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

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.

Download Full-text

Mis-splicing of Mdm2 leads to Increased P53-Activity and Craniofacial Defects in a MFDM Eftud2 Mutant Mouse Model

10.1101/2020.09.22.308205 ◽

2020 ◽

Author(s):

Marie-Claude Beauchamp ◽

Anissa Djedid ◽

Eric Bareke ◽

Fjodor Merkuri ◽

Rachel Aber ◽

...

Keyword(s):

Cell Death ◽

Neural Crest ◽

Target Genes ◽

Mutant Mouse ◽

Neural Crest Cell ◽

Exon Skipping ◽

Homozygous Deletion ◽

Craniofacial Defects ◽

Retained Introns ◽

Mandibulofacial Dysostosis

SummaryEFTUD2, a GTPase and core component of the splicesome, 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 leads to neural crest cell death and malformations in the brain and craniofacial region of embryos. RNAseq analysis of embryonic mutant heads revealed a significant increase in exon skipping, in retained introns and enriched levels of Mdm2 transcripts lacking exon 3. Mutants also had increased nuclear P53, higher expression of P53-target genes, and increased cell death. Their craniofacial development was significantly improved when treated with Pifithrin-α, an inihibitor of P53. We propose that craniofacial defects caused by mutations of EFTUD2 are a result of mis-splicing of Mdm2 and P53-associated cell death. Hence, drugs that reduce P53 activity may help prevent craniofacial defects associated with spliceosomopathies.

Download Full-text

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.

Download Full-text

MicroRNA-135a Protects Against Ethanol-Induced Apoptosis in Neural Crest Cells and Craniofacial Defects in Zebrafish by Modulating the Siah1/p38/p53 Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.583959 ◽

2020 ◽

Vol 8 ◽

Author(s):

Fuqiang Yuan ◽

Yang Yun ◽

Huadong Fan ◽

Yihong Li ◽

Lanhai Lu ◽

...

Keyword(s):

Neural Crest ◽

Neural Crest Cells ◽

P53 Pathway ◽

Induced Apoptosis ◽

Craniofacial Defects

Download Full-text

E-cigarette aerosol exposure can cause craniofacial defects in Xenopus laevis embryos and mammalian neural crest cells

PLoS ONE ◽

10.1371/journal.pone.0185729 ◽

2017 ◽

Vol 12 (9) ◽

pp. e0185729 ◽

Cited By ~ 17

Author(s):

Allyson E. Kennedy ◽

Suraj Kandalam ◽

Rene Olivares-Navarrete ◽

Amanda J. G. Dickinson

Keyword(s):

Xenopus Laevis ◽

Neural Crest ◽

Neural Crest Cells ◽

Aerosol Exposure ◽

Craniofacial Defects ◽

Xenopus Laevis Embryos

Download Full-text

Med23 Regulates Sox9 Expression during Craniofacial Development

Journal of Dental Research ◽

10.1177/0022034520969109 ◽

2020 ◽

pp. 002203452096910

Author(s):

S. Dash ◽

S. Bhatt ◽

K.T. Falcon ◽

L.L. Sandell ◽

P.A. Trainor

Keyword(s):

Cleft Palate ◽

Neural Crest ◽

Messenger Rna ◽

Target Genes ◽

Genetic Diagnosis ◽

Neural Crest Cell ◽

Neural Crest Cells ◽

Craniofacial Anomalies ◽

Sox9 Expression

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.

Download Full-text

MAPRE2 mutations result in altered human cranial neural crest migration, underlying craniofacial malformations in CSC-KT syndrome

Scientific Reports ◽

10.1038/s41598-021-83771-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Cedric Thues ◽

Jorge S. Valadas ◽

Liesbeth Deaulmerie ◽

Ann Geens ◽

Amit K. Chouhan ◽

...

Keyword(s):

Neural Crest ◽

Induced Pluripotent Stem Cell ◽

Neural Crest Cells ◽

Branchial Arch ◽

Cellular Migration ◽

Cranial Neural Crest ◽

Craniofacial Malformations ◽

Neural Crest Migration

AbstractCircumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies.

Download Full-text

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling

PLoS Genetics ◽

10.1371/journal.pgen.1009579 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009579

Author(s):

Mary E. Swartz ◽

C. Ben Lovely ◽

Johann K. Eberhart

Keyword(s):

Neural Crest ◽

Phenotypic Variability ◽

Choanal Atresia ◽

Neural Crest Cells ◽

Bmp Signaling ◽

Loss Of Function ◽

Shh Signaling ◽

Shh Pathway ◽

Transgenic Embryos ◽

Craniofacial Defects

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant’s phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Download Full-text

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

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.73.1 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Samantha Brugmann ◽

Kelsey H. Elliott ◽

Xiaoting Chen ◽

Joseph Salomone ◽

Praneet Chaturvedi ◽

...

Keyword(s):

Neural Crest ◽

Target Genes ◽

Neural Crest Cells

Download Full-text

Early Craniofacial Defects in Zebrafish that Have Reduced Function of a Wnt-Interacting Extracellular Matrix Protein, Tinagl1

The Cleft Palate-Craniofacial Journal ◽

10.1597/15-283 ◽

2017 ◽

Vol 54 (4) ◽

pp. 381-390 ◽

Cited By ~ 9

Author(s):

Hannah Neiswender ◽

Sammy Navarre ◽

David J. Kozlowski ◽

Ellen K. Lemosy

Keyword(s):

Neural Crest ◽

Cleft Lip ◽

Matrix Protein ◽

Bone Development ◽

Neural Crest Cells ◽

Extracellular Matrix Protein ◽

Cranial Neural Crest ◽

Pharyngeal Arches ◽

Cranial Neural Crest Cells ◽

Craniofacial Defects

Objective Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. Results Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. Conclusions These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.

Download Full-text