Fig. 7

Knockdown of Scrt2, Atf4 and Rfx3 showed similar phenotypic alterations during cerebellum development. In utero transfection with shRNA against Scrt2, Atf4 and Rfx3 was performed at E12.5 and harvested at E18.5 (a-d, f-h) and E15.5 (e). The harvested cerebellar tissue was sectioned and immunofluorescent staining with EGFP (all) and Calbindin (e and f) antibody was performed. a) Control EGFP transfection – medial section. b) Control EGFP transfection – lateral section. c) Scrt2 shRNA transfection – medial section. d) Scrt2 shRNA transfection – lateral section. Note yellow arrows indicate location of developmental abnormality and tissue atrophy. The Scrt2 shRNA transfected cerebella seems to exhibit developmental retardation such as lack of foliation at E18.5. e) Double color immunofluorescent staining with EGFP and Calbindin antibody on Scrt2 shRNA transfected cerebellar tissue at E18.5Most of the EGFP+ Scrt2 shRNA transfected cells have disappeared and only their processes (punctate EGFP signals) remain. f) Double color immunofluorescent staining with EGFP and Calbindin antibody on Scrt2 shRNA transfected cerebellar tissue at E15.5. Many of the EGFP+ Scrt2 shRNA transfected cells are positive for calbindin staining as well, indicated by thin white arrows. g) Rfx3 shRNA transfection – lateral section. H) Atf4 shRNA transfection – medial section . In utero transfection against Rfx3 (g) and Atf4 (h) shRNA showed a similar, but less dramatic phenotype of tissue atrophy and developmental delay as in the Scrt2 knockdown. EGL – External granular layer, NE – Neuroepithelium, PCP – Purkinje cell plate, RL – Rhombic lip