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Constitutively expressed Protocadherin-α regulates the coalescence and elimination of homotypic olfactory axons through its cytoplasmic region.

Hasegawa S, Hirabayashi T, Kondo T, Inoue K, Esumi S, Okayama A, Hamada S, Yagi T - Front Mol Neurosci (2012)

Bottom Line: Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity.These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity.The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

View Article: PubMed Central - PubMed

Affiliation: KOKORO-Biology Group and CREST-JST, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University Osaka, Japan.

ABSTRACT
Olfactory sensory neuron (OSN) axons coalesce into specific glomeruli in the olfactory bulb (OB) according to their odorant receptor (OR) expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α) family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

No MeSH data available.


Related in: MedlinePlus

PcdhaΔ(2–c2)/Δ(2–c2) deletion mutant mice. (A) Wild-type Pcdh-α genes consist of variable-region (α1 to α12, αc1 and αc2) and constant-region (CR1–CR3) exons. The individual variable exons are transcribed from their own promoters. A Pcdh-α transcript is produced from one variable exon and three or four constant exons by splicing. In the PcdhaΔ(2–c2)/Δ(2–c2) mice, exons α2–αc2 were deleted, leaving only exon α1 in the variable region. (B) RT-PCR analysis of brain extracts of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) Δ(2–c2/Δ2–c2) mice. (C) qRT-PCR analysis of α1 transcripts in the brain of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. ***P < 0.0001, vs. WT and vs. Pcdha+/Δ(2–c2). Data are shown as the mean ± S.D. (D) Immunoblotting analysis of brain lysates with an anti-Pcdhα CR antibody. (E) Expression of α1 and αCR transcripts in OSNs of the OE were examined by in situ hybridization histochemistry. Constitutive expression of α1 transcripts was seen in the OSNs of PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm. (F) Pcdh-α immunoreactivity with an anti-Pcdhα CR antibody was strong in the OSN axons and glomeruli of both WT (+/+) and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm.
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Figure 6: PcdhaΔ(2–c2)/Δ(2–c2) deletion mutant mice. (A) Wild-type Pcdh-α genes consist of variable-region (α1 to α12, αc1 and αc2) and constant-region (CR1–CR3) exons. The individual variable exons are transcribed from their own promoters. A Pcdh-α transcript is produced from one variable exon and three or four constant exons by splicing. In the PcdhaΔ(2–c2)/Δ(2–c2) mice, exons α2–αc2 were deleted, leaving only exon α1 in the variable region. (B) RT-PCR analysis of brain extracts of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) Δ(2–c2/Δ2–c2) mice. (C) qRT-PCR analysis of α1 transcripts in the brain of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. ***P < 0.0001, vs. WT and vs. Pcdha+/Δ(2–c2). Data are shown as the mean ± S.D. (D) Immunoblotting analysis of brain lysates with an anti-Pcdhα CR antibody. (E) Expression of α1 and αCR transcripts in OSNs of the OE were examined by in situ hybridization histochemistry. Constitutive expression of α1 transcripts was seen in the OSNs of PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm. (F) Pcdh-α immunoreactivity with an anti-Pcdhα CR antibody was strong in the OSN axons and glomeruli of both WT (+/+) and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm.

Mentions: Abnormal glomerular morphology in prenatal and neonatal PcdhaΔCR/ΔCR mice. (A) Ectopic glomerulus-like structures of M71-expressing OSNs were found in neonatal PcdhaΔCR/ΔCR mice by whole-mount observation. X-gal-stained lateral M71 glomeruli in whole-mounted OBs from WT (a, b) or PcdhaΔCR/ΔCR(ΔCR/ΔCR) (c, d) mice at P0. In PcdhaΔCR/ΔCR mice, abnormal axonal projections from the olfactory nerve were often detected (arrowheads). Melanocytes (arrows) were visible on some of the whole-mount preparations of the olfactory bulbs. Scale bars, 500 μm. (B) Sectional analysis of the coalescence of WT (a–f) and PcdhaΔCR2/ΔCR2 (g–l) P2 axons on embryonic day (E) 17.5. Serial sections of OBs were double-labeled with anti-β-galactosidase (for P2, green) and anti-NCAM (red) antibodies. There were more P2 glomerulus-like structures (arrowheads) in the PcdhaΔCR2/ΔCR2 (ΔCR2/ΔCR2) mice (See Figure 3A). Scale bar, 100 μm. (C) OBs in WT (a–c) and PcdhaΔCR/ΔCR mice (g–i) were double-labeled with anti-NCAM (red) and anti-MAP2 (green) antibodies. Due to the orientation shown in panels (d) and (j), in PcdhaΔCR/ΔCR mice, the OSN axons appeared to extend beyond the normal confines of the glomerular layer and often terminated as an intensely stained spatially restricted and condensed structure (j and l, arrows). In addition, the primary axons terminated in less clearly defined glomeruli in PcdhaΔCR/ΔCR (ΔCR/ΔCR) than in WT (+/+) mice. Primary glomerular structures could be detected in WT (d, f, asterisks) but not PcdhaΔCR/ΔCR mice (j, l) (See Figures 6B,C). Immunostaining with anti-MAP2 (green) antibody did not show significant differences between WT (e) and PcdhaΔCR/ΔCR (k) mice. Scale bar, 100 μm.


Constitutively expressed Protocadherin-α regulates the coalescence and elimination of homotypic olfactory axons through its cytoplasmic region.

