Effects Of Conditional Deletion Of Shp2 Under HGFAP Promoter On Structure And Function Of Cerebral Cortex, Cerebellum And Brain Stem In New Born Mice

Background:Shp2 tyrosine phosphatase is a widely expressed protein which is involved in a variety of cell signaling events. Recent studies also revealed its key roles in the development and function of the mammalian central nervous system through modulation of various neural tissue and cell types. Radial glias are important neural cells widely present in the mammalian central nervous system, and our understanding of the unique roles these cells has significantly expanded in the last decade. However, the role of Shp2 in modulation of radial glia and its underlineing mechanisms have not been well illustrated. Previous studies revealed that hGFAP promoter directs Cre recombinase to radial glia. Thus studies that delete Shp2 in radial glia using the hGFAP-Cre conditional knockout technique would largely expand our knowledge in understanding the underlining molecular mechanisms of central nervous system function and development.Objectives:(1) Generation and confirmation of hGFAP promoter-directed Shp2 conditional knockout (CKO) mice.(2) Characterization of Shp2 CKO new born mice on survival, morphogenic development and sensory-motor development.(3) Characterization of Shp2 CKO new born mice on morphological and histological architecture of the central nervous system and its possible molecular mechanisms.Methods:hGFAP-Cre transgenic mice were bred with Shp2flox/flox mice to generate hGFAP-Ce/ shp2fiox/fiox(Shp2 CKO) mice. DNA, mRNA and protein extracted from brain and heart tissue were sent for further genotyping, and real-time PCR, Western blot and immunochemicalhistology were also conducted for confirmation of effectiveness of the CKO. Shp2 CKO new borm mice as well as its littermate were carefully observed for appearance, survival date, body and whole brain weight growth. Behavioral analysis including nest finding, attraction to auditory stimulus, righting reflex and wire hanging were done to evaluate sensory-motor reflex development. Then brains of Shp2 CKO mice and its control littermates were fixed and sectioned. H-E staining and toluidine blue staining were done for observation of histological architecture of cerebral cortex and cerebellum. Immunohistochemistry, immunofluorescence and TUNEL assay were conducted for further observation of neuron, glia, cell proliferation and apoptosis in cerebral cortex, cerebellum and brain stem. Protein samples were further extracted from microdissected cerebral cortex and expression level of Akt, Erk signaling were examined by Western blot assay.Results:Part ⅠhGFAP-Ce/Shp2flox/flox (Shp2 CKO) mice were successfully generated and the efficacy of conditional knockout was then confirmed on DNA, mRNA and protein levels.Part ⅡShp2 CKO mice exhibited early postnatal lethality, body and brain growth retardation and impaired sensory-motor development.Part ⅢShp2 CKO mice exhibited dysplasia of cerebral cortex with reduced neuron genesis, increased gliogenesis, reduced proliferation in the ventricular zone and mass cortical apoptosis. Shp2 CKO also led to glial defects in cerebellum and abnormal gliogenesis in brain stem. Partial inhibition of Erk signaling wre observed in Shp2 CKO cerebral cortex.Conclusions:We successfully generated Shp2 CKO mice under the hGFAP promoter. Shp2 plays key roles in central nervous system development and function by modulation of radial glia function.