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M6a is expressed in the murine neural retina and regulates neurite extension.

Zhao J, Iida A, Ouchi Y, Satoh S, Watanabe S - Mol. Vis. (2008)

Bottom Line: M6a is expressed mainly in the nervous system, and its expression and function in mammalian retina have not been described.M6a expression was completely paralleled by that of the synaptic marker, synaptophysin.These results suggest that M6a plays a role in retinal development by regulating neurites, and it may also function to modulate synaptic activities in the adult retina.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: Glycoprotein m6a (M6a) is a cell-surface glycoprotein that belongs to the myelin proteolipid protein family. M6a is expressed mainly in the nervous system, and its expression and function in mammalian retina have not been described. Using proteomics analysis of mouse retinal membrane fractions, we identified M6a as a retinal membrane protein that is strongly expressed at embryonic stages. Our aim was to reveal the function of M6a in development of mouse retina in this work.

Methods: Detailed expression pattern of M6a was examined by immunostaining using frozen sections of mouse retina obtained at various developmental stages. For functional analysis of M6a in mouse retinal development, we performed retorovirus-mediated overexpression of M6a in mouse retinal explant culture. Then, cell differentiation, proliferation and structural maturation of the cells were examined.

Results: M6a transcripts were strongly expressed in embryonic retina. After completion of retinal differentiation, the level of expression decreased as mouse development progressed. Immunohistochemistry showed that in the immature mouse retina, M6a was strongly expressed in the axons of retinal ganglion cells. After birth, M6a expression was confined to the inner plexiform layer, and finally, to the inner and outer plexiform layers of adult mouse retina. M6a expression was completely paralleled by that of the synaptic marker, synaptophysin. Mouse retinal progenitor cells that overexpressed M6a following retrovirus-mediated gene transfer were subjected to in vitro explant or monolayer cultures. The neurite outgrowth of M6a-overexpressing retinal cells was strikingly enhanced, although M6a did not affect differentiation and proliferation.

Conclusions: These results suggest that M6a plays a role in retinal development by regulating neurites, and it may also function to modulate synaptic activities in the adult retina.

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Effects of forced expression of M6a on retinal neurite extension. A,B: Neurite extension of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal monolayer cultures. Retinal explants were infected with retorovirus particles that encode M6a and EGFP, and the cells were dissociated and subjected to monolayer culturing. After 11 days of culture, the cultures were harvested and stained with anti-GFP antibody. Neurite length was examined by measuring the EGFP-positive neurites on photographs taken under a fluorescence microscope. The length distribution percentages for the neurites are shown in A. The average lengths of neurites longer than 10 μm are shown in B. More than 100 cells were counted for each sample, and essentially the same results were obtained in three independent experiments. C,D: Neurite extension of virus-transduced EGFP-positive cells in retinal reaggregation cultures. Morphology of the M6a-EGFP virus-transfected retinal cells in reaggregation culture (C). The percentage of cells with neurite extensions of 30-100 μm or 100-200 μm in the M6a and control EGFP expressed retinal cells (D). Asterisk is p<0.05 by the Student's t-test.
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f3: Effects of forced expression of M6a on retinal neurite extension. A,B: Neurite extension of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal monolayer cultures. Retinal explants were infected with retorovirus particles that encode M6a and EGFP, and the cells were dissociated and subjected to monolayer culturing. After 11 days of culture, the cultures were harvested and stained with anti-GFP antibody. Neurite length was examined by measuring the EGFP-positive neurites on photographs taken under a fluorescence microscope. The length distribution percentages for the neurites are shown in A. The average lengths of neurites longer than 10 μm are shown in B. More than 100 cells were counted for each sample, and essentially the same results were obtained in three independent experiments. C,D: Neurite extension of virus-transduced EGFP-positive cells in retinal reaggregation cultures. Morphology of the M6a-EGFP virus-transfected retinal cells in reaggregation culture (C). The percentage of cells with neurite extensions of 30-100 μm or 100-200 μm in the M6a and control EGFP expressed retinal cells (D). Asterisk is p<0.05 by the Student's t-test.

Mentions: Since M6a has been implicated in neurite extension in brain, we investigated whether M6a was also involved in neurite extension in the retina, using monolayer cultures of retinas infected with retroviruses that encode control EGFP or M6a-IRES-EGFP. Neurite extension in monolayer cultures was examined by measuring the length of neurites from photographs taken under a fluorescence microscope. To distinguish neural cells from glial cells, we stained the retinal cells with antibody against GS, which is a glial cell marker, and evaluated the neurite lengths of the GS-negative and EGFP-positive cells. In both the control and M6a-expressing samples, approximately 60% of the cells extended neurites by 0–10 μm of neurite. However, when we compared the cell population distribution categorized by neurite length, we discovered that M6a-expressing cells extended longer neurites than did control virus-infected cells (Figure 3A). The average neurite lengths were greater than 10 μm for the M6a-overexpressing cells (39.2 μm) and control cells (25.5 μm; Figure 3B). We next confirmed these results with a different method of culture. We prepared reaggregation cultures by mixing dissociated virus-infected retinal explants prepared from E17.5 and dissociated retinal cells isolated from the same brood. Eight days later, percentage of M6a-overexpressing retinal cells bearing neurite over 30 μm length was about twice-times higher than that of control EGFP expressing cells (Figure 3C,D). This indicates that M6a plays a role in promoting the neurite extension during retinal development.


M6a is expressed in the murine neural retina and regulates neurite extension.

