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

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

Additional file 1: of Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern

Statistical analyses. Table S1A: Statistical analyses of comparisons of gc accumulation in lobules 3, 7 and 10 between 2 time-points. Table S1B: Statistical analyses of comparisons of gc accumulation between lobules 3, 7 and 10 at each time-point. Table S2. Theoretical calculation of stage for maximum gc production. Table S3. Statistical analysis of comparisons of the levels of proliferation and differentiation between lobules at each time-point. Table S4A: Statistical analysis of the comparisons of the levels of gc production between lobules at P6, P10 and P14 in WTs and in En1 -/+ ;En2 -/- mutants (mt). Table S4B: Statistical analysis of the comparisons of the levels of gc production in each lobule between WTs and En1 -/+ ;En2 -/- mutants. Table S4C: Statistical analysis of the comparisons of the ratio between gc production in lobule 7 and other lobules between WTs and En1 -/+ ;En2 -/- mutants. Table S5A: Statistical analysis of the comparisons, at P6 or P10, of the levels of proliferation and differentiation between lobules and between control (ctr) and En1 -/+ ;En2 -/- mutant (mt). Table S5B: Statistical analysis of the comparisons between genotypes of the ratios between lobule 7 and other lobules. Table S6A: Statistical analysis of the comparisons of EGL thickness between lobules and between control (ctr) and En1 -/+ ;En2 -/- mutant (mt) at P6. Table S6B: Statistical analysis of the comparisons of EGL thickness between lobules and between control (ctr) and En1 -/+ ;En2 -/- mutant (mt) at P10. (DOC 113 kb

Additional file 2: of Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern

Figure S1: Rate of accumulation of gcs over time in each lobule. Figure S2: Foliation is greatly delayed and the pattern is highly variable in En1 +/- ;En2 -/- mutants. Figure S3: Model of how changes in fissure formation and production of gcs in the anterior cerebellum can alter the morphology of lobules. (PDF 648 kb

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

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