Craniofacial defects in mice lacking BMP type I receptor Alk2 in neural crest cells

Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.

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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

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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.

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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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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.

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Augmented BMP signaling commits cranial neural crest cells to a chondrogenic fate by suppressing autophagic β-catenin degradation

Science Signaling ◽

10.1126/scisignal.aaz9368 ◽

2021 ◽

Vol 14 (665) ◽

pp. eaaz9368

Author(s):

Jingwen Yang ◽

Megumi Kitami ◽

Haichun Pan ◽

Masako Toda Nakamura ◽

Honghao Zhang ◽

...

Keyword(s):

Neural Crest ◽

Neural Crest Cells ◽

Bmp Signaling ◽

Fibrodysplasia Ossificans Progressiva ◽

Type I ◽

Cranial Neural Crest ◽

Connective Tissues ◽

Multipotent Stem Cells ◽

Morphogenetic Protein ◽

Cranial Neural Crest Cells

Cranial neural crest cells (CNCCs) are a population of multipotent stem cells that give rise to craniofacial bone and cartilage during development. Bone morphogenetic protein (BMP) signaling and autophagy have been individually implicated in stem cell homeostasis. Mutations that cause constitutive activation of the BMP type I receptor ACVR1 cause the congenital disorder fibrodysplasia ossificans progressiva (FOP), which is characterized by ectopic cartilage and bone in connective tissues in the trunk and sometimes includes ectopic craniofacial bones. Here, we showed that enhanced BMP signaling through the constitutively activated ACVR1 (ca-ACVR1) in CNCCs in mice induced ectopic cartilage formation in the craniofacial region through an autophagy-dependent mechanism. Enhanced BMP signaling suppressed autophagy by activating mTORC1, thus blocking the autophagic degradation of β-catenin, which, in turn, caused CNCCs to adopt a chondrogenic identity. Transient blockade of mTORC1, reactivation of autophagy, or suppression of Wnt–β-catenin signaling reduced ectopic cartilages in ca-Acvr1 mutants. Our results suggest that BMP signaling and autophagy coordinately regulate β-catenin activity to direct the fate of CNCCs during craniofacial development. These findings may also explain why some patients with FOP develop ectopic bones through endochondral ossification in craniofacial regions.

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Neural crest cells require Meis2 for patterning the mandibular arch via the Sonic hedgehog pathway

Biology Open ◽

10.1242/bio.052043 ◽

2020 ◽

Vol 9 (6) ◽

pp. bio052043

Author(s):

Jaroslav Fabik ◽

Katarina Kovacova ◽

Zbynek Kozmik ◽

Ondrej Machon

Keyword(s):

Neural Crest ◽

Sonic Hedgehog ◽

Neural Crest Cells ◽

Ectopic Ossification ◽

Pharyngeal Arches ◽

Altered Expression ◽

Neural Crest Origin ◽

Cranial Neural Crest Cells ◽

Craniofacial Defects ◽

Molecular Signals

ABSTRACTCranial neural crest cells (cNCCs) originate in the anterior neural tube and populate pharyngeal arches in which they contribute to formation of bone and cartilage. This cell population also provides molecular signals for the development of tissues of non-neural crest origin, such as the tongue muscles, teeth enamel or gland epithelium. Here we show that the transcription factor Meis2 is expressed in the oral region of the first pharyngeal arch (PA1) and later in the tongue primordium. Conditional inactivation of Meis2 in cNCCs resulted in loss of Sonic hedgehog signalling in the oropharyngeal epithelium and impaired patterning of PA1 along the lateral–medial and oral–aboral axis. Failure of molecular specification of PA1, illustrated by altered expression of Hand1/2, Dlx5, Barx1, Gsc and other markers, led to hypoplastic tongue and ectopic ossification of the mandible. Meis2-mutant mice thus display craniofacial defects that are reminiscent of several human syndromes and patients with mutations in the Meis2 gene.

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