Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( <i>arhgef10</i>) Causes Locomotor Abnormalities in Zebrafish Larvae.

Yi Zhang; Ming Xing AN; Chen Gong; Yang Yang LI; Yu Tong Wang; Meng Lin; Rong LI; Chan Tian

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Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae.

Author: Yi ZHANG ^{1
,

2
,

3
,

4} ; Ming Xing AN ^{1
,

2
,

3
,

4} ; Chen GONG ^{1
,

2
,

3
,

4} ; Yang Yang LI ^{1
,

2
,

3
,

4} ; Yu Tong WANG ^{1
,

2
,

3
,

4} ; Meng LIN ^{1
,

2
,

3
,

4} ; Rong LI ^{1
,

2
,

3
,

4} ; Chan TIAN ^{1
,

2
,

3
,

5
,

6}
Author Information

1. Department of Obstetrics and Gynecology, Center for Medical Genetics, School of Basic Medical Sciences, Third Hospital, Peking University, Beijing 100191, China
2. Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing 100191, China
3. Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China
4. National Clinical Research Center for Obstetrics and Gynecology, Beijing 100191, China.
5. National Clinical Research Center for Obstetrics and Gynecology, Beijing 100191, China
6. Department of Medical Genetics, Center for Medical Genetics, Peking University Health Science Center, Beijing 100191, China.
Publication Type:Journal Article
Keywords: CRISPR/Cas9; Copy loss; Haploinsufficiency; Zebrafish; arhgef10
MeSH: Animals; Annexin A5; Apoptosis; Blotting, Western; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cell Line; Cell Proliferation; Cells, Cultured; Flow Cytometry; Genotype; Humans; In Situ Hybridization; Larva/physiology*; Phenotype; RNA/isolation & purification*; Real-Time Polymerase Chain Reaction/standards*; Rho Guanine Nucleotide Exchange Factors/metabolism*; Sincalide/analysis*; Spectrophotometry/methods*; Zebrafish/physiology*
From: Biomedical and Environmental Sciences 2022;35(1):35-44
CountryChina
Language:English
Abstract: OBJECTIVE:To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.
METHODS:Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.
RESULTS:WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.
CONCLUSION:Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.