Regulation Of Cerebellar Granule Cell Precursor Cells Development And Medulloblastoma Formation By Atoh1 Enhancer

The cerebellum is located in the hindbrain,above the brain stem and below the occipital lobe.Granule cells in cerebellum account for about 60%-70%of the total number of neurons in the whole brain,and are the most numerous type of neurons in the nervous system.The cerebellum is responsible for regulating movement and balance,but also for some higher cognitive functions,such as emotion,language,and social interaction.Anatomically,the cerebellar cortex has a unique three-layered structure and relatively conservative developmental characteristics among different species,making it an ideal paradigm for studying the development of the nervous system.However,the mechanism of cerebellar granule cell regulation is still unclear.Atonal b HLH transcription factor 1（Atoh1）is a basic helix-loop-helix transcription factor,which contains a protein-binding domain and a DNA-binding domain.The protein-binding domain consists of twoαhelices and is one of the main regulators of the differentiation of granule precursor cells during cerebellar development.In this study,we first analyzed the temporal and spatial expression patterns of Atoh1 m RNA in the cerebellum using RNA scope technology.Then,the expression of Atoh1 gene in Atoh1transgenic（Atoh1-cre;td Tomato/+）and Atoh1 promoter-driven td Tomato gene knockout（Atoh1-3*V5-P2A-td Tomato/+）reporter mice was analyzed systematically.The results showed that Atoh1 m RNA was highly expressed in the rhombic lip and external granule cell layer of embryonic cerebellum.At the protein level,Atoh1 was mainly expressed in the exterior layer of the rhombic lip and the proliferative outer granular cell layer in td Tomato knock-in mice.In Atoh1 transgenic reporter mice,td Tomato-positive cells were highly expressed in the rhombic lip,in the external granule cell layer and in the inner granular cell layer throughout the development of granular cells.These results suggested that Atoh1 was mainly expressed in the proliferative region of granule precursor cells,including the rhombic lip and the external granule cell layer.Previous studies have found that there is a key enhancer in the 3’UTR region of Atoh1 gene that regulate the expression of Atoh1,but its effect on the development of granule precursor cells and the specific regulatory mechanism are still unclear.Therefore,in order to clarify the function of this enhancer,we further analyzed the cerebellar developmental phenotype of Atoh1 enhance-deficient mouse strains constructed by our collaborators.After Atoh1 enhancer deletion,the expression of Atoh1 m RNA in cerebellum decreased,the body weight and cerebellum weight of mice decreased significantly,and the cerebellum stratification and lobulation were disordered.Next,we further analyzed the cerebellar granule precursor cells,we found that the outer granule cell layer became thinner and the distribution of granule precursor cells was disordered.Immunofluorescence staining showed that the granule precursor cells did not show significant apoptosis after Atoh1 enhancer deletion,but the proliferation ability of granule precursor cells was significantly reduced compared with wild-type mice at different developmental stages.Therefore,cerebellar degeneration after Atoh1 enhancer deletion may be caused by the decreased proliferation of granule precursor cells.In addition,medulloblastoma is the most common type of cerebellar malignant tumor,which is mainly caused by abnormal malignant proliferation of cerebellar granule precursor cells.Many signal pathways regulating cerebellar neurogenesis are significantly disregulated in cerebellar medulloblastoma tissues.At present,the key signaling molecular mechanisms regulating cerebellar neurogenesis with medulloblastoma have not been fully clarified.Previous studies have shown that Atoh1 plays an important regulatory role in the regulation of medulloblastoma,but the role of Atoh1 enhancer in this process remains unclear.Therefore,we constructed a mouse model of Atoh1enhancer deletion in Shh subtype medulloblastoma(Smo M2^{h GFAP};Atoh1 E0^+/-),the results showed that,compared with Smo M2^{h GFAP} mice,the cerebellar tumor volume and weight of mice with Atoh1 enhancer knockout were significantly reduced,and the expression level of Atoh1 was also significantly decreased.Compared with tumor mice,the proliferation ability of granule progenitor cells in tumor mice after Atoh1 enhancer knockout was significantly decreased.In conclusion,in this study,the spatial and temporal expression pattern of Atoh1 in cerebellar development was analyzed at m RNA and protein levels in mouse models constructed with two different genetic strategies,and the precise expression pattern of Atoh1 in cerebellar development was further identified,providing direct evidence for further understanding of its regulatory role in cerebellar granulosum cell development.In addition,based on the mouse model of Atoh1 enhancer deletion combined with Shh subtype cerebellar medulloblastoma,we also found that Atoh1 enhancer not only plays an important regulatory role in the physiological development of cerebellar granule progenitor cells,but also participates in the regulation of the occurrence and development of Shh subtype cerebellar medulloblastoma.Atoh1 enhancer deletion can significantly delay the malignant proliferative phenotype of cerebellar medulloblastoma.The above work provides a new experimental and theoretical basis for further revealing and understanding the development of cerebellar granule precursor cells and its regulatory mechanism in the occurrence and development of medulloblastoma.