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, 108 (12), 5015-20

Constitutive Activation of B-Raf in the Mouse Germ Line Provides a Model for Human Cardio-Facio-Cutaneous Syndrome

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Constitutive Activation of B-Raf in the Mouse Germ Line Provides a Model for Human Cardio-Facio-Cutaneous Syndrome

Jelena Urosevic et al. Proc Natl Acad Sci U S A.

Abstract

RASopathies are a class of developmental syndromes that result from congenital mutations in key elements of the RAS/RAF/MEK signaling pathway. A well-recognized RASopathy is the cardio-facio-cutaneous (CFC) syndrome characterized by a distinctive facial appearance, heart defects, and mental retardation. Clinically diagnosed CFC patients carry germ-line mutations in four different genes, B-RAF, MEK1, MEK2, and K-RAS. B-RAF is by far the most commonly mutated locus, displaying mutations that most often result in constitutive activation of the B-RAF kinase. Here, we describe a mouse model for CFC generated by germ-line expression of a B-RafLSLV600E allele. This targeted allele allows low levels of expression of B-RafV600E, a constitutively active B-Raf kinase first identified in human melanoma. B-Raf+/LSLV600E mice are viable and display several of the characteristic features observed in CFC patients, including reduced life span, small size, facial dysmorphism, cardiomegaly, and epileptic seizures. These mice also show up-regulation of specific catecholamines and cataracts, two features detected in a low percentage of CFC patients. In addition, B-Raf+/LSLV600E mice develop neuroendocrine tumors, a pathology not observed in CFC patients. These mice may provide a means of better understanding the pathophysiology of at least some of the clinical features present in CFC patients. Moreover, they may serve as a tool to evaluate the potential therapeutic efficacy of B-RAF inhibitors and establish the precise window at which they could be effective against this congenital syndrome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Activation of the Mek/Erk pathway in B-Raf+/LSLV600E mice. (A) Protein extracts obtained from B-Raf+/+ and B-Raf+/LSLV600E adult brain and heart tissues as well as E13.5 embryos were submitted to Western blot analyses using antibodies to B-Raf, pMek1/2, Mek1, pErk1/2, and Erk1/2. GAPDH was used as loading control. (B) Protein extracts obtained from B-Raf+/+ and B-Raf+/LSLV600E adult brain and heart tissues were incubated with a monoclonal antibody against B-Raf, and the resulting immunoprecipitates were assayed for kinase activity using Mek1 as a substrate. The levels of pMek1 were determined by blotting with specific polyclonal antibodies against pMek1. The same membrane was used for Western blot analyses using B-Raf antibody as loading control. Arrowheads indicate the migration of the corresponding proteins.
Fig. 2.
Fig. 2.
B-Raf+/LSLV600E mice display decreased survival and growth rates. (A) Survival of B-Raf+/+ mice of all genetic backgrounds (open circles; n = 40) and B-Raf+/LSLV600E mice of various genetic background including B6/CD1 (open triangles; n = 44), B6/129 (gray triangles; n = 33), and B6 (solid triangles; n = 18). (B) Body weights of B-Raf+/+ male and female mice of B6/129 and B6/CD1 genetic backgrounds (open circles) compared with those of B-Raf+/LSLV600E male and female mice of B6/129 (gray triangles) and B6/CD1 (open triangles) genetic backgrounds. Error bars represent SEMs.
Fig. 3.
Fig. 3.
Cranial defects in B-Raf+/LSLV600E mice. (A) Representative side views of heads (Upper) and sagittal CT images (Lower) of adult B-Raf+/+ and B-Raf+/LSLV600E mice in B6/129 genetic background illustrating the more rounded skull vault of the mutant mice (arrowheads). (B) Overlay of two representative sagittal CT sections of a B6/129 B-Raf+/+ mouse (white) and a B6/129 B-Raf+/LSLV600E littermate (red). (C) Same as A, but images correspond to mice in a B6/CD1 genetic background. Note the cataracts in the eye of the two B-Raf+/LSLV600E mice. (D) Overlay of two representative sagittal CT sections of a B6/CD1 B-Raf+/+ mouse (white) and a B6/CD1 B-Raf+/LSLV600E littermate (red). (E) 2D analysis of sagittal CT projections of 10-wk-old adult B-Raf+/+ (n = 6) and B-Raf+/LSLV600E (n = 5) littermates (B6/129 genetic background). The anatomical position of a set of 10 homologous landmarks in a sagittal section of an adult B-Raf+/+ mouse is indicated by red circles. (F) P values of differences between WT and mutant mice by Hotteling's T2 test for each landmark coordinate (x and y) depicted in E after generalized procrustes superimposition. NS, not significant.
Fig. 4.
Fig. 4.
Increased number of GFAP positive cells in hippocampal and cortical areas. (A and B) GFAP staining of hippocampal areas from 8-wk-old B-Raf+/+ and B-Raf+/LSLV600E littermates (B6/CD1 background). (C and D) Amplified image of the quadrant indicated in A and B. (Scale bar, 1,000 μm.)
Fig. 5.
Fig. 5.
Heart defects in B-Raf+/LSLV600E mice. (A) Histological analysis of heart chambers and aortic valves of 8-wk-old B-Raf+/+ and B-Raf+/LSLV600E littermates. (Left) H&E staining of atrial cardiomyocytes. (Scale bar, 100 μm.) (Center) H&E staining of ventricular cardiomyocytes. (Scale bar, 50 μm.) (Right) H&E staining of aortic valves (arrowheads). (Scale bar, 500 μm.) (B) Sirius red staining of ventricular cardiomyocytes of B-Raf+/+ and B-Raf+/LSLV600E littermates. (Scale bar, 100 μm.) (C) Relative cardiomyocyte size. (D) Relative number of cardiomyocytes per area. (E–G) PET analysis of heart functions including (E) end systolic volume, (F) end diastolic volume, and (G) ejection fraction. B-Raf+/+ (open bars) and B-Raf+/LSLV600E (solid bards) mice (n = 6) had a B6 (A–D) or B6/CD1 (E–G) genetic background. For each mouse, three photos of the ventricular area were taken using the same magnification (20×). The number of cardiomyocytes was determined by counting the nuclei. The area occupied by the cardiomyocytes was determined using Image J software. Relative values represented in C and D were obtained by normalizing with those values obtained from control B-Raf+/+ animals. Error bars represent SEMs. *P < 0.05; **P < 0.01.
Fig. 6.
Fig. 6.
B-Raf+/LSLV600E mice develop chromaffin cell tumors. (A Left) H&E staining of a representative paraganglioma (PG) located adjacent to the renal artery (RA). Dotted line serves to separate both structures. (A Right) A pheochromocytoma depicting a mitotic figure (arrowhead). (B) Immunohistochemical staining for chromogranin A and synaptophysin of a representative pheochromocytoma. (C) H&E staining of micrometastases (m) detected in the lung of a pheochromocytoma-bearing mouse. Note the difference in nuclear and cellular size between normal lung parenchyma and a group of cells forming micrometastases. (Scale bars, 50 μm.)

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