CRAF-deficiency in postnatal mice.
(A) Lethality-analysis of CRAF ko embryos around birth (embryonic day 16.5, E16.5-newborn mice at postnatal day 0/1, P0/P1). White bars show total numbers of CRAF ko animals at different developmental stages (E16.5-P0/P1). Dark bars indicate the number of dead mice. No lethal CRAF ko embryos could be observed at E16.5. CRAF ko (E16.5 total), n = 6 (from n = 39 littermates); CRAF ko (E18 total), n = 25 (from n = 145 littermates); CRAF ko (E18 lethal), n = 11, indicating 47% lethality of CRAF ko at E18; CRAF ko (P0/P1 total), n = 109 (from n = 873 littermates); CRAF ko (P0/P1 lethal), n = 73 indicating 67% lethality of CRAF ko at P0/P1. (B) Kaplan-Meier survival curve of postnatal CRAF ct (dark line) and CRAF ko (pointed line) mice. Mice were daily monitored. CRAF ct, n = 763; CRAF ko, n = 110. (C) Western blot analysis of CRAF (C-20) expression in dissected brain areas (hp, hippocampus; pc, prefrontal cortex; cb, cerebellum; bo, olfactory bulb) of postnatal (P30) CRAF+/+ control (CRAF ct) and CRAF-deficient (CRAF-/-, CRAF ko) mice. ß-actin serves as loading control. (D) Phenotypic analysis of CRAF ko mice compared to CRAF ct siblings. (Upper line) CRAF ko embryos (right panel) are anyhow smaller and appear more shiny compared to CRAF ct siblings (left panel) at E16.5. (2nd line) From E18 (left panel) on CRAF ko animals (white arrow) are clearly discernible due to their reduced body size compared to CRAF ct siblings. The placenta is shown on top of animals. (2nd line, right panel) CRAF ko mice were later born with open eyes (white arrow and Inset). (3rd line left) Postnatal CRAF ko mice are strongly reduced in body size at the age of P10 (white arrow) with less hair and pink skin color shining through the coat. (3rd line right) Postnatal CRAF ko mice at the age of P30 showed reduced body size (white arrow) compared to CRAF ct siblings (right). (Lower panel left) Postnatal CRAF ko mice with eye open at birth (EOB) phenotype at the age of P12 compared to CRAF ct sibling with closed eyes. (Lower panel right) CRAF ko mouse at the age of P30 with eyelid defect (white arrow). (E) Quantitative body weight analysis of CRAF ct (dark bars) and CRAF ko (white bars) siblings (n = 234) from postnatal day P1 until P43. Data are mean ± s.e.m.; n = 234. Significant differences are shown in p-value p< 0.05 (*), p< 0.01 (**), p< 0.001 (***). (F) Quantitative brain weight analysis of CRAF ct (dark bars) and CRAF ko (white bars) siblings (n = 234) from postnatal day P1 until P43. Data are mean ± s.e.m.; significant differences are shown in p-value p< 0.05 (*), p< 0.01 (**), p< 0.001 (***). (G) Cerebellar abnormalities in postnatal CRAF ko mice. (Upper panel) Representative sagittal sections of Nissl stained cerebellum of CRAF ct (left) and CRAF ko (right) mice at the age of P30. White arrows in CRAF ko (upper panel right) indicate alterations in lobule X (LX) related to reduced length, failure of proper build central core stream and sulcus compared to CRAF ct (left, white arrows). Also LIII appeared strongly reduced in size in CRAF ko (upper panel right) compared to CRAF ct (left). Scale bars = 1mm. (Lower panel) Representative sagittal sections of Calbindin stained cerebellum LX of CRAF ct (left) and CRAF ko (right) mice at the age of P30. Calbindin-stained Purkinje cell neurons of CRAF ko mice are irregular located in LX. Scale bars = 60μm. Higher magnification of Calbindin-staining reveals elongated and less arborescent dendrite formation of Purkinje cell neurons through the molecular layer. Scale bars = 60μm. (H) Analysis of CRAF expression by immunohistochemistry. Representative sagittal sections of P10 (upper panels) and P30 (lower panels) CRAF ct (left panels) and CRAF ko (right panels) hippocampus stained for CRAF (C-20). White arrowheads indicate CRAF stained cell body of granule neurons in the granule cell layer (GCL) of the hippocampal dentate gyrus in CRAF ct (left panels). CRAF ko sections exhibit no CRAF-expression (right panel). Sections were counterstained with hematoxylin. Scale bar = 50μm.