Limits...
The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Gα12/13 and RhoA.

Ackerman SD, Garcia C, Piao X, Gutmann DH, Monk KR - Nat Commun (2015)

Bottom Line: In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants.This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential.Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by Gα12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

No MeSH data available.


Related in: MedlinePlus

gpr56 mutant spinal cord axons are hypomyelinated(a) Schematic representation of a 5 dpf zebrafish. Larvae were cut between segments 5 and 6 (red dashed line) and prepared for TEM. Axis shows orientation of the embryo (D, dorsal; V, ventral; A, anterior; P, posterior) (b) Diagram of a 5 dpf zebrafish cross-section, dorsal is up (D), ventral is down (V). In this image, the spinal cord is in orange and includes neuronal cell bodies (blue) and myelinated axons (green). Ventral region used for quantification is boxed in green, dorsal region used for quantification boxed in magenta. Muscle in purple. (c-k) Representative TEM images from the ventral spinal cord of WT (c-eN=6), gpr56stl13/stl13 mutant larvae (f-h, N=5) and gpr56stl14/stl14 mutant larvae (i-k, N=4) at 5 dpf. Higher magnifications of c, f, and i are shown in d-e, g-h, and j-k, respectively. (c-d, f-g, i-j) Myelinated axons are shaded green, unmyelinated large caliber axons (≥ 500 nm) are shaded orange. (e, h, k) Images from panels d, g, and j without pseudocolor. (l) Quantification of the percent of myelinated axons in the ventral spinal cord of gpr56stl13/stl13 (p<.019) and gpr56stl14/stl14 (p<.039) compared to WT controls. (m) Quantification of the total number of axonsin the ventral spinal cord of gpr56stl13/stl13 (p<.5) and gpr56stl14/stl14 (p<.78) compared to WT controls. (n-o) We also did not observe a significant difference in the number of myelin wraps per myelinated axon in mutants compared to control on the large caliber Mauthner axon (m) or on smaller caliber myelinated axons. (c,f,i) Scale bar, 1 µm. (d-e, g-h, j-k) Scale bar, 500 nm. (l-o) Quantification performed on a stereotyped 14 µm2 region (b) in the ventral spinal cord. Student’s t-test used to test for statistical significance and error bars shown as ± s.d. NS, not significant. Data represents two technical replicates.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4302765&req=5

Figure 3: gpr56 mutant spinal cord axons are hypomyelinated(a) Schematic representation of a 5 dpf zebrafish. Larvae were cut between segments 5 and 6 (red dashed line) and prepared for TEM. Axis shows orientation of the embryo (D, dorsal; V, ventral; A, anterior; P, posterior) (b) Diagram of a 5 dpf zebrafish cross-section, dorsal is up (D), ventral is down (V). In this image, the spinal cord is in orange and includes neuronal cell bodies (blue) and myelinated axons (green). Ventral region used for quantification is boxed in green, dorsal region used for quantification boxed in magenta. Muscle in purple. (c-k) Representative TEM images from the ventral spinal cord of WT (c-eN=6), gpr56stl13/stl13 mutant larvae (f-h, N=5) and gpr56stl14/stl14 mutant larvae (i-k, N=4) at 5 dpf. Higher magnifications of c, f, and i are shown in d-e, g-h, and j-k, respectively. (c-d, f-g, i-j) Myelinated axons are shaded green, unmyelinated large caliber axons (≥ 500 nm) are shaded orange. (e, h, k) Images from panels d, g, and j without pseudocolor. (l) Quantification of the percent of myelinated axons in the ventral spinal cord of gpr56stl13/stl13 (p<.019) and gpr56stl14/stl14 (p<.039) compared to WT controls. (m) Quantification of the total number of axonsin the ventral spinal cord of gpr56stl13/stl13 (p<.5) and gpr56stl14/stl14 (p<.78) compared to WT controls. (n-o) We also did not observe a significant difference in the number of myelin wraps per myelinated axon in mutants compared to control on the large caliber Mauthner axon (m) or on smaller caliber myelinated axons. (c,f,i) Scale bar, 1 µm. (d-e, g-h, j-k) Scale bar, 500 nm. (l-o) Quantification performed on a stereotyped 14 µm2 region (b) in the ventral spinal cord. Student’s t-test used to test for statistical significance and error bars shown as ± s.d. NS, not significant. Data represents two technical replicates.

