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Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

Hashimoto Y, Muramatsu K, Kunii M, Yoshimura S, Yamada M, Sato T, Ishida Y, Harada R, Harada A - FASEB J. (2012)

Bottom Line: The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome.The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown.The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

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Related in: MedlinePlus

Morphological features of mutant clones selected using morphology-based screening. Expression of VAChT-EGFP in the parental PC12-VAChT cells (A), mutant clones (B, left panels), and reverted clones (B, right panels). Fluorescence of VAChT-EGFP is diminished (P26), almost absent (P05 and P173), or aggregated in the cell body (P12). Clone P207 has multibranched neurites and intense VAChT-EGFP fluorescence at the tips of these neurites. After Cre/loxP-mediated recombination (B, right panels), these mutant clones became indistinguishable from the parental PC12 cells (PC12-VAChT). Scale bars = 100 μm.
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Figure 3: Morphological features of mutant clones selected using morphology-based screening. Expression of VAChT-EGFP in the parental PC12-VAChT cells (A), mutant clones (B, left panels), and reverted clones (B, right panels). Fluorescence of VAChT-EGFP is diminished (P26), almost absent (P05 and P173), or aggregated in the cell body (P12). Clone P207 has multibranched neurites and intense VAChT-EGFP fluorescence at the tips of these neurites. After Cre/loxP-mediated recombination (B, right panels), these mutant clones became indistinguishable from the parental PC12 cells (PC12-VAChT). Scale bars = 100 μm.

Mentions: The VAChT is expressed on SLMVs in PC12 cells. Thus, for the morphology-based screening, we established a PC12 cell line, PC12-VAChT, which stably expressed VAChT-EGFP in addition to mCAT1 (Fig. 3A). Table 2 shows the summary of these results. We introduced the gene trap retrovirus into the PC12-VAChT cell line and selected 180 colonies using G418 and puromycin. For each clone, we examined the branching of neurites after NGF stimulation as well as the intensity and distribution of VAChT-EGFP using a fluorescence microscope. A total of 24 clones that displayed abnormal morphologies and/or abnormal VAChT distribution were selected for further experiments. The typical clones are shown in the left panels of Fig. 3B. In some cases (clones P05, P26, and P173), the amount of SLMVs was reduced; in other cases (clones P12 and P207), the distribution pattern of SLMVs or the morphology of PC12 was changed.


Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

Hashimoto Y, Muramatsu K, Kunii M, Yoshimura S, Yamada M, Sato T, Ishida Y, Harada R, Harada A - FASEB J. (2012)

Morphological features of mutant clones selected using morphology-based screening. Expression of VAChT-EGFP in the parental PC12-VAChT cells (A), mutant clones (B, left panels), and reverted clones (B, right panels). Fluorescence of VAChT-EGFP is diminished (P26), almost absent (P05 and P173), or aggregated in the cell body (P12). Clone P207 has multibranched neurites and intense VAChT-EGFP fluorescence at the tips of these neurites. After Cre/loxP-mediated recombination (B, right panels), these mutant clones became indistinguishable from the parental PC12 cells (PC12-VAChT). Scale bars = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475256&req=5

Figure 3: Morphological features of mutant clones selected using morphology-based screening. Expression of VAChT-EGFP in the parental PC12-VAChT cells (A), mutant clones (B, left panels), and reverted clones (B, right panels). Fluorescence of VAChT-EGFP is diminished (P26), almost absent (P05 and P173), or aggregated in the cell body (P12). Clone P207 has multibranched neurites and intense VAChT-EGFP fluorescence at the tips of these neurites. After Cre/loxP-mediated recombination (B, right panels), these mutant clones became indistinguishable from the parental PC12 cells (PC12-VAChT). Scale bars = 100 μm.
Mentions: The VAChT is expressed on SLMVs in PC12 cells. Thus, for the morphology-based screening, we established a PC12 cell line, PC12-VAChT, which stably expressed VAChT-EGFP in addition to mCAT1 (Fig. 3A). Table 2 shows the summary of these results. We introduced the gene trap retrovirus into the PC12-VAChT cell line and selected 180 colonies using G418 and puromycin. For each clone, we examined the branching of neurites after NGF stimulation as well as the intensity and distribution of VAChT-EGFP using a fluorescence microscope. A total of 24 clones that displayed abnormal morphologies and/or abnormal VAChT distribution were selected for further experiments. The typical clones are shown in the left panels of Fig. 3B. In some cases (clones P05, P26, and P173), the amount of SLMVs was reduced; in other cases (clones P12 and P207), the distribution pattern of SLMVs or the morphology of PC12 was changed.

Bottom Line: The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome.The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown.The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

ABSTRACT
The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

Show MeSH
Related in: MedlinePlus