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. 2002 Nov 12;99(23):15200-5.
doi: 10.1073/pnas.222362099. Epub 2002 Oct 23.

Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development

Alessandro Ieraci  1 , Paolo E Forni, Carola Ponzetto
Affiliations

Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development

Alessandro Ieraci et al. Proc Natl Acad Sci U S A. .

Abstract

Cerebellar development occurs mainly postnatally and implies cell proliferation and migration. Hepatocyte growth factor (HGF) and Met are involved in mediating these responses in other tissues and are coexpressed in the cerebellum. Here we show that Met is localized in granule cell precursors and that cultures of these cells respond to HGF with proliferation. To study the role of HGF and Met in the cerebellum in vivo, we produced a viable hypomorphic Met mutant by knocking in the met locus a point mutation to abrogate the receptor Grb2-binding site. A similar mutant was previously described as perinatal lethal. In this "first-generation" knock-in the recombinant locus retained the Neo cassette (Met(grb2/grb2neo+)). In the knock-in presented here Neo was Loxed and excised by Cre recombinase, which led to higher tissue levels of Met(grb2) protein, sufficient to rescue viability. In Met(grb2/grb2neo-) mice the size of the cerebellum was reduced and foliation defects were evident, especially in the central and posterior half of the vermis. Proliferation of granule precursors in vivo was 25% lower than in controls. In cultures of mutant granule cells HGF-induced microtubule-associated protein kinase activation was reduced and transient. Behavioral tests indicated a balance impairment in Met(grb2/grb2neo-) mice. Altogether these data indicate that normal cerebellar development and, possibly, function, require HGF and Met, and that proliferation of granule cells in the cerebellum critically depends on full HGF/Met signaling.

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Figures

Fig 1.
Fig 1.
Expression and localization of Met in the cerebellum, and proliferative response of granule cell cultures to HGF. (A) Western blot of mouse cerebellum and liver extracts at P8 (p145 m-Met: mouse Met, specifically detected by anti-mouse Met antibody). Neg. control: cerebellum from genetically modified mice expressing a mouse-human Met-chimera (see Fig. 2). (B) Anti-m-Met immunostaining revealing expression in the EGL. (C) Phase contrast showing cerebellar morphology. (E) Pretreatment of the antibody with cognate peptide. (F) Double staining with anti-Met (red) and anti-proliferating cell nuclear antigen (green) showing coexpression. (B, C, and E, ×20; F, ×40.) (D) BrdUrd incorporation in HGF- or IGF-1-treated cerebellar granule cell cultures from P6 mice. Three experiments were performed in quadruplicate and values are shown as mean ± SEM.
Fig 2.
Fig 2.
Knock-in of YVNV→YVHV signaling mutation (Metgrb2) in the met locus. (A) Schematic representation of the knock-in strategy. Black boxes represent exons. N indicates NdeI sites. The Neo cassette was flanked by LoxP sites (triangles). Asterisk indicates the location of the mutation. Arrows indicate primers used for screening of ES (1 and 2) and Neo− alleles (1 and 5), and for genotyping mice heterozygous for the Metgrb2 mutation (3 and 4). (B) Southern blot of NdeI digests of genomic DNA isolated from double-selected R1 ES cell clones [+/+, WT ES; +/−, control recombinant ES cells used for production of first-generation Metgrb2 mice (see text); 1–3, recombinant ES clones]. (C) Western blots of total protein extracts from E13.5 WT, Metgrb2/grb2neo+, and Metgrb2/grb2neo− embryos. h-Met, antibody specific for human Met; Actin, anti-actin (control for protein loading).
Fig 3.
Fig 3.
Macroscopic analysis of Metgrb2/grb2neo− cerebellum. (A and B) Cerebellum from WT (A) and Metgrb2/grb2neo− mutant (B) at P23. (C and D) Sagittal sections of cerebellum from P23 WT (C) and mutant mice (D) stained with cresyl violet (×1.6). Cerebellar fissures and sulci: plt, posteriolateral; uvu, uvular; scd, secondary; ppy, prepyramidal; itc, intercrural; dcl, declival; pri, primary; pcu, preculminate; pct, precentral. (E) Fissure depth was analyzed by using the image pro plus system (Media Cybernetics, Silver Spring, MD). Six mutants and five controls were analyzed. Values are shown as mean ± SEM. t test: *, P < 0.02; **, P < 0.01; ***, P < 0.001. (F and G) Calbindin staining of Purkinje cells at P8 in control (F) and Metgrb2/grb2neo− mice (×40).
Fig 4.
Fig 4.
BrdUrd incorporation in the cerebellum of Metgrb2/grb2neo− mutants. (A) P8 mice were injected with BrdUrd and killed 2 h later. Sections were stained by immunohistochemistry using anti-BrdUrd antibodies. BrdUrd-positive cells were counted in at least six regions of the EGL (3,000–4,000 cells per cerebellum). Five mutant and three control mice were analyzed and values are shown as mean ± SD. t test: P < 0.0001. (B and C) P7 mice were injected with BrdUrd and killed 72 h later (×20). Black, BrdUrd-positive cells; light blue, hematoxylin-stained nuclei; ML, molecular layer; PL, Purkinje layer; IGL, internal granule layer.
Fig 5.
Fig 5.
HGF-dependent MAP-kinase phosphorylation in Metgrb2/grb2neo−. (A and C) Representative Western blots of extracts of cerebellar granule cell cultures from P6 WT and mutant mice, stimulated with HGF (20 ng/ml) (A) or BDNF (100 ng/ml) (C) for the indicated times. Blots were probed with antiphospho-MAP kinase, stripped, and reprobed with an antibody against β-actin for normalization. (B and D) Densitometric analysis of the Western blots. Scanned films were analyzed with the IMAGE MASTER TOTAL LAB V100 program (Amersham Pharmacia) and plotted with Microsoft EXCEL. Values were obtained from eight different experiments and are presented as mean ± SEM.
Fig 6.
Fig 6.
Cerebellar function in Metgrb2/grb2neo− mutant mice. WT and mutant mice were tested for equilibrium and coordination by the thin-rod test. The time spent on the rod up to a limit of 60 s was measured. Nine animals for each genotype were tested in a total of 15 sessions. Values are shown as mean ± SEM.
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