Mentions: To further delineate how impaired Gpr56 function affects oligodendrocyte myelination, we performed transmission electron microscopy (TEM) to measure the extent and quality of myelin present in the ventral spinal cord of gpr56 mutants (gpr56stl13/stl13: N=5, gpr56stl14/stl14: N=4, WT: N=6) during development (Fig. 3a–k). We found a significant reduction (gpr56stl13/stl13, p<.019; gpr56stl14/stl14, p<.039, Student’s t-test) in the percent of myelinated axons in mutant larvae compared to WT controls at 5 dpf (Fig. 3l), but total axon number was unchanged (gpr56stl13/stl13, p<.5; gpr56stl14/stl14, p<.78, Student’s t-test Fig. 3m). We also did not observe a change in the number of myelin wraps surrounding those axons that were myelinated in gpr56stl13/stl13 mutants compared to controls (Fig. 3n–o; non-Mauthner: gpr56stl13/stl13: p<.29, gpr56stk14/stl14: p<.6; Mauthner: gpr56stl13/stl13: p<.25, gpr56stk14/stl14: p<.76, Student’s t-test). In contrast to the hypomyelination phenotype shown in the ventral spinal cord of gpr56 mutants at 5 dpf (Fig. 3f–k), we did not observe any effect on oligodendrocyte myelination in the dorsal spinal cord at this developmental stage (Supplementary Fig. 5). By 21 dpf, however, we observed a statistically significant decrease in the percent of myelinated axons in the ventral (p<.007, Student’s t-test) and dorsal (p<.002) spinal cord of gpr56stl13/stl13 (N=4) mutants relative to WT siblings (N=2), with no change in axon number (Supplementary Fig. 6, dorsal spinal cord: p<.89, ventral spinal spinal: p<.96, Student’s t-test). We suspect that we were unable to detect a difference in the dorsal spinal cord at larval stages because so few axons are myelinated in this region at 5 dpf. Interestingly, we also observed many oligodendrocytes with distended endoplasmic reticula in gpr56stl13/stl13 mutants at 21 dpf, which was rarely seen in controls, supporting the hypothesis that the protein encoded by gpr56stl13/stl13 does not traffic properly from the ER to the plasma membrane (Supplementary Fig. 6). To determine if myelination is simply delayed in gpr56stl13/stl13 mutants, we analyzed myelin ultrastructure in the spinal cord of gpr56stl13/stl13 animals at 6 months of age (Supplementary Fig. 7). At 6 months, we likewise found a statistically significant decrease in the percent of myelinated axons in gpr56stl13/stl13 mutants (N=4) compared to WT controls (N=3) in both the ventral (Supplementary Fig. 7g, axon diameter of .2–.5 µm (p<.021), .5–1 µm (p<.005), and 1–2 µm (p<.029), Student’s t-test) and dorsal spinal cord (Supplementary Fig. 7n, axon diameter of .2−.5 µm (p<.003) and .5–1 µm (p<.008), Student’s t-test), without any change in myelin thickness for axons that were myelinated (Supplementary Fig. 7h,o, linear regression analysis). Axon number was also unchanged in mutants compared to controls (Supplementary Fig. 7f,m, dorsal spinal cord: p<.11, ventral spinal cord: p<.34, Student’s t-test). Collectively, our analysis demonstrates that altered Gpr56 function causes developmental CNS hypomyelination that persists in the adult spinal cord.


