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The Gs-linked receptor GPR3 inhibits the proliferation of cerebellar granule cells during postnatal development.

Tanaka S, Shaikh IM, Chiocca EA, Saeki Y - PLoS ONE (2009)

Bottom Line: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation.In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression.These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain.

Methodology/principal findings: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit.

Conclusions/significance: These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

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Increased proliferation of granule precursor cells in the IGL of a GPR3−/− mouse.In panel a, BrdU was administered to P14 GPR3−/− (right panel) or wild type mice (left panel) 4 hours prior to sacrifice. Sections (20 µm) were incubated with a rat anti-BrdU monoclonal antibody and FITC-labelled anti-rat secondary antibody. Fluorescent cells are indicated by arrows in the IGL of GPR3−/− mice but not in wild-type mice. As expected, meningeal cells on cerebellar surface also showed BrdU incorporation. In panel b, parallel sections were stained with an anti-ki67 antibody to detect proliferating cells in IGL. Positive Ki67 cells were visualized in IGL of GPR3−/− vs. wild-type mice. In panel c, merged images of Ki67 positive cells and βtubulin positive are shown in the right subpanel. White arrowheads point to doubly positive cells. Scale bar = 100 µm (panel a,b) and 20 µm (panel c).
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pone-0005922-g004: Increased proliferation of granule precursor cells in the IGL of a GPR3−/− mouse.In panel a, BrdU was administered to P14 GPR3−/− (right panel) or wild type mice (left panel) 4 hours prior to sacrifice. Sections (20 µm) were incubated with a rat anti-BrdU monoclonal antibody and FITC-labelled anti-rat secondary antibody. Fluorescent cells are indicated by arrows in the IGL of GPR3−/− mice but not in wild-type mice. As expected, meningeal cells on cerebellar surface also showed BrdU incorporation. In panel b, parallel sections were stained with an anti-ki67 antibody to detect proliferating cells in IGL. Positive Ki67 cells were visualized in IGL of GPR3−/− vs. wild-type mice. In panel c, merged images of Ki67 positive cells and βtubulin positive are shown in the right subpanel. White arrowheads point to doubly positive cells. Scale bar = 100 µm (panel a,b) and 20 µm (panel c).

Mentions: To determine if GPR3 gene expression was associated with an anti-proliferative effect in the postnatal cerebellum, we utilized the 5-bromo-2′ deoxyuridine (BrdU) pulse chase labeling technique. When BrdU was injected 4 hours prior to the sacrifice of P14 mice, the number of BrdU-positive cells was increased in the IGL of GPR3−/− versus wild-type type (Figure 4a). Quantitatively, this increase was significant in all IGL areas of cerebellum except for 10Cb (Table 1). Interestingly, there was no significant difference in the number of BrdU-positive cells in the molecular layer (ML) or white matter (WM) of the cerebellum between wild type and GPR3−/− mice. Similar results were also obtained using P12 GPR3−/− or wild type mice (data not shown).


The Gs-linked receptor GPR3 inhibits the proliferation of cerebellar granule cells during postnatal development.

Tanaka S, Shaikh IM, Chiocca EA, Saeki Y - PLoS ONE (2009)

Increased proliferation of granule precursor cells in the IGL of a GPR3−/− mouse.In panel a, BrdU was administered to P14 GPR3−/− (right panel) or wild type mice (left panel) 4 hours prior to sacrifice. Sections (20 µm) were incubated with a rat anti-BrdU monoclonal antibody and FITC-labelled anti-rat secondary antibody. Fluorescent cells are indicated by arrows in the IGL of GPR3−/− mice but not in wild-type mice. As expected, meningeal cells on cerebellar surface also showed BrdU incorporation. In panel b, parallel sections were stained with an anti-ki67 antibody to detect proliferating cells in IGL. Positive Ki67 cells were visualized in IGL of GPR3−/− vs. wild-type mice. In panel c, merged images of Ki67 positive cells and βtubulin positive are shown in the right subpanel. White arrowheads point to doubly positive cells. Scale bar = 100 µm (panel a,b) and 20 µm (panel c).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691605&req=5

pone-0005922-g004: Increased proliferation of granule precursor cells in the IGL of a GPR3−/− mouse.In panel a, BrdU was administered to P14 GPR3−/− (right panel) or wild type mice (left panel) 4 hours prior to sacrifice. Sections (20 µm) were incubated with a rat anti-BrdU monoclonal antibody and FITC-labelled anti-rat secondary antibody. Fluorescent cells are indicated by arrows in the IGL of GPR3−/− mice but not in wild-type mice. As expected, meningeal cells on cerebellar surface also showed BrdU incorporation. In panel b, parallel sections were stained with an anti-ki67 antibody to detect proliferating cells in IGL. Positive Ki67 cells were visualized in IGL of GPR3−/− vs. wild-type mice. In panel c, merged images of Ki67 positive cells and βtubulin positive are shown in the right subpanel. White arrowheads point to doubly positive cells. Scale bar = 100 µm (panel a,b) and 20 µm (panel c).
Mentions: To determine if GPR3 gene expression was associated with an anti-proliferative effect in the postnatal cerebellum, we utilized the 5-bromo-2′ deoxyuridine (BrdU) pulse chase labeling technique. When BrdU was injected 4 hours prior to the sacrifice of P14 mice, the number of BrdU-positive cells was increased in the IGL of GPR3−/− versus wild-type type (Figure 4a). Quantitatively, this increase was significant in all IGL areas of cerebellum except for 10Cb (Table 1). Interestingly, there was no significant difference in the number of BrdU-positive cells in the molecular layer (ML) or white matter (WM) of the cerebellum between wild type and GPR3−/− mice. Similar results were also obtained using P12 GPR3−/− or wild type mice (data not shown).

Bottom Line: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation.In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression.These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

View Article: PubMed Central - PubMed

Affiliation: Dardinger Laboratory for Neuro-oncology and Neurosciences, Department of Neurological Surgery, The Ohio State University, Columbus, OH, USA.

ABSTRACT

Background: During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain.

Methodology/principal findings: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit.

Conclusions/significance: These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.

Show MeSH
Related in: MedlinePlus