Jean-Pierre Saint-Jeannet

Background: The development of the vertebrate face is regulated by a spatiotemporally precise, multistep developmental program initiated at the neural plate border by the specification of a unique population of cells, the neural crest. These cells will undergo epithelial‐to‐mesenchymal transition, migration in the pharyngeal arches, and differentiation into the skeletal elements of the face. Results: Evidence from multiple organisms indicates that these processes are evolutionarily conserved and governed by core signaling pathways including BMP, Wnt, FGF, and retinoic acid (RA) that differentially regulate the expression of several families of transcription factors that are the effectors of the craniofacial developmental programs. Conclusions: In this review, we summarize the molecular mechanisms that regulate the different steps in neural crest and vertebrate craniofacial development and discuss the role of RA gradients and homeostasis in this context and the craniofacial pathologies associated with RA dysregulation.

Background: Mandibulofacial dysostosis (MFD) is a congenital disorder characterized by defects in facial bones of neural crest origin. Nager syndrome combines many features of MFD with limb defects. Mutations in SF3B4 , a gene located on chromosome 1 that encodes a protein of the spliceosome, were identified as a cause for Nager syndrome in approximately 60% of patients. Methods: A region of chromosome 9 (9q32) that contains 35 genes has also been linked to Nager syndrome and may account for some affected individuals for which the causes of the disease have not been identified. Because Nager syndrome belongs to a rapidly growing list of craniofacial syndromes caused by pathogenic variants of splicing factors, we focused our attention on two genes in the 9q32 region that encode factors involved in pre‐mRNA processing, PRPF4 and PTBP3 , and analyzed their role in craniofacial development in Xenopus embryos. Results: Loss‐of‐function experiments in Xenopus laevis embryos indicate that Ptbp3 is required while Prpf4 is dispensable for neural crest gene expression at the neurula stage, a phenotype that is partially rescued by expression of human PTBP3. At the tadpole stage, Ptbp3‐depleted embryos have severely hypoplastic craniofacial cartilages, phenocopying the defects observed in Sf3b4 morphant tadpoles. Furthermore, ptbp3 expression is significantly increased in Xenopus tropicalis sf3b4 Null embryos as compared to wild‐type siblings. Conclusion: We propose that dysregulation of PTBP3 expression may cause Nager syndrome in a subset of patients who do not have a mutation in SF3B4 .

Background: In vitro modeling is a powerful approach to investigate the pathomechanisms driving human congenital conditions. Here, we use human embryonic stem cells (hESCs) to model Nager and Rodriguez syndromes, two craniofacial conditions characterized by hypoplastic neural crest‐derived craniofacial bones, caused by pathogenic variants of SF3B4, a core component of the spliceosome.Results: We observed that siRNA‐mediated knockdown of SF3B4 interferes with the production of hESC‐derived neural crest cells, as seen by a marked reduction in neural crest gene expression. This phenotype is associated with an increase in neural crest cell apoptosis and premature neuronal differentiation.Conclusions: Altogether, these results point to a role of SF3B4 in neural crest cell survival, maintenance, and differentiation. We propose that the dysregulation of these processes may contribute to Nager/Rodriguez syndrome‐associated craniofacial defects.

Nager and Rodriguez syndromes are rare craniofacial and limb disorders characterized by midface retrusion, micrognathia, absent thumbs and radial hypoplasia. These disorders result from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4), a component of the pre-mRNA spliceosomal machinery. Although the spliceosome is present and functions in all cells of the body, most spliceosomopathies – including Nager and Rodriguez syndromes – are cell- or tissue-specific in their pathology. To understand the pathomechanism underlying these conditions, we generated a Xenopus tropicalis sf3b4 mutant line using CRISPR/Cas9 gene-editing technology. Homozygous deletion of sf3b4 is detrimental to the development of cranial neural crest (NC)-derived cartilage progenitors. Temporal RNA-sequencing analyses of mutant embryos identified an increase in exon-skipping events, followed by important transcriptional changes associated with an enrichment for terms consistent with defects in NC cell migration and survival. We propose that disruption of these processes may underly the pathogenesis of Nager and Rodriguez syndromes.

Natriuretic peptides and their receptors have been implicated in a broad range of physiological processes, regulating blood pressure, cardiac hypertrophy and fibrosis, fat metabolism, and long bone growth. They mediate their action through the modulation of intracellular levels of cGMP and cAMP. Here we report a completely novel function for natriuretic peptide signaling in the control of cell fate in the embryonic ectoderm. Using a combination of morpholino-based knockdowns, pharmacological inhibitors and rescue assays in Xenopus embryos we show that natriuretic peptide receptor 3 (Npr3), natriuretic peptide receptor 1 (Npr1) and the natriuretic peptide Nppc cooperate in the regulation of neural crest and cranial placode formation, two cell populations that make a major contribution to the craniofacial skeleton and paired sensory organs in vertebrates. Npr3 plays an especially pivotal role in this process by differentially regulating these two developmental programs. Our findings suggest that Npr3 has a dual function in the embryonic ectoderm, it (i) reduces ligand-mediated activation of Npr1 to generate cGMP levels compatible with neural crestformation, and (ii) modulates cAMP levels via inhibition of adenylyl cyclase to promote cranial placode fate. Therefore the intracellular modulation of these two-second messengers participates in the segregation of these cell populations during embryogenesis.

Nager syndrome is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4) a component of the pre-mRNA spliceosomal machinery. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, most spliceosomopathies – including Nager syndrome – are cell/tissue-specific in their pathology. In Nager syndrome patients, it is the neural crest (NC)-derived craniofacial skeletal structures that are primarily affected. To understand the pathomechanism underlying this condition, we generated a Xenopus tropicalis sf3b4 mutant line using the CRISPR/Cas9 gene editing technology. Here we describe the sf3b4 mutant phenotype at neurula, tail bud, and tadpole stages, and performed temporal RNA-sequencing analysis to characterize the splicing events and transcriptional changes underlying this phenotype. Our data show that while loss of one copy of sf3b4 is largely inconsequential in Xenopus tropicalis , homozygous deletion of sf3b4 causes major splicing defects and massive gene dysregulation, which disrupt cranial NC cell migration and survival, thereby pointing at an essential role of Sf3b4 in craniofacial development.

In vitro modeling is a powerful approach to investigate the pathomechanisms driving human congenital conditions. Here we use human embryonic stem cells (hESCs) to model Nager and Rodriguez syndromes, two craniofacial conditions characterized by hypoplastic neural crest-derived craniofacial bones, caused by pathogenic variants of SF3B4, a core component of the spliceosome. We observed that siRNA-mediated knockdown of SF3B4 interferes with the production of hESC-derived neural crest cells, as seen by a marked reduction in neural crest gene expression. This phenotype is associated with an increase in neural crest cell apoptosis and premature neuronal differentiation. Altogether these results point at a role of SF3B4 in neural crest cell survival, maintenance, and differentiation. We propose that the dysregulation of these processes may contribute to Nager/Rodriguez syndrome associated craniofacial defects.