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

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.


The axons of newly produced gcs stack inside to out in the molecular layer. a-b Adjacent sagittal midline sections of a P28 Atoh1-tau Cb after Tm administration at P10 stained for (a) X-gal to visualize the nuclei of the marked cells, (b) GFP fluorescent immunostaining (green) to visualize the axons of the marked granule cells. The anteroposterior zones have been color coded (anterior zone: green, central zone: blue, posterior zone: purple and nodular zone: yellow). c-h sagittal midline sections of lobule 3 of P28 Atoh1-tau Cb stained for GFP to reveal the position of the axons of the marked granule cells after Tm administration at the indicated ages showing that the proportion of molecular layer labeled diminishes as Tm is administered later
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Fig1: The axons of newly produced gcs stack inside to out in the molecular layer. a-b Adjacent sagittal midline sections of a P28 Atoh1-tau Cb after Tm administration at P10 stained for (a) X-gal to visualize the nuclei of the marked cells, (b) GFP fluorescent immunostaining (green) to visualize the axons of the marked granule cells. The anteroposterior zones have been color coded (anterior zone: green, central zone: blue, posterior zone: purple and nodular zone: yellow). c-h sagittal midline sections of lobule 3 of P28 Atoh1-tau Cb stained for GFP to reveal the position of the axons of the marked granule cells after Tm administration at the indicated ages showing that the proportion of molecular layer labeled diminishes as Tm is administered later

Mentions: Tm was injected every two days from P2 to P14 to Atoh1-Tau mice, 1 day for each mouse, and the fate-mapped cells were analyzed at P28. As expected, the cell bodies (detected with X-gal staining) of the marked cells were scattered throughout the IGL (Fig. 1a), whereas the labeled axons (immunostained for GFP) were stacked on top of unlabeled axons defining an outer portion of labeled ML and inner portion of unlabeled ML (Fig. 1b-h). We did indeed observe that the height of the outer ML that was labeled progressively decreased as Tm was administered later in Cb development (Fig. 1c-h). We measured the area of labeled ML as a proportion of the total area of ML in each lobule on three midline sections from animals that received 50 μg/g (n = 1) or 200 μg/g (n = 2) of Tm at P8. As expected, we did not observe a significant difference in the proportion of ML area labeled in each lobule between animals that received different doses of Tm, except for lobule 10 where curiously the proportion of ML area labeled was slightly greater in the 1 Atoh1-Tau animal induced with 50 μg/g compared to the two animals induced with 200 μg/g Tm (33.9 % compared to 29.9 % (t-test, p = 0.0015) or 30.8 % (t-test, p = 0.0045)). We then compared the density of labeled cell nuclei (Fig. 2a) to the proportion of labeled ML area (Fig. 2b) in corresponding regions of each lobule, 2 adults were injected with 50 μg/g Tm at P8 in order to distinguish individual labeled cells. We indeed found a high correlation between the density of labeled cell nuclei in particular lobules and the proportion of labeled ML area in the two animals analyzed (Spearman correlation coefficient r = 0.8897, p < 0.0001, Fig. 2c, and r = 0.7055, p = 0.0048, not shown). Thus, the proportion of labeled ML area represents the proportion of axons in the ML produced from the time of induction until the completion of gc production.Fig. 1


Differential timing of granule cell production during cerebellum development underlies generation of the foliation pattern
The axons of newly produced gcs stack inside to out in the molecular layer. a-b Adjacent sagittal midline sections of a P28 Atoh1-tau Cb after Tm administration at P10 stained for (a) X-gal to visualize the nuclei of the marked cells, (b) GFP fluorescent immunostaining (green) to visualize the axons of the marked granule cells. The anteroposterior zones have been color coded (anterior zone: green, central zone: blue, posterior zone: purple and nodular zone: yellow). c-h sagittal midline sections of lobule 3 of P28 Atoh1-tau Cb stained for GFP to reveal the position of the axons of the marked granule cells after Tm administration at the indicated ages showing that the proportion of molecular layer labeled diminishes as Tm is administered later
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Related In: Results  -  Collection

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Fig1: The axons of newly produced gcs stack inside to out in the molecular layer. a-b Adjacent sagittal midline sections of a P28 Atoh1-tau Cb after Tm administration at P10 stained for (a) X-gal to visualize the nuclei of the marked cells, (b) GFP fluorescent immunostaining (green) to visualize the axons of the marked granule cells. The anteroposterior zones have been color coded (anterior zone: green, central zone: blue, posterior zone: purple and nodular zone: yellow). c-h sagittal midline sections of lobule 3 of P28 Atoh1-tau Cb stained for GFP to reveal the position of the axons of the marked granule cells after Tm administration at the indicated ages showing that the proportion of molecular layer labeled diminishes as Tm is administered later
Mentions: Tm was injected every two days from P2 to P14 to Atoh1-Tau mice, 1 day for each mouse, and the fate-mapped cells were analyzed at P28. As expected, the cell bodies (detected with X-gal staining) of the marked cells were scattered throughout the IGL (Fig. 1a), whereas the labeled axons (immunostained for GFP) were stacked on top of unlabeled axons defining an outer portion of labeled ML and inner portion of unlabeled ML (Fig. 1b-h). We did indeed observe that the height of the outer ML that was labeled progressively decreased as Tm was administered later in Cb development (Fig. 1c-h). We measured the area of labeled ML as a proportion of the total area of ML in each lobule on three midline sections from animals that received 50 μg/g (n = 1) or 200 μg/g (n = 2) of Tm at P8. As expected, we did not observe a significant difference in the proportion of ML area labeled in each lobule between animals that received different doses of Tm, except for lobule 10 where curiously the proportion of ML area labeled was slightly greater in the 1 Atoh1-Tau animal induced with 50 μg/g compared to the two animals induced with 200 μg/g Tm (33.9 % compared to 29.9 % (t-test, p = 0.0015) or 30.8 % (t-test, p = 0.0045)). We then compared the density of labeled cell nuclei (Fig. 2a) to the proportion of labeled ML area (Fig. 2b) in corresponding regions of each lobule, 2 adults were injected with 50 μg/g Tm at P8 in order to distinguish individual labeled cells. We indeed found a high correlation between the density of labeled cell nuclei in particular lobules and the proportion of labeled ML area in the two animals analyzed (Spearman correlation coefficient r = 0.8897, p < 0.0001, Fig. 2c, and r = 0.7055, p = 0.0048, not shown). Thus, the proportion of labeled ML area represents the proportion of axons in the ML produced from the time of induction until the completion of gc production.Fig. 1

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&minus;/+;En2&minus;/&minus;), 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.