Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice

Beverdam, Annemiek; Brouwer, Antje; Reijnen, Mark; Korving, Jeroen; Meijlink, Frits

doi:10.1242/dev.128.20.3975

Cited by 152 publications

(25 citation statements)

References 32 publications

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“…Notably, mutations of some TF genes of Md-set have not yet been associated with jawbone disorders in humans. These synopses appear to contradict not only the RNA-seq, qPCR, and IF data for OB and raw jaw/tibia bones reported here but also the described mouse jawbone phenotypes for various mutations, such as Alx3 and Alx4 (Beverdam et al 2001), Dlx4 (Wu et al 2015), Msx1 (Nassif et al 2014), and Msx2 (Aïoub et al 2007). These observations support that jawbone abnormalities might also be present but not diagnosed in human disorders and highlight the need for systematic exploration of the jawbones while investigating skeletal and dental diseases.…”

Section: Discussioncontrasting

confidence: 87%

Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology

et al. 2022

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Craniofacial and jaw bones have unique physiological specificities when compared to axial and appendicular bones. However, the molecular profile of the jaw osteoblast (OB) remains incomplete. The present study aimed to decipher the bone site-specific profiles of transcription factors (TFs) expressed in OBs in vivo. Using RNA sequencing analysis, we mapped the transcriptome of confirmed OBs from 2 different skeletal sites: mandible (Md) and tibia (Tb). The OB transcriptome contains 709 TF genes: 608 are similarly expressed in Md-OB and Tb-OB, referred to as “OB-core”; 54 TF genes are upregulated in Md-OB, referred to as “Md-set”; and 18 TF genes are upregulated in Tb-OB, referred to as “Tb-set.” Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Md-set and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest vs. mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders, and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

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Section: Discussioncontrasting

confidence: 87%

Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology

et al. 2022

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“…In mouse and zebrafish, Alx1, Alx3, and Alx4 have been shown to be expressed in spatiotemporally restricted regions of the craniofacial mesenchyme (Zhao et al , ; Qu et al , ; ten Berge et al , ; Beverdam & Meijlink, ; Dee et al , ; Lours‐Calet et al , ). Evidence of gene compensation has previously been reported in animal studies (Beverdam et al , ; Dee et al , ). In studies of sea urchins, Alx4 was shown to be directly regulated by Alx1 (Rafiq et al , ; Khor et al , ).…”

Section: Discussionmentioning

confidence: 68%

ALX 1‐ related frontonasal dysplasia results from defective neural crest cell development and migration

Pini

Kueper

et al. 2020

EMBO Mol Med

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A pedigree of subjects presented with frontonasal dysplasia ( FND ). Genome sequencing and analysis identified a p.L165F missense variant in the homeodomain of the transcription factor ALX 1 which was imputed to be pathogenic. Induced pluripotent stem cells ( iPSC ) were derived from the subjects and differentiated to neural crest cells ( NCC ). NCC derived from ALX 1 L165F/L165F iPSC were more sensitive to apoptosis, showed an elevated expression of several neural crest progenitor state markers, and exhibited impaired migration compared to wild‐type controls. NCC migration was evaluated in vivo using lineage tracing in a zebrafish model, which revealed defective migration of the anterior NCC stream that contributes to the median portion of the anterior neurocranium, phenocopying the clinical presentation. Analysis of human NCC culture media revealed a change in the level of bone morphogenic proteins ( BMP ), with a low level of BMP 2 and a high level of BMP 9. Soluble BMP 2 and BMP 9 antagonist treatments were able to rescue the defective migration phenotype. Taken together, these results demonstrate a mechanistic requirement of ALX 1 in NCC development and migration.

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“…Species-specific differences in alx1 gene functions may also reflect divergence in genetic compensation strategies employed by these organisms. In mouse, alx4 compensates for loss of alx3 since alx3;alx4 double mutants develop with severe orofacial clefts and single mutants do not ( Beverdam et al, 2001 ; Lakhwani et al, 2010 ). In zebrafish, single alx4a and alx4b mutants develop normally, while alx3 mutants exhibit medial ANC defects ( Mitchell et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…In humans, ALX1 mutations are linked to severe frontonasal dysplasia (FND) and extreme microphthalmia, and ALX3 and ALX4 genes are associated with a phenotypic spectrum of hypertelorism and nasal-tip duplications ( Pini et al, 2020 ; Uz et al, 2010 ). These functions of alx genes are conserved in other vertebrates, but the pathogenic developmental mechanisms leading to the observed craniofacial malformations remain largely unknown ( Beverdam et al, 2001 ; Dee et al, 2013 ; Forsthoefel, 1963 ; Lakhwani et al, 2010 ; Lyons et al, 2016 ; Pini et al, 2020 ; Qu et al, 1999 ; Zhao et al, 1996 ). In mice, disruption of alx1 results in anencephaly, precluding analysis of its role in facial and ocular morphogenesis, while compound mutants of alx3 and alx4 present with FND-like defects ( Beverdam et al, 2001 ; Lakhwani et al, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

Zebrafish models of alx-linked frontonasal dysplasia reveal a role for Alx1 and Alx3 in the anterior segment and vasculature of the developing eye

et al. 2022

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The cellular and genetic mechanisms that coordinate formation of facial sensory structures with surrounding skeletal and soft tissue elements remain poorly understood. Alx1, a homeobox transcription factor, is a key regulator of midfacial morphogenesis. ALX1 mutations in humans are linked to severe congenital anomalies of the facial skeleton (frontonasal dysplasia, FND) with malformation or absence of eyes and orbital contents (micro- and anophthalmia). Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. Here we show that zebrafish alx1; alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity. Collectively, these data demonstrate a conserved role for zebrafish Alx genes in controlling ocular and facial development, and a novel role in protecting these key midfacial structures from ethanol toxicity during embryogenesis. These data also reveal novel roles for Alx genes in ocular anterior segment formation and vascular development, and suggest retinal deficits in Alx mutants may be secondary to aberrant ocular vascularization and anterior segment defects. This study establishes robust zebrafish models for interrogating conserved genetic mechanisms that coordinate facial and ocular development, and for exploring gene-environment interactions relevant to fetal alcohol syndrome.

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Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice

Cited by 152 publications

References 32 publications

Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology

Transcriptional Regulation of Jaw Osteoblasts: Development to Pathology

ALX 1‐ related frontonasal dysplasia results from defective neural crest cell development and migration

Zebrafish models of alx-linked frontonasal dysplasia reveal a role for Alx1 and Alx3 in the anterior segment and vasculature of the developing eye

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