Effects And Regulation Mechanisms Of Rp58 In Development Of Mouse Pontine Nuclei

Background:The pontine nuclei are the largest nuclei in brainstem,and play an important role in signal transmission between cerebral cortex and cerebellum.The pontine nuclei belong to precerebellar nuclei,which also contain external cuneate nucleus,lateral reticular nucleus,and inferior olivary nuclei.These nuclei entirely arise from the proliferative neuroepithelium of lower rhombic lip located in hindbrain.Because of its complicated mechanism,efforts are needed to fully understand the regulation network of pontine nuclei development.Previous studies have shown that Rp58,a transcription factor highly expressed in cortex,hippocampus,and cerebellum,plays an important role in the development of these neural structures.Although the expression of Rp58 is crucial for neural system development,whether Rp58 is involved in regulating the pontine nuclei development is still not reported.Objective:（1）To investigate the spatiotemporal expression patterns of transcriptional factor Rp58 in the development of mouse pontine nuclei;（2）To find out whether deletion of Rp58 affects the development of mouse pontine nuclei and other precerebellar nuclei;（3）To explore the mechanisms of Rp58 in the regulation of mouse pontine nuclei development.Methods:（1）B6 mice were used as research objects and the brain tissues were collected from embryonic and postnatal mice.Rp58 and Barhl1 RNA in situ hybridization staining were performed to explore the expression of Rp58 in various stages of pontine nuclei development.（2）The transgenic technology was performed to construct the Rp58 knockout mouse model and Rp58^fl/fl-Nestin Cre conditional knockout mouse model.RNA in situ hybridization and immunofluorescence staining were applied to explore whether the development of pontine nuclei was affected at embryonic and postnatal stages.Furthermore,the development of other components of precerebellar nuclei was observed to study if it was affected by the loss of Rp58.（3）Math1 and Barhl1 RNA in situ hybridization and Cleaved Casepase3 immunofluorescence were performed to figure out the mechanism of pontine nuclei dysplasia caused by Rp58 knockout from three aspects:the size of progenitor pool,neuron migration and cell apoptosis.Rp58^fl/fl-Math1Cre ER mouse model was established,in which Rp58 was induced to be deleted in Math1 positive cells,to explore whether the function of Rp58 in regulating pontine nuclei development is cell autonomous.Results:（1）Rp58 was highly expressed in the progenitor cells of the pontine nuclei and other precerebellar nuclei at E10,and continuously expressed in migration and maturation of pontine nuclei at E14,E16,E18and P8.（2）The DNA gel electrophoresis and Western-blot verified that Rp58 knockout mouse model and Rp58^fl/fl-Nestin Cre conditional knockout mouse model were constructed successfully.After Rp58 was completely knocked out,the pontine nuclei dysplasia can be detected as early as E15.The size of pontine nuclei decreased 34%and 37%at E16and E18;55%and 41%of BARHL1 positive pontine nuclei neurons were reduced at same stages.In terms of Rp58^fl/fl-Nestin Cre conditional knockout mice,the disappearance of pontine nuclei was observed under the anatomical microscope.The Nissl staining and RNA in situ hybridization showed that the pontine nuclei almost disappeared in Rp58^fl/fl-Nestin Cre conditional knockout mice at P8 and P17.The size of external cuneate nucleus and lateral tegmental nucleus were reduced at embryonic stages and almost disappeared at postnatal stages in this mice model,while development of inferior olive nuclei was not affected.（3）At E12,no difference was found in the progenitors of pontine nuclei located in lower rhombic lip between Rp58^+/+and Rp58^-/-mice.Ectopic migration neurons were observed in the Rp58^-/-mice at E16.Compared with the control group,there was a 3.24-fold increasing signal of Cleaved Caspase3 in the pontine nuclei of Rp58^fl/fl-Nestin Cre conditional knockout mice at P0.A defect of pontine nuclei development was obserbed in Rp58^fl/fl-Math1Cre ER conditional knockout mice.Conclusion:（1）This study firstly revealed that Rp58 is continuously expressed in the process of pontine nuclei development.（2）The expression of Rp58 is essential for development of the pontine nuclei,while the lost of Rp58 leads to depletion of pontine nuclei structure.Rp58is required in the development of other mossy fiber nuclei,such as external cuneate nucleus and lateral tegmental nucleus,while the development of inferior olive nuclei was not regulated by Rp58.（3）The expression of Rp58 is necessary for pontine nuclei neuron migration and survival.The function of Rp58 is cell autonomous.