Actinin-associated LIM protein-deficient mice maintain normal development and structure of skeletal muscle

Abstract

The actinin-associated LIM protein, ALP, is the prototype of a large family of proteins containing an N-terminal PDZ domain and a C-terminal LIM domain. These PDZ-LIM proteins are components of the muscle cytoskeleton and occur along the Z lines owing to interaction of the PDZ domain with the spectrin-like repeats of alpha-actinin. Because PDZ and LIM domains are typically found in proteins that mediate cellular signaling, PDZ-LIM proteins are suspected to participate in muscle development. Interestingly the ALP gene occurs at 4q35 near the heterochromatic region mutated in facioscapulohumeral muscular dystrophy, indicating a possible role for ALP in this disease. Here, we describe the generation and analysis of mice lacking the ALP gene. Surprisingly, the ALP knockout mice show no gross histological abnormalities and maintain sarcolemmal integrity as determined by serum pyruvate kinase assays. The absence of a dystrophic phenotype in these mice suggests that down-regulation of ALP does not participate in facioscapulohumeral muscular dystrophy. These data suggest that ALP does not participate in muscle development or that an alternative PDZ-LIM protein can compensate for the lack of ALP.

FIG. 1

Genomic replacement of the mouse ALP exon 1. (A) The restriction maps of the wild-type ALP locus, the pPNT-ALP targeting vector, and the properly targeted locus are shown. Recombination (shown as large Xs) between the targeting vector and the wild-type locus eliminated the HSV TK gene and produced the knockout allele. A BamHI site was engineered to allow the wild-type and knockout genomic loci to be distinguished. (B) Genomic Southern blots of ES cell clones. Use of the 5′ probe for Southern analysis of a BamHI digest yielded a 20-kb band from the wild-type allele and a 11.5-kb band from the mutant allele. Use of the 3′ probe for Southern blots of PstI digests yielded a 4-kb band from the wild-type allele and a 3-kb band from the mutant allele. Clones 7 and 29 show proper targeting in both BamHI (left) and PstI (right) digests.

FIG. 2

Analysis of ALP genotype and gene expression in ALP knockout mice. (A) PCR genotyping of ALP mutant mice. DNA from wild-type (+/+), heterozygous (+/−), or homozygous (−/−) mice was amplified by PCR using an ALP-specific primer (P1), a neo-specific primer (P2), and a common primer (P3). The wild-type ALP allele produced a 250-bp DNA fragment with P1 and P3 (top), and the mutated allele produced a 290-bp DNA fragment with P2 and P3 (bottom). (B) RT-PCR analysis of ALP gene expression. First-strand cDNA from heterozygous (+/−) or homozygous (−/−) mice was amplified by PCR using ALP-specific primers that lie outside the first coding exon and flank the alternatively spliced region. Skeletal muscle ALP (ALP-SK) yielded a 948-bp fragment, and cardiac muscle-ALP (ALP-H) yielded a 702-bp fragment with this primer pair. Primers that amplify a 528-bp DNA fragment of tubulin were used to control for input cDNA. (C) Crude gastrocnemius muscle homogenates from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice were analyzed by Western blotting with antibodies to ALP and α-actinin.

FIG. 3

Histochemical analysis of ALP knockout mouse muscle. (A) Cryosections of gastrocnemius muscle from ALP^+/+ and ALP^−/− mice were analyzed with hematoxylin and eosin (H&E) staining. Sections from knockout mouse tissue resembled the wild-types and showed regularly sized and shaped myofibers and predominantly peripherally localized nuclei. (B) Immunofluorescent staining shows that ALP staining at Z lines is abolished in muscle sections from an ALP^−/− mouse. (C) The localization of α-actinin at skeletal muscle Z lines remains normal in an ALP^−/− mouse.