Hasegawa S, Hirabayashi T, Kondo T, Inoue K, Esumi S, Okayama A, Hamada S, Yagi T - Front Mol Neurosci (2012)

PcdhaΔ(2–c2)/Δ(2–c2) deletion mutant mice. (A) Wild-type Pcdh-α genes consist of variable-region (α1 to α12, αc1 and αc2) and constant-region (CR1–CR3) exons. The individual variable exons are transcribed from their own promoters. A Pcdh-α transcript is produced from one variable exon and three or four constant exons by splicing. In the PcdhaΔ(2–c2)/Δ(2–c2) mice, exons α2–αc2 were deleted, leaving only exon α1 in the variable region. (B) RT-PCR analysis of brain extracts of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) Δ(2–c2/Δ2–c2) mice. (C) qRT-PCR analysis of α1 transcripts in the brain of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. ***P < 0.0001, vs. WT and vs. Pcdha+/Δ(2–c2). Data are shown as the mean ± S.D. (D) Immunoblotting analysis of brain lysates with an anti-Pcdhα CR antibody. (E) Expression of α1 and αCR transcripts in OSNs of the OE were examined by in situ hybridization histochemistry. Constitutive expression of α1 transcripts was seen in the OSNs of PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm. (F) Pcdh-α immunoreactivity with an anti-Pcdhα CR antibody was strong in the OSN axons and glomeruli of both WT (+/+) and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm.
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Figure 6: PcdhaΔ(2–c2)/Δ(2–c2) deletion mutant mice. (A) Wild-type Pcdh-α genes consist of variable-region (α1 to α12, αc1 and αc2) and constant-region (CR1–CR3) exons. The individual variable exons are transcribed from their own promoters. A Pcdh-α transcript is produced from one variable exon and three or four constant exons by splicing. In the PcdhaΔ(2–c2)/Δ(2–c2) mice, exons α2–αc2 were deleted, leaving only exon α1 in the variable region. (B) RT-PCR analysis of brain extracts of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) Δ(2–c2/Δ2–c2) mice. (C) qRT-PCR analysis of α1 transcripts in the brain of WT (+/+), Pcdha+/Δ(2–c2) [+/Δ(2–c2)], and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. ***P < 0.0001, vs. WT and vs. Pcdha+/Δ(2–c2). Data are shown as the mean ± S.D. (D) Immunoblotting analysis of brain lysates with an anti-Pcdhα CR antibody. (E) Expression of α1 and αCR transcripts in OSNs of the OE were examined by in situ hybridization histochemistry. Constitutive expression of α1 transcripts was seen in the OSNs of PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm. (F) Pcdh-α immunoreactivity with an anti-Pcdhα CR antibody was strong in the OSN axons and glomeruli of both WT (+/+) and PcdhaΔ(2–c2)/Δ(2–c2) [Δ(2–c2)/Δ(2–c2)] mice. Scale bar, 100 μm.
Mentions: Abnormal glomerular morphology in prenatal and neonatal PcdhaΔCR/ΔCR mice. (A) Ectopic glomerulus-like structures of M71-expressing OSNs were found in neonatal PcdhaΔCR/ΔCR mice by whole-mount observation. X-gal-stained lateral M71 glomeruli in whole-mounted OBs from WT (a, b) or PcdhaΔCR/ΔCR(ΔCR/ΔCR) (c, d) mice at P0. In PcdhaΔCR/ΔCR mice, abnormal axonal projections from the olfactory nerve were often detected (arrowheads). Melanocytes (arrows) were visible on some of the whole-mount preparations of the olfactory bulbs. Scale bars, 500 μm. (B) Sectional analysis of the coalescence of WT (a–f) and PcdhaΔCR2/ΔCR2 (g–l) P2 axons on embryonic day (E) 17.5. Serial sections of OBs were double-labeled with anti-β-galactosidase (for P2, green) and anti-NCAM (red) antibodies. There were more P2 glomerulus-like structures (arrowheads) in the PcdhaΔCR2/ΔCR2 (ΔCR2/ΔCR2) mice (See Figure 3A). Scale bar, 100 μm. (C) OBs in WT (a–c) and PcdhaΔCR/ΔCR mice (g–i) were double-labeled with anti-NCAM (red) and anti-MAP2 (green) antibodies. Due to the orientation shown in panels (d) and (j), in PcdhaΔCR/ΔCR mice, the OSN axons appeared to extend beyond the normal confines of the glomerular layer and often terminated as an intensely stained spatially restricted and condensed structure (j and l, arrows). In addition, the primary axons terminated in less clearly defined glomeruli in PcdhaΔCR/ΔCR (ΔCR/ΔCR) than in WT (+/+) mice. Primary glomerular structures could be detected in WT (d, f, asterisks) but not PcdhaΔCR/ΔCR mice (j, l) (See Figures 6B,C). Immunostaining with anti-MAP2 (green) antibody did not show significant differences between WT (e) and PcdhaΔCR/ΔCR (k) mice. Scale bar, 100 μm.

Bottom Line: Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity.These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity.The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

View Article: PubMed Central - PubMed

Affiliation: KOKORO-Biology Group and CREST-JST, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University Osaka, Japan.

ABSTRACT
Olfactory sensory neuron (OSN) axons coalesce into specific glomeruli in the olfactory bulb (OB) according to their odorant receptor (OR) expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α) family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α's cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

No MeSH data available.


Related in: MedlinePlus