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Updated: Mar 19, 2026
Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons
Published on: April 18, 2017
Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by
Huang-Wei Lien1, Rey-Yue Yuan2, Chih-Ming Chou3
1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan.
Abstract:
Cyclins play a central role in cell-cycle regulation; in mammals, the D family of cyclins consists of cyclin D1, D2, and D3. In Xenopus, only homologs of cyclins D1 and D2 have been reported, while a novel cyclin, cyclin Dx (ccndx), was found to be required for the maintenance of motor neuron progenitors during embryogenesis. It remains unknown whether zebrafish possess cyclin D3 or cyclin Dx. In this study, we identified a zebrafish ccndx gene encoding a protein which can form a complex with Cdk4. Through whole-mount in situ hybridization, we observed that zccndx mRNA is expressed in the motor neurons of hindbrain and spinal cord during development. Analysis of a 4-kb promoter sequence of the zccndx gene revealed the presence of HRE sites, which can be regulated by HIF2α. Morpholino knockdown of zebrafish Hif2α and cyclin Dx resulted in the abolishment of isl1 and oligo2 expression in the precursors of motor neurons, and also disrupted axon growth. Overexpression of cyclin Dx mRNA in Hif2α morphants partially rescued zccndx expression. Taken together, our data indicate that zebrafish cyclin Dx plays a role in maintaining the precursors of motor neurons.
Insights
Zebrafish cyclin Dx (ccndx) is crucial for motor neuron development, maintaining progenitor cells and supporting axon growth. Its regulation involves HIF2α, highlighting a conserved pathway in vertebrate neurogenesis.
Area of Science:
- Developmental Biology
- Molecular Biology
- Neuroscience
Background:
- Cyclins regulate the cell cycle; the D family includes D1, D2, and D3 in mammals.
- Xenopus has cyclin D1/D2 homologs, and a novel cyclin Dx (ccndx) essential for motor neuron progenitor maintenance.
- The presence of cyclin D3 and cyclin Dx in zebrafish was previously unknown.
Purpose of the Study:
- To identify and characterize the zebrafish cyclin Dx (ccndx) gene.
- To investigate the role of zebrafish cyclin Dx in motor neuron development.
- To explore the regulatory relationship between zebrafish cyclin Dx and HIF2α.
Main Methods:
- Identification of the zebrafish ccndx gene and characterization of its protein product.
- Whole-mount in situ hybridization to determine zccndx mRNA expression patterns.
- Promoter sequence analysis to identify hypoxia-response elements (HREs).
- Morpholino-based knockdown of Hif2α and cyclin Dx, followed by analysis of motor neuron markers and axon growth.
- Rescue experiments involving cyclin Dx mRNA overexpression in Hif2α morphants.
Main Results:
- A zebrafish ccndx gene was identified, encoding a protein that complexes with Cdk4.
- zccndx mRNA expression was observed in developing motor neurons of the hindbrain and spinal cord.
- The zccndx promoter contains HIF2α-regulated HRE sites.
- Knockdown of Hif2α or cyclin Dx abolished isl1 and oligo2 expression and disrupted axon growth in motor neuron precursors.
- Overexpression of cyclin Dx partially rescued zccndx expression in Hif2α morphants.
Conclusions:
- Zebrafish cyclin Dx (ccndx) plays a significant role in maintaining motor neuron precursors.
- The study reveals a functional link between zebrafish cyclin Dx and HIF2α in motor neuron development.
- These findings contribute to understanding the conserved mechanisms of neurogenesis in vertebrates.
