover, we identified ectopic X-inactivation in SOX9-positive chondroprogenitors during wild-type forelimb development in both sexes. Inhibition of X-inactivation disrupts proximodistal limb patterning ex vivo. These findings establish X-inactivation as a signaling-dependent developmental mechanism linking chromosome-scale gene alterations to skeletal fate specification.

Analysis of Diamond Blackfan anemia syndrome (DBAS) cohorts and animal models has not revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we used an established Rpl5Skax23-Jus/+ murine DBAS model to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of patients with DBAS bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at embryonic day 12.5 (E12.5), we demonstrate downregulation of erythroid differentiation pathways consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in Scd1 expression in the subset of E12.5 mutant embryos at risk for erythroid failure. Stearoyl–coenzyme A desaturase 1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult Rpl5Skax23-Jus/+ mice, mutant mice exhibited delayed erythroid recovery, whereas pretreatment with an stearoyl–coenzyme A desaturase 1 inhibitor resulted in improved erythropoiesis in both wild-type and mutant mice. This analysis suggests a potential role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that requires further study as a potential target for drug development.