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Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern

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ABSTRACT

Background: The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies.

Methods and results: In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1−/+;En2−/−), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice.

Conclusions: Our results reveal that gc production is differentially regulated in each zone of the cerebellar vermis, and our mutant analysis indicates that the dynamics of gc production plays a role in determining the 3D structure of the Cb.

Electronic supplementary material: The online version of this article (doi:10.1186/s13064-016-0072-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

En1+/−;En2−/− mutant gcp proliferation and differentiation are altered primarily in the anterior and nodular zones. The level of gc proliferation (a) and differentiation (b) were calculated as in Fig. 4c and d, respectively, at P6 and P10 in lobules 3 (AZ: green), 7 (CZ: blue) and 10 (NZ: 10) for En2+/− control and En1+/−;En2−/− mutant mice. p-values of the Dunnet’s post-hoc multiple comparisons tests following ANOVA comparing the levels of proliferation and differentiation between lobules at each time-point are shown when significant (see Additional file 1: Table S5A). c EGL thickness in μm is shown for the same lobules at P6 and P10 (n = 3 mice for each measurement). The outer EGL is represented in colors and the inner EGL in grey. p-values of Tukey’s (for lobule comparisons) and Sidak’s (for genotype comparisons) post-hoc multiple comparisons tests following ANOVA are shown when significant (See Additional file 1: Tables S6)
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Fig8: En1+/−;En2−/− mutant gcp proliferation and differentiation are altered primarily in the anterior and nodular zones. The level of gc proliferation (a) and differentiation (b) were calculated as in Fig. 4c and d, respectively, at P6 and P10 in lobules 3 (AZ: green), 7 (CZ: blue) and 10 (NZ: 10) for En2+/− control and En1+/−;En2−/− mutant mice. p-values of the Dunnet’s post-hoc multiple comparisons tests following ANOVA comparing the levels of proliferation and differentiation between lobules at each time-point are shown when significant (see Additional file 1: Table S5A). c EGL thickness in μm is shown for the same lobules at P6 and P10 (n = 3 mice for each measurement). The outer EGL is represented in colors and the inner EGL in grey. p-values of Tukey’s (for lobule comparisons) and Sidak’s (for genotype comparisons) post-hoc multiple comparisons tests following ANOVA are shown when significant (See Additional file 1: Tables S6)

Mentions: At P2, we did not find significant differences in the S-phase indices between lobules 3, 7 and 10 (Fig. 4c, Additional file 1: Table S2). However, differentiation (quitting fraction) tended to be higher in lobule 3 (41.5 %) compared to 7 (34.1 %), with borderline significance (p = 0.0541, Fig. 4d, Additional file 1: Table S3). These results suggest differentiation rather than proliferation rates might be a more important determinant of the lower production of gcs between P2 and P4 in lobule 7. At P6, the level of differentiation was lowest in lobule 7, especially compared to lobule 10 (Fig. 4d, Additional file 1: Table S3, see also Fig. 8b). The proliferation index of gcps in lobule 7 at P6 was significantly higher than in lobule 10 (p = 0.0458, Fig. 4c, see also Fig. 8a), and similar to lobule 3. At P10 the level of differentiation was significantly higher in lobule 3 compared to 7 (p = 0.0025, see also Fig. 8b). In contrast, the level of proliferation was higher in lobule 7 than in 10 with borderline significance (p = 0.0540) and appeared higher than in 3 (although not significantly) (Fig. 4c, Additional file 1: Table S3, see also Fig. 8a). The results at P10 are consistent with a more active (proliferative) EGL in lobule 7 (CZ) at later time-points compared to lobules 3 and 10, and greater differentiation in lobule 3 (AZ). In addition, when a 30’ pulse of BrdU was given at P14 or P16, many BrdU labeled cells were detected in lobules 6b and 7, whereas almost no EGL cells were present in lobule 3 (Fig. 5) or 10 (not shown). Therefore the EGL remains active at P14-P16 in the lobules of the CZ, whereas the gcps are depleted in lobules of the other zones by P14. This result is consistent with our finding that gc production is almost complete at P14 in lobules of the AZ, PZ and NZ whereas more than 10 % of gcs are produced after P14 in the CZ (Fig. 3).Fig. 5


Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern
En1+/−;En2−/− mutant gcp proliferation and differentiation are altered primarily in the anterior and nodular zones. The level of gc proliferation (a) and differentiation (b) were calculated as in Fig. 4c and d, respectively, at P6 and P10 in lobules 3 (AZ: green), 7 (CZ: blue) and 10 (NZ: 10) for En2+/− control and En1+/−;En2−/− mutant mice. p-values of the Dunnet’s post-hoc multiple comparisons tests following ANOVA comparing the levels of proliferation and differentiation between lobules at each time-point are shown when significant (see Additional file 1: Table S5A). c EGL thickness in μm is shown for the same lobules at P6 and P10 (n = 3 mice for each measurement). The outer EGL is represented in colors and the inner EGL in grey. p-values of Tukey’s (for lobule comparisons) and Sidak’s (for genotype comparisons) post-hoc multiple comparisons tests following ANOVA are shown when significant (See Additional file 1: Tables S6)
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Fig8: En1+/−;En2−/− mutant gcp proliferation and differentiation are altered primarily in the anterior and nodular zones. The level of gc proliferation (a) and differentiation (b) were calculated as in Fig. 4c and d, respectively, at P6 and P10 in lobules 3 (AZ: green), 7 (CZ: blue) and 10 (NZ: 10) for En2+/− control and En1+/−;En2−/− mutant mice. p-values of the Dunnet’s post-hoc multiple comparisons tests following ANOVA comparing the levels of proliferation and differentiation between lobules at each time-point are shown when significant (see Additional file 1: Table S5A). c EGL thickness in μm is shown for the same lobules at P6 and P10 (n = 3 mice for each measurement). The outer EGL is represented in colors and the inner EGL in grey. p-values of Tukey’s (for lobule comparisons) and Sidak’s (for genotype comparisons) post-hoc multiple comparisons tests following ANOVA are shown when significant (See Additional file 1: Tables S6)
Mentions: At P2, we did not find significant differences in the S-phase indices between lobules 3, 7 and 10 (Fig. 4c, Additional file 1: Table S2). However, differentiation (quitting fraction) tended to be higher in lobule 3 (41.5 %) compared to 7 (34.1 %), with borderline significance (p = 0.0541, Fig. 4d, Additional file 1: Table S3). These results suggest differentiation rather than proliferation rates might be a more important determinant of the lower production of gcs between P2 and P4 in lobule 7. At P6, the level of differentiation was lowest in lobule 7, especially compared to lobule 10 (Fig. 4d, Additional file 1: Table S3, see also Fig. 8b). The proliferation index of gcps in lobule 7 at P6 was significantly higher than in lobule 10 (p = 0.0458, Fig. 4c, see also Fig. 8a), and similar to lobule 3. At P10 the level of differentiation was significantly higher in lobule 3 compared to 7 (p = 0.0025, see also Fig. 8b). In contrast, the level of proliferation was higher in lobule 7 than in 10 with borderline significance (p = 0.0540) and appeared higher than in 3 (although not significantly) (Fig. 4c, Additional file 1: Table S3, see also Fig. 8a). The results at P10 are consistent with a more active (proliferative) EGL in lobule 7 (CZ) at later time-points compared to lobules 3 and 10, and greater differentiation in lobule 3 (AZ). In addition, when a 30’ pulse of BrdU was given at P14 or P16, many BrdU labeled cells were detected in lobules 6b and 7, whereas almost no EGL cells were present in lobule 3 (Fig. 5) or 10 (not shown). Therefore the EGL remains active at P14-P16 in the lobules of the CZ, whereas the gcps are depleted in lobules of the other zones by P14. This result is consistent with our finding that gc production is almost complete at P14 in lobules of the AZ, PZ and NZ whereas more than 10 % of gcs are produced after P14 in the CZ (Fig. 3).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: The mouse cerebellum (Cb) has a remarkably complex foliated three-dimensional (3D) structure, but a stereotypical cytoarchitecture and local circuitry. Little is known of the cellular behaviors and genes that function during development to determine the foliation pattern. In the anteroposterior axis the mammalian cerebellum is divided by lobules with distinct sizes, and the foliation pattern differs along the mediolateral axis defining a medial vermis and two lateral hemispheres. In the vermis, lobules are further grouped into four anteroposterior zones (anterior, central, posterior and nodular zones) based on genetic criteria, and each has distinct lobules. Since each cerebellar afferent group projects to particular lobules and zones, it is critical to understand how the 3D structure of the Cb is acquired. During cerebellar development, the production of granule cells (gcs), the most numerous cell type in the brain, is required for foliation. We hypothesized that the timing of gc accumulation is different in the four vermal zones during development and contributes to the distinct lobule morphologies.

Methods and results: In order to test this idea, we used genetic inducible fate mapping to quantify accumulation of gcs in each lobule during the first two postnatal weeks in mice. The timing of gc production was found to be particular to each lobule, and delayed in the central zone lobules relative to the other zones. Quantification of gc proliferation and differentiation at three time-points in lobules representing different zones, revealed the delay involves a later onset of maximum differentiation and prolonged proliferation of gc progenitors in the central zone. Similar experiments in Engrailed mutants (En1−/+;En2−/−), which have a smaller Cb and altered foliation pattern preferentially outside the central zone, showed that gc production, proliferation and differentiation are altered such that the differences between zones are attenuated compared to wild-type mice.

Conclusions: Our results reveal that gc production is differentially regulated in each zone of the cerebellar vermis, and our mutant analysis indicates that the dynamics of gc production plays a role in determining the 3D structure of the Cb.

Electronic supplementary material: The online version of this article (doi:10.1186/s13064-016-0072-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus