This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Morphological, behavioral and molecular studies on CRISPR/Cas9-mediated knockout of the NOMO1 gene in zebrafish
Qiang Li
Children's Hospital of Fudan University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8388-0968
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Multiple clinical genome-wide analysis identified that chromosome 16p13.11 is a hotspot associated with neuropsychiatric disorders such as autism, schizophrenia and epilepsy. Nodal modulator 1 (NOMO1), located on human chromosome 16p13.11, was considered as a candidate gene with neuropsychiatric disorders. However, it is unknown whether the nomo1 deficiency causes neurological abnormalities, and the molecular mechanisms and pathogenesis of the NOMO1 gene remain unclear. To study the effects of nomo1 deficiency on brain development and neuropsychiatric system, a nomo1 knockout zebrafish model was established.
Methods: We developed a viable vertebrate model of nomo1 loss-of-function using CRISPR/Cas9 technology and characterized nomo1 mutant zebrafish. Phenotypic and functional studies of developing nomo1 mutant zebrafish, including morphological measurements, behavioral assays, and functional mechanistic analyses, were performed.
Results: Morphological differences in the phenotype of nomo1-/- zebrafish gradually became less noticeable during development, however, the enlarged interstitial spaces in midbrain and hindbrain were detected in nomo1 mutant zebrafish. Meanwhile, the nomo1 deficiency caused the change of expression levels in neurotransmitters of γ-aminobutyrate, glutamate and serotonin. Interestingly, the nomo1 loss-of-function zebrafish model exhibited social defects and repetitive behaviors in juvenile, which represented autism-like behaviors. The transcriptome analysis showed different gene expression patterns in mutant zebrafish at the genetic level. Further results revealed that the neuroactive drug PTZ recovered the decreased locomotor phenotype in larval mutant zebrafish.
Conclusions: In this study, we established a nomo1 vertebrate animal model using CRISPR/Cas9 gene editing approach. The loss-of-function of nomo1 displayed autism-like behaviors and altered levels of the γ-aminobutyrate, glutamate and serotonin in zebrafish, which provide evidence that nomo1 as a candidate gene for autism. The versatility of zebrafish model is contributed to studying NOMO1-related disorders and conducting drug screening in future.
Limitations: Further studies are needed to determine whether an intervention with a neuroactive drug in nomo1-/- zebrafish to alter the behavioral phenotype is applicable to the behavior of human patients.
Keywords
Nomo1, CRISPR/Cas9, Zebrafish, Autism, Social behavior, Neurotransmitter, Functional mechanism
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Table.S1Primersareusedinthisstudy.docx
Table. S1. The primer sequences are used in this study.
- Figure.S2Theforebrainmarkersonmutantzebrafish.jpg
Fig. S2. The forebrain marker expression and DA levels were no different between WT and mutant zebrafish. (A) Expression of neurological genes in nomo1-/- mutant zebrafish at 48 hpf determined by RT-qPCR. Data are shown as the mean ± SEM. (B-E’) Mutant zebrafish at 48 hpf were analyzed by WISH to detect the site of related gene expression. Scale bar: 100 μm.
- Figure.S3Thekinpreferencebehavior.jpg
Fig. S3. There is no difference in kin recognition behavior among WT and nomo1-/- zebrafish. (A) Schematic diagram of the kin recognition experiment. (B-C) The ratio of distance moved/time spent in the kin preference region in nomo1-/- zebrafish was not different from that determined for WT zebrafish (N = 15 for each group). Data are presented as mean ± SEM.
- Table.S2ThelistedDEGsandpvalues.xlsx
- Figure.S1Morphologicalanalysisofnomo1zebrafish.jpg
- Video.S1WTsocialpreferencebehavior.mp4
- Video.S3WTshoalingbehavior.mp4
- Video.S2nomo1socialpreferencebehavior.mp4
- Video.S5backandforthmotions.mp4
- Video.S4nomo1shoalingbehavior.mp4
- Video.S6stereotypicmovement.mp4
- Video.S7largecircularmovement.mp4
- Video.S8WTkinpreferencebehavior.mp4
- Video.S9nomo1kinpreferencebehavior.mp4
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 2
Posted 07 Jan, 2021
No community comments so far
No comments provided
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
Learn more about our company.
This is a preprint. It has not completed peer review.
Research
Morphological, behavioral and molecular studies on CRISPR/Cas9-mediated knockout of the NOMO1 gene in zebrafish
Qiang Li
Children's Hospital of Fudan University
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8388-0968
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 2
Posted 07 Jan, 2021
No community comments so far
No comments provided
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Multiple clinical genome-wide analysis identified that chromosome 16p13.11 is a hotspot associated with neuropsychiatric disorders such as autism, schizophrenia and epilepsy. Nodal modulator 1 (NOMO1), located on human chromosome 16p13.11, was considered as a candidate gene with neuropsychiatric disorders. However, it is unknown whether the nomo1 deficiency causes neurological abnormalities, and the molecular mechanisms and pathogenesis of the NOMO1 gene remain unclear. To study the effects of nomo1 deficiency on brain development and neuropsychiatric system, a nomo1 knockout zebrafish model was established.
Methods: We developed a viable vertebrate model of nomo1 loss-of-function using CRISPR/Cas9 technology and characterized nomo1 mutant zebrafish. Phenotypic and functional studies of developing nomo1 mutant zebrafish, including morphological measurements, behavioral assays, and functional mechanistic analyses, were performed.
Results: Morphological differences in the phenotype of nomo1-/- zebrafish gradually became less noticeable during development, however, the enlarged interstitial spaces in midbrain and hindbrain were detected in nomo1 mutant zebrafish. Meanwhile, the nomo1 deficiency caused the change of expression levels in neurotransmitters of γ-aminobutyrate, glutamate and serotonin. Interestingly, the nomo1 loss-of-function zebrafish model exhibited social defects and repetitive behaviors in juvenile, which represented autism-like behaviors. The transcriptome analysis showed different gene expression patterns in mutant zebrafish at the genetic level. Further results revealed that the neuroactive drug PTZ recovered the decreased locomotor phenotype in larval mutant zebrafish.
Conclusions: In this study, we established a nomo1 vertebrate animal model using CRISPR/Cas9 gene editing approach. The loss-of-function of nomo1 displayed autism-like behaviors and altered levels of the γ-aminobutyrate, glutamate and serotonin in zebrafish, which provide evidence that nomo1 as a candidate gene for autism. The versatility of zebrafish model is contributed to studying NOMO1-related disorders and conducting drug screening in future.
Limitations: Further studies are needed to determine whether an intervention with a neuroactive drug in nomo1-/- zebrafish to alter the behavioral phenotype is applicable to the behavior of human patients.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7