The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Gα12/13 and RhoA.

Ackerman SD, Garcia C, Piao X, Gutmann DH, Monk KR - Nat Commun (2015)

gpr56 mutant spinal cord axons are hypomyelinated(a) Schematic representation of a 5 dpf zebrafish. Larvae were cut between segments 5 and 6 (red dashed line) and prepared for TEM. Axis shows orientation of the embryo (D, dorsal; V, ventral; A, anterior; P, posterior) (b) Diagram of a 5 dpf zebrafish cross-section, dorsal is up (D), ventral is down (V). In this image, the spinal cord is in orange and includes neuronal cell bodies (blue) and myelinated axons (green). Ventral region used for quantification is boxed in green, dorsal region used for quantification boxed in magenta. Muscle in purple. (c-k) Representative TEM images from the ventral spinal cord of WT (c-eN=6), gpr56stl13/stl13 mutant larvae (f-h, N=5) and gpr56stl14/stl14 mutant larvae (i-k, N=4) at 5 dpf. Higher magnifications of c, f, and i are shown in d-e, g-h, and j-k, respectively. (c-d, f-g, i-j) Myelinated axons are shaded green, unmyelinated large caliber axons (≥ 500 nm) are shaded orange. (e, h, k) Images from panels d, g, and j without pseudocolor. (l) Quantification of the percent of myelinated axons in the ventral spinal cord of gpr56stl13/stl13 (p<.019) and gpr56stl14/stl14 (p<.039) compared to WT controls. (m) Quantification of the total number of axonsin the ventral spinal cord of gpr56stl13/stl13 (p<.5) and gpr56stl14/stl14 (p<.78) compared to WT controls. (n-o) We also did not observe a significant difference in the number of myelin wraps per myelinated axon in mutants compared to control on the large caliber Mauthner axon (m) or on smaller caliber myelinated axons. (c,f,i) Scale bar, 1 µm. (d-e, g-h, j-k) Scale bar, 500 nm. (l-o) Quantification performed on a stereotyped 14 µm2 region (b) in the ventral spinal cord. Student’s t-test used to test for statistical significance and error bars shown as ± s.d. NS, not significant. Data represents two technical replicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4302765&req=5

Figure 3: gpr56 mutant spinal cord axons are hypomyelinated(a) Schematic representation of a 5 dpf zebrafish. Larvae were cut between segments 5 and 6 (red dashed line) and prepared for TEM. Axis shows orientation of the embryo (D, dorsal; V, ventral; A, anterior; P, posterior) (b) Diagram of a 5 dpf zebrafish cross-section, dorsal is up (D), ventral is down (V). In this image, the spinal cord is in orange and includes neuronal cell bodies (blue) and myelinated axons (green). Ventral region used for quantification is boxed in green, dorsal region used for quantification boxed in magenta. Muscle in purple. (c-k) Representative TEM images from the ventral spinal cord of WT (c-eN=6), gpr56stl13/stl13 mutant larvae (f-h, N=5) and gpr56stl14/stl14 mutant larvae (i-k, N=4) at 5 dpf. Higher magnifications of c, f, and i are shown in d-e, g-h, and j-k, respectively. (c-d, f-g, i-j) Myelinated axons are shaded green, unmyelinated large caliber axons (≥ 500 nm) are shaded orange. (e, h, k) Images from panels d, g, and j without pseudocolor. (l) Quantification of the percent of myelinated axons in the ventral spinal cord of gpr56stl13/stl13 (p<.019) and gpr56stl14/stl14 (p<.039) compared to WT controls. (m) Quantification of the total number of axonsin the ventral spinal cord of gpr56stl13/stl13 (p<.5) and gpr56stl14/stl14 (p<.78) compared to WT controls. (n-o) We also did not observe a significant difference in the number of myelin wraps per myelinated axon in mutants compared to control on the large caliber Mauthner axon (m) or on smaller caliber myelinated axons. (c,f,i) Scale bar, 1 µm. (d-e, g-h, j-k) Scale bar, 500 nm. (l-o) Quantification performed on a stereotyped 14 µm2 region (b) in the ventral spinal cord. Student’s t-test used to test for statistical significance and error bars shown as ± s.d. NS, not significant. Data represents two technical replicates.
Mentions: To further delineate how impaired Gpr56 function affects oligodendrocyte myelination, we performed transmission electron microscopy (TEM) to measure the extent and quality of myelin present in the ventral spinal cord of gpr56 mutants (gpr56stl13/stl13: N=5, gpr56stl14/stl14: N=4, WT: N=6) during development (Fig. 3a–k). We found a significant reduction (gpr56stl13/stl13, p<.019; gpr56stl14/stl14, p<.039, Student’s t-test) in the percent of myelinated axons in mutant larvae compared to WT controls at 5 dpf (Fig. 3l), but total axon number was unchanged (gpr56stl13/stl13, p<.5; gpr56stl14/stl14, p<.78, Student’s t-test Fig. 3m). We also did not observe a change in the number of myelin wraps surrounding those axons that were myelinated in gpr56stl13/stl13 mutants compared to controls (Fig. 3n–o; non-Mauthner: gpr56stl13/stl13: p<.29, gpr56stk14/stl14: p<.6; Mauthner: gpr56stl13/stl13: p<.25, gpr56stk14/stl14: p<.76, Student’s t-test). In contrast to the hypomyelination phenotype shown in the ventral spinal cord of gpr56 mutants at 5 dpf (Fig. 3f–k), we did not observe any effect on oligodendrocyte myelination in the dorsal spinal cord at this developmental stage (Supplementary Fig. 5). By 21 dpf, however, we observed a statistically significant decrease in the percent of myelinated axons in the ventral (p<.007, Student’s t-test) and dorsal (p<.002) spinal cord of gpr56stl13/stl13 (N=4) mutants relative to WT siblings (N=2), with no change in axon number (Supplementary Fig. 6, dorsal spinal cord: p<.89, ventral spinal spinal: p<.96, Student’s t-test). We suspect that we were unable to detect a difference in the dorsal spinal cord at larval stages because so few axons are myelinated in this region at 5 dpf. Interestingly, we also observed many oligodendrocytes with distended endoplasmic reticula in gpr56stl13/stl13 mutants at 21 dpf, which was rarely seen in controls, supporting the hypothesis that the protein encoded by gpr56stl13/stl13 does not traffic properly from the ER to the plasma membrane (Supplementary Fig. 6). To determine if myelination is simply delayed in gpr56stl13/stl13 mutants, we analyzed myelin ultrastructure in the spinal cord of gpr56stl13/stl13 animals at 6 months of age (Supplementary Fig. 7). At 6 months, we likewise found a statistically significant decrease in the percent of myelinated axons in gpr56stl13/stl13 mutants (N=4) compared to WT controls (N=3) in both the ventral (Supplementary Fig. 7g, axon diameter of .2–.5 µm (p<.021), .5–1 µm (p<.005), and 1–2 µm (p<.029), Student’s t-test) and dorsal spinal cord (Supplementary Fig. 7n, axon diameter of .2−.5 µm (p<.003) and .5–1 µm (p<.008), Student’s t-test), without any change in myelin thickness for axons that were myelinated (Supplementary Fig. 7h,o, linear regression analysis). Axon number was also unchanged in mutants compared to controls (Supplementary Fig. 7f,m, dorsal spinal cord: p<.11, ventral spinal cord: p<.34, Student’s t-test). Collectively, our analysis demonstrates that altered Gpr56 function causes developmental CNS hypomyelination that persists in the adult spinal cord.

Bottom Line: In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants.This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential.Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by Gα12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

No MeSH data available.


Related in: MedlinePlus