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Targeted ablation of NrCAM or ankyrin-B results in disorganized lens fibers leading to cataract formation.

Moré MI, Kirsch FP, Rathjen FG - J. Cell Biol. (2001)

Bottom Line: The NgCAM-related cell adhesion molecule (NrCAM) is an immunoglobulin superfamily member of the L1 subgroup that interacts intracellularly with ankyrins.The disorganization of fiber cells becomes histologically distinct during late embryonic development and includes abnormalities of the cytoskeleton and of connexin50-containing gap junctions.Also, these studies provide genetic evidence of an interaction between NrCAM and ankyrin-B.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany.

ABSTRACT
The NgCAM-related cell adhesion molecule (NrCAM) is an immunoglobulin superfamily member of the L1 subgroup that interacts intracellularly with ankyrins. We reveal that the absence of NrCAM causes the formation of mature cataracts in the mouse, whereas significant pathfinding errors of commissural axons at the midline of the spinal cord or of proprioceptive axon collaterals are not detected. Cataracts, the most common cause of visual impairment, are generated in NrCAM-deficient mice by a disorganization of lens fibers, followed by cellular disintegration and accumulation of cellular debris. The disorganization of fiber cells becomes histologically distinct during late embryonic development and includes abnormalities of the cytoskeleton and of connexin50-containing gap junctions. Furthermore, analysis of lenses of ankyrin-B mutant mice also reveals a disorganization of lens fibers at postnatal day 1, indistinguishable from that generated by the absence of NrCAM, indicating that NrCAM and ankyrin-B are required to maintain contact between lens fiber cells. Also, these studies provide genetic evidence of an interaction between NrCAM and ankyrin-B.

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NrCAM gene disruption. (a) Map of the targeting vector, and of part of the genomic locus before and after homologous recombination with the targeting vector. The position of exons 5, 6, and 8 as well as the exon numbering was adopted from the human NrCAM locus (Dry et al., 2001). neo, PGKneopA cassette; tk, MC1TKpA cassette; H, HindIII; K, KpnI; S, SacI; P, PauI; B, BamHI; N, NheI; Sp, SpeI. Only relevant restriction sites are shown. The positions of the external Southern blot probe, as well as the PCR primers, are indicated. (b) Southern blot of DNA from tail biopsies of wild-type, heterozygous, and homozygous mice, using the restriction enzymes BamHI and KpnI. (c) PCR of genomic DNA using the primers Mimo71 and Moré1 (c), and the primers neoL1 and Moré1. (d) Immunoblot blot analysis using whole brain from adult wild-type and NrCAM−/− mice using polyclonal antibody 463 against NrCAM. The NrCAM band is completely absent in the mutants. Additional bands are due to nonspecific binding activity of the antibody. To demonstrate equal loading, a parallel blot was analyzed with polyclonal antibodies to L1. Molecular weight markers are indicated on the left. Immunohistological studies using neural tissue as well as immunoblots of brain and lens tissue using a polyclonal antibody provided by M. Grumet also reveal that NrCAM is absent (not shown).
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fig1: NrCAM gene disruption. (a) Map of the targeting vector, and of part of the genomic locus before and after homologous recombination with the targeting vector. The position of exons 5, 6, and 8 as well as the exon numbering was adopted from the human NrCAM locus (Dry et al., 2001). neo, PGKneopA cassette; tk, MC1TKpA cassette; H, HindIII; K, KpnI; S, SacI; P, PauI; B, BamHI; N, NheI; Sp, SpeI. Only relevant restriction sites are shown. The positions of the external Southern blot probe, as well as the PCR primers, are indicated. (b) Southern blot of DNA from tail biopsies of wild-type, heterozygous, and homozygous mice, using the restriction enzymes BamHI and KpnI. (c) PCR of genomic DNA using the primers Mimo71 and Moré1 (c), and the primers neoL1 and Moré1. (d) Immunoblot blot analysis using whole brain from adult wild-type and NrCAM−/− mice using polyclonal antibody 463 against NrCAM. The NrCAM band is completely absent in the mutants. Additional bands are due to nonspecific binding activity of the antibody. To demonstrate equal loading, a parallel blot was analyzed with polyclonal antibodies to L1. Molecular weight markers are indicated on the left. Immunohistological studies using neural tissue as well as immunoblots of brain and lens tissue using a polyclonal antibody provided by M. Grumet also reveal that NrCAM is absent (not shown).

Mentions: To study the function of NrCAM in an in vivo situation, we generated a mouse strain deficient for this gene. To do this, we first characterized the NrCAM locus surrounding the ATG using a mouse embryonic stem (ES) cell BAC clone. A long and a short arm was subcloned from the BAC clone to construct the targeting vector to destroy the start codon within exon 4 (Fig. 1 a). Electroporated ES cells were screened for homologous recombination and used to create chimeric mice via blastocyst injection. The correct integration of the targeting vector and a deletion of the ATG-containing exon was confirmed by Southern hybridization as shown in Fig. 1 b and using the PauI-NheI fragment (region deleted in the mutant) as probe (not shown). Alternatively, the replacement of the ATG-containing exon by the neo cassette in the mutant mice was shown by PCR (Fig. 1 c). To detect NrCAM at the protein level, we generated polyclonal antibodies against a glutathione S-transferase fusion protein of the FNIII domains 1–4 of NrCAM. Immunohistochemical studies and an immunoblot from brains of both wild-type and NrCAM-deficