Tsc1 haploinsufficiency leads to Pax2 dysregulation in the developing murine cerebellum

Tuberous sclerosis complex 1 (TSC1) is a tumour suppressor gene that inhibits the mechanistic target of rapamycin (mTOR) pathway. Mutations in TSC1 lead to a rare complex disorder of the same name, in which up to 50% of patients present with autism spectrum disorder (ASD). ASD is a highly prevalent, early-onset neurodevelopmental disorder, characterized by social deficits and repetitive behaviours, although the type and severity of symptoms show wide variability across individuals. Amongst different brain areas proposed to play a role in the development of ASD, the cerebellum is commonly reported to be altered, and cerebellar-specific deletion of Tsc1 in mice is sufficient to induce an ASD-like phenotype. Given that the mTOR pathway is crucial for proper cell replication and migration, this suggests that dysregulation of this pathway, particularly during critical phases of cerebellar development, could contribute to the establishment of ASD. Here, we used a mouse model of TSC to investigate gene and protein expression during embryonic and early postnatal periods of cerebellar development. We found that, at E18 and P7, mRNA levels of the cerebellar inhibitory interneuron marker Pax2 were dysregulated. This was accompanied by changes in the expression of mTOR pathway-related genes and downstream phosphorylation of S6. Differential gene correlation analysis revealed dynamic changes in correlated gene pairs across development, with an overall loss of correlation between mTOR- and cerebellar-related genes in Tsc1 mutants compared to controls. We corroborated the genetic findings by characterizing the mTOR pathway and cerebellar development on protein and cellular levels with Western blot and immunohistochemistry. We found that Pax2-expressing cells were hypertrophic at E18 while, at P7, their number was increased and maturation into parvalbumin-expressing cells delayed. Our findings indicate that E18 and P7 are crucial time points in cerebellar development in mice that are particularly susceptible to mTOR pathway dysregulation.

CEP120-mediated KIAA0753 recruitment onto centrioles is required for timely neuronal differentiation and germinal zone exit in the developing cerebellum

Joubert syndrome (JS) is a recessive ciliopathy in which all affected individuals have congenital cerebellar vermis hypoplasia. Here, we report that CEP120, a JS-associated protein involved in centriole biogenesis and cilia assembly, regulates timely neuronal differentiation and the departure of granule neuron progenitors (GNPs) from their germinal zone during cerebellar development. Our results show that depletion of Cep120 perturbs GNP cell cycle progression, resulting in a delay of cell cycle exit in vivo. To dissect the potential mechanism, we investigated the association between CEP120 interactome and the JS database and identified KIAA0753 (a JS-associated protein) as a CEP120-interacting protein. Surprisingly, we found that CEP120 recruits KIAA0753 to centrioles, and that loss of this interaction induces accumulation of GNPs in the germinal zone and impairs neuronal differentiation. Importantly, the replenishment of wild-type CEP120 rescues the above defects, whereas expression of JS-associated CEP120 mutants, which hinder KIAA0753 recruitment, does not. Together, our data reveal a close interplay between CEP120 and KIAA0753 for the germinal zone exit and timely neuronal differentiation of GNPs during cerebellar development, and mutations in CEP120 and KIAA0753 may participate in the heterotopia and cerebellar hypoplasia observed in JS patients.

Fetal cerebellar development: 3D morphometric analysis of fetal postmortem MRI among Chinese fetuses.

The development of the cerebellum starts from early gestational period and extends postnatal. Because of its protracted development, the cerebellum is susceptible to developmental anomalies such as Dandy-Walker malformations, Blakes pouch cysts and vermin hypoplasia. Measurements of fetal cerebellar parameters of a normal growing fetus in each week of gestation is important for setting up morphometric standards and hence used as clinical reference data. Any deviation from the normal cerebellar parameters alerts the clinicians for the possibility of presence of cerebellar malformations. Study objective: The objective of this study was to assess the fetal cerebellar growth by quantifying the following parameters; fetal cerebellar volume, anterior-posterior diameter and superior-inferior diameter. Methods: We used 3T and 7T MRI to scan the postmortem fetal brains at different stages of development and subsequently analyze the images using ITK-SNAP software. Results: The mean superior-inferior cerebellar diameter was found to be 19.12+2. 70mm.The linear(y=bo+b1t) model was the best fit (r2=0.996, F=32022.961) to describe the relationship between the gestational age and the superior-inferior diameter(y=5.89+0.49t). There was significant correlation between the superior-inferior cerebellar diameter and the gestation age, Pearson correlation coefficient of 0.999, r=0.001. The median cerebellar volume was 8607.7mm, the mean rank high among males(78.12) as compared to female(68.25). There was no statistically significant difference of the cerebellar volume between males and females (u=2193.5,p=0.16). The quadratic(y=bo+b1t+b2t2) model was the best fit regression equation (r2=0.994,F=10791.157) describing the relationship between the cerebellar volume and the gestational age. The median anterior-posterior diameter was 12.45 mm. There was significant correlation between anterior-posterior diameter and the gestational age with Spearmans rho of (0.997, p=0.01). The linear model was the best fit the best fit model (y=bo+b1t) describing the relationship between anterior-posterior diameter and the gestational age(y=3.31+0.5t) r2=0.998, F=70646.838. Conclusion: Significant correlation between the superior-inferior cerebellar diameters, the anterior-posterior cerebellar diameter and the gestational age was found. These two linear parameters follow the first-degree polynomial in relation to the gestational age. The cerebellar volume follows the second-degree polynomial as it increases with the gestational age and correlate significantly with the gestational age. This study has provided new insight to the development of the cerebellum, and setup a benchmark data of which the deviation from it will alert the clinicians for the possibility of presence of cerebellar malformations. Key words: Cerebellar Development, Cranial Magnetic Resonance imaging, Cerebellar Malformations

Temporal analysis of enhancers during mouse brain development reveals dynamic regulatory function and identifies novel regulators of cerebellar development.

In this study, we identified active enhancers in the mouse cerebellum at embryonic and postnatal stages establishing the first catalog of enhancers active during embryonic cerebellum development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Our study provides insight into the dynamics of gene expression regulation by enhancers in the developing brain and delivers a rich resource of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.