Zhao J, Iida A, Ouchi Y, Satoh S, Watanabe S - Mol. Vis. (2008)

Effects of forced expression of M6a on retinal neurite extension. A,B: Neurite extension of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal monolayer cultures. Retinal explants were infected with retorovirus particles that encode M6a and EGFP, and the cells were dissociated and subjected to monolayer culturing. After 11 days of culture, the cultures were harvested and stained with anti-GFP antibody. Neurite length was examined by measuring the EGFP-positive neurites on photographs taken under a fluorescence microscope. The length distribution percentages for the neurites are shown in A. The average lengths of neurites longer than 10 μm are shown in B. More than 100 cells were counted for each sample, and essentially the same results were obtained in three independent experiments. C,D: Neurite extension of virus-transduced EGFP-positive cells in retinal reaggregation cultures. Morphology of the M6a-EGFP virus-transfected retinal cells in reaggregation culture (C). The percentage of cells with neurite extensions of 30-100 μm or 100-200 μm in the M6a and control EGFP expressed retinal cells (D). Asterisk is p<0.05 by the Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2529470&req=5

f3: Effects of forced expression of M6a on retinal neurite extension. A,B: Neurite extension of virus-transduced enhanced green fluorescent protein (EGFP)-positive cells in retinal monolayer cultures. Retinal explants were infected with retorovirus particles that encode M6a and EGFP, and the cells were dissociated and subjected to monolayer culturing. After 11 days of culture, the cultures were harvested and stained with anti-GFP antibody. Neurite length was examined by measuring the EGFP-positive neurites on photographs taken under a fluorescence microscope. The length distribution percentages for the neurites are shown in A. The average lengths of neurites longer than 10 μm are shown in B. More than 100 cells were counted for each sample, and essentially the same results were obtained in three independent experiments. C,D: Neurite extension of virus-transduced EGFP-positive cells in retinal reaggregation cultures. Morphology of the M6a-EGFP virus-transfected retinal cells in reaggregation culture (C). The percentage of cells with neurite extensions of 30-100 μm or 100-200 μm in the M6a and control EGFP expressed retinal cells (D). Asterisk is p<0.05 by the Student's t-test.
Mentions: Since M6a has been implicated in neurite extension in brain, we investigated whether M6a was also involved in neurite extension in the retina, using monolayer cultures of retinas infected with retroviruses that encode control EGFP or M6a-IRES-EGFP. Neurite extension in monolayer cultures was examined by measuring the length of neurites from photographs taken under a fluorescence microscope. To distinguish neural cells from glial cells, we stained the retinal cells with antibody against GS, which is a glial cell marker, and evaluated the neurite lengths of the GS-negative and EGFP-positive cells. In both the control and M6a-expressing samples, approximately 60% of the cells extended neurites by 0–10 μm of neurite. However, when we compared the cell population distribution categorized by neurite length, we discovered that M6a-expressing cells extended longer neurites than did control virus-infected cells (Figure 3A). The average neurite lengths were greater than 10 μm for the M6a-overexpressing cells (39.2 μm) and control cells (25.5 μm; Figure 3B). We next confirmed these results with a different method of culture. We prepared reaggregation cultures by mixing dissociated virus-infected retinal explants prepared from E17.5 and dissociated retinal cells isolated from the same brood. Eight days later, percentage of M6a-overexpressing retinal cells bearing neurite over 30 μm length was about twice-times higher than that of control EGFP expressing cells (Figure 3C,D). This indicates that M6a plays a role in promoting the neurite extension during retinal development.

Bottom Line: M6a is expressed mainly in the nervous system, and its expression and function in mammalian retina have not been described.M6a expression was completely paralleled by that of the synaptic marker, synaptophysin.These results suggest that M6a plays a role in retinal development by regulating neurites, and it may also function to modulate synaptic activities in the adult retina.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

ABSTRACT

Purpose: Glycoprotein m6a (M6a) is a cell-surface glycoprotein that belongs to the myelin proteolipid protein family. M6a is expressed mainly in the nervous system, and its expression and function in mammalian retina have not been described. Using proteomics analysis of mouse retinal membrane fractions, we identified M6a as a retinal membrane protein that is strongly expressed at embryonic stages. Our aim was to reveal the function of M6a in development of mouse retina in this work.

Methods: Detailed expression pattern of M6a was examined by immunostaining using frozen sections of mouse retina obtained at various developmental stages. For functional analysis of M6a in mouse retinal development, we performed retorovirus-mediated overexpression of M6a in mouse retinal explant culture. Then, cell differentiation, proliferation and structural maturation of the cells were examined.

Results: M6a transcripts were strongly expressed in embryonic retina. After completion of retinal differentiation, the level of expression decreased as mouse development progressed. Immunohistochemistry showed that in the immature mouse retina, M6a was strongly expressed in the axons of retinal ganglion cells. After birth, M6a expression was confined to the inner plexiform layer, and finally, to the inner and outer plexiform layers of adult mouse retina. M6a expression was completely paralleled by that of the synaptic marker, synaptophysin. Mouse retinal progenitor cells that overexpressed M6a following retrovirus-mediated gene transfer were subjected to in vitro explant or monolayer cultures. The neurite outgrowth of M6a-overexpressing retinal cells was strikingly enhanced, although M6a did not affect differentiation and proliferation.

Conclusions: These results suggest that M6a plays a role in retinal development by regulating neurites, and it may also function to modulate synaptic activities in the adult retina.

Show MeSH
Related in: MedlinePlus