Hand2 Functions to Synergistically Activate Gli Target Genes in Mandibular Neural Crest Cells

The etiology and pathogenesis of craniofacial birth defects are multifactorial and include both genetic and environmental factors. Despite the identification of numerous genes associated with congenital craniofacial anomalies, our understanding of their etiology remains incomplete, and many affected individuals have an unknown genetic diagnosis. Here, we show that conditional loss of a Mediator complex subunit protein, Med23 in mouse neural crest cells ( Med23 fx/fx; Wnt1-Cre), results in micrognathia, glossoptosis, and cleft palate, mimicking the phenotype of Pierre Robin sequence. Sox9 messenger RNA and protein levels are both upregulated in neural crest cell–derived mesenchyme surrounding Meckel’s cartilage and in the palatal shelves in Med23 fx/fx; Wnt1-Cre mutant embryos compared to controls. Consistent with these observations, we demonstrate that Med23 binds to the promoter region of Sox9 and represses Sox9 expression in vitro. Interestingly, Sox9 binding to β-catenin is enhanced in Med23 fx/fx; Wnt1-Cre mutant embryos, which, together with downregulation of Col2a1 and Wnt signaling target genes, results in decreased proliferation and altered jaw skeletal differentiation and cleft palate. Altogether, our data support a cell-autonomous requirement for Med23 in neural crest cells, potentially linking the global transcription machinery through Med23 to the etiology and pathogenesis of craniofacial anomalies such as micrognathia and cleft palate.

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Mutation in Eftud2 causes craniofacial defects in mice via mis-splicing of Mdm2 and increased P53

Human Molecular Genetics ◽

10.1093/hmg/ddab051 ◽

2021 ◽

Author(s):

Marie-Claude Beauchamp ◽

Anissa Djedid ◽

Eric Bareke ◽

Fjodor Merkuri ◽

Rachel Aber ◽

...

Keyword(s):

Neural Crest ◽

Target Genes ◽

Exon Skipping ◽

Neural Crest Cells ◽

Homozygous Deletion ◽

Craniofacial Malformations ◽

P53 Pathway ◽

Alternatively Spliced ◽

Craniofacial Defects ◽

Mandibulofacial Dysostosis

Abstract EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and increased levels of an alternatively spliced Mdm2 transcript lacking exon 3. Exon skipping in Mdm2 was also increased in O9-1 mouse neural crest cells after siRNA knock-down of Eftud2 and in MFDM patient cells. Moreover, we found increased nuclear P53, higher expression of P53-target genes and increased cell death. Finally, overactivation of the P53 pathway in Eftud2 knockdown cells was attenuated by overexpression of non-spliced Mdm2, and craniofacial development was improved when Eftud2-mutant embryos were treated with Pifithrin-α, an inhibitor of P53. Thus, our work indicates that the P53-pathway can be targeted to prevent craniofacial abnormalities and shows a previously unknown role for alternative splicing of Mdm2 in the etiology of MFDM.

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TWIST1 interacts with adherens junction proteins during neural tube formation and regulates fate transition in cranial neural crest cells

10.1101/2021.08.22.457283 ◽

2021 ◽

Author(s):

Jessica W Bertol ◽

Shelby Johnston ◽

Rabia Ahmed ◽

Victoria K Xie ◽

Lissette Cruz ◽

...

Keyword(s):

Neural Crest ◽

Cell Fate ◽

Neural Tube ◽

Target Genes ◽

Structural Integrity ◽

Neural Crest Cells ◽

Neural Tube Closure ◽

Cranial Neural Crest ◽

Mesenchymal Transition ◽

Cranial Neural Crest Cells

Cell fate determination is a necessary and tightly regulated process for producing different cell types and structures during development. Cranial neural crest cells (CNCCs) are unique to vertebrate embryos and emerge from the neural fold borders into multiple cell lineages that differentiate into bone, cartilage, neurons, and glial cells. We previously reported that Irf6 genetically interacts with Twist1 during CNCC-derived tissue formation. Here, we investigated the mechanistic role of Twist1 and Irf6 at early stages of craniofacial development. Our data indicates that TWIST1 interacts with a/b/g-CATENINS during neural tube closure, and Irf6 is involved in the structural integrity of the neural tube. Twist1 suppresses Irf6 and other epithelial genes in CNCCs during epithelial-to-mesenchymal transition (EMT) process and cell migration. Conversely, a loss of Twist1 leads to a sustained expression of epithelial and cell adhesion markers in migratory CNCCs. Disruption of TWIST1 phosphorylation in vivo leads to epidermal blebbing, edema, neural tube defects, and CNCC-derived structural abnormalities. Altogether, this study describes an uncharacterized function of Twist1 and Irf6 in the neural tube and CNCCs and provides new target genes of Twist1 involved in cytoskeletal remodeling. Furthermore, the association between DNA variations within TWIST1 putative enhancers and human facial morphology is also investigated.

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Snrpb, the gene mutated in CCMS, is haploinsufficient and required in neural crest cells for proper splicing of developmentally important genes essential for craniofacial morphogenesis

10.1101/2021.08.07.455514 ◽

2021 ◽

Author(s):

Sabrina Shameen Alam ◽

Shruti Kumar ◽

Marie-Claude Beauchamp ◽

Eric Bareke ◽

Alexia Boucher ◽

...

Keyword(s):

Neural Crest ◽

Target Genes ◽

Intron Retention ◽

Exon Skipping ◽

Neural Crest Cells ◽

Heterozygous Deletion ◽

Craniofacial Malformations ◽

Pharyngeal Arches ◽

The Face ◽

Morphological Defects

Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). However, the underlying pathophysiology of CCMS remains a mystery. We generated mouse embryos with heterozygous deletion of Snrpb and showed that they arrest shortly after implantation. We also showed that heterozygous deletion of Snrpb in the developing brain and neural crest cells models many of the craniofacial malformations found in CCMS, and results in death shortly after birth. Abnormalities in these mutant embryos ranged from cleft palate to a complete absence of the ventral portion of the face and are due to apoptosis of the neural crest cells in the frontonasal prominence and pharyngeal arches. RNAseq analysis of mutant embryonic heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5' splice strength. Mutant embryonic heads had increased exon-skipping in Mdm2 and Mdm4 negative regulators of the P53-pathway and a increased nuclear P53 and P53-target genes. However, removing one or both copies of P53 in Snrpb heterozygous mutant neural crest cells did not rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations.

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hReg-CNCC reconstructs a regulatory network in human cranial neural crest cells and annotates variants in a developmental context

Communications Biology ◽

10.1038/s42003-021-01970-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Zhanying Feng ◽

Zhana Duren ◽

Ziyi Xiong ◽

Sijia Wang ◽

Fan Liu ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Neural Crest ◽

Genetic Variants ◽

Regulatory Network ◽

Target Genes ◽

Neural Crest Cells ◽

Molecular Networks ◽

Cranial Neural Crest ◽

Cranial Neural Crest Cells

AbstractCranial Neural Crest Cells (CNCC) originate at the cephalic region from forebrain, midbrain and hindbrain, migrate into the developing craniofacial region, and subsequently differentiate into multiple cell types. The entire specification, delamination, migration, and differentiation process is highly regulated and abnormalities during this craniofacial development cause birth defects. To better understand the molecular networks underlying CNCC, we integrate paired gene expression & chromatin accessibility data and reconstruct the genome-wide human Regulatory network of CNCC (hReg-CNCC). Consensus optimization predicts high-quality regulations and reveals the architecture of upstream, core, and downstream transcription factors that are associated with functions of neural plate border, specification, and migration. hReg-CNCC allows us to annotate genetic variants of human facial GWAS and disease traits with associated cis-regulatory modules, transcription factors, and target genes. For example, we reveal the distal and combinatorial regulation of multiple SNPs to core TF ALX1 and associations to facial distances and cranial rare disease. In addition, hReg-CNCC connects the DNA sequence differences in evolution, such as ultra-conserved elements and human accelerated regions, with gene expression and phenotype. hReg-CNCC provides a valuable resource to interpret genetic variants as early as gastrulation during embryonic development. The network resources are available at https://github.com/AMSSwanglab/hReg-CNCC.

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