Functional Study of <i>hoxa1a</i> Regulating Craniofacial Bone Development in Zebrafish

doi:10.13523/j.cb.2105054

China Biotechnology

2021, Vol. 41

Issue (9): 20-26 DOI: 10.13523/j.cb.2105054

Functional Study of hoxa1a Regulating Craniofacial Bone Development in Zebrafish

WU Xiu-zhi^1,^2,³,WANG Hong-jie^1,^2,³,ZU Yao^1,^2,^3,^**(

)

1 Key Laboratory of Exploration and Utilization of Aquatic Germplasm Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China
2 International Joint Research Center for Marine Biosciences, Shanghai Ocean University, Shanghai 201306, China
3 National Aquatic Animal Pathogen Bank, Shanghai Ocean University, Shanghai 201306, China

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Abstract

hox genes encode a family of highly conserved transcription factors. Human HOXA1 mutation causes ABDS (athabascan brainstem dysgenesis syndrome), which leads to craniofacial bone deformity induced facial defect and paralysis. In this paper, zebrafish was used to study the functional mechanism of the homologous gene hoxa1a. Firstly, hoxa1a gene was edited by using CRISPR/Cas9 technology, which resulted in gene mutation. The T7E1 assay showed F₀ digestion efficiency was 70% on average. Then F₁ was screened and it was found that hoxa1a heterozygote generated 8 bp insertion and 7 bp deletion. Furthermore, the heterozygotes were crossed and hoxa1a homozygous F₂ mutant was obtained, which was confirmed by sequencing. At 5 dpf, homozygous mutants of hoxa1a showed craniofacial dysplasia. The results of alcian blue cartilage staining and alizarin red hard bone staining demonstrated that the hoxa1a mutant had abnormal skull development, fracture of ethmoid plate, and defect of gill arch development. In this study, ABDS disease model in zebrafish was successfully constructed and the results indicate that hoxa1a mutation might cause abnormal craniofacial skeletal development, which lays a foundation for its mechanism study and provides a new idea for the pathogenesis of human ABDS disease.

Key words： hoxa1a gene ABDS Craniofacial skeletal development CRISPR/Cas9

Received: 29 May 2021 Published: 30 September 2021

ZTFLH:

Q812

Corresponding Authors: Yao ZU E-mail: yzu@shou.edu.cn

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Cite this article:

WU Xiu-zhi,WANG Hong-jie,ZU Yao. Functional Study of hoxa1a Regulating Craniofacial Bone Development in Zebrafish. China Biotechnology, 2021, 41(9): 20-26.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2105054 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I9/20

Fig.1 Using CRISPR/Cas9 system to edit zebrafish hoxa1a gene (a) CRISPR/Cas9 knockout pattern (b) Knockout efficiency of F₀ was 70% on average (c) Screening for F₁ zebrafish with inherited mutation using T7E1 (d) F₁ heterozygous sequence alignment, screening +8 bp heterozygous and -7 bp heterozygous

Fig.2 A homozygous hoxa1a^-/- zebrafish with abnormal facial development was successfully established (a) Comparison of sequencing peak maps of wildtype, hoxa1a^+/- and hoxa1a^-/- zebrafish. The red arrows indicate the position at which the mutation starts, and the red boxes indicate the inserted bases (b) Phenotype observations showed that the wildtype and heterozygous mutant had normal faces, while the homozygous mutant faces skeletal were abnormal at 5 dpf. As shown by the red arrow, Scale bar: 100 μm

Fig.3 Amino acids sequence and protein structure of hoxa1a mutation

Fig.4 Alizarin red staining of adult hoxa1a^-/- zebrafish showed defective skull development and rupture of ethmoid plate (a),(b) Alizarin red staining of wildtype at 3 months showed normal ethmoid plate (c),(d) Alizarin red staining of homozygous hoxa1a mutant at 3 months showed rupture of ethmoid plate, as shown by the red arrow (e),(f) Alizarin red staining of wildtype 1 year old wildtype zebrafish showed normal ethmoid plates (g),(h) Alizarin Red Staining of homozygous hoxa1a mutant zebrafish of 1 year old indicated a rupture of the ethmoid plate, as shown by the red arrow. And the hoxa1a^-/- had abnormal mouth shape, as shown by the green arrow. a, c, e, g scale bar: 0.5 mm; b, d, f, h scale bar: 1 mm

Fig.5 The loss of hoxa1a resulted in defective facial cartilage development and ethmoid plate rupture in juvenile zebrafish (a),(b) The results of cartilage staining of wildtype juvenile zebrafish at 1 month showed normal ethmoid plate and normal development of pharyngeal arch (c),(d) The cartilage staining results of hoxa1a^-/- at 1 month showed that the ethmoid plate was broken, as shown by the black arrow. The gill arch was lightly stained and was abnormal development, as shown by the red arrow. There was also a loss of cartilage at the anterior end of the first pharyngeal arch, as shown by the green arrow. Scale bar: 0.5 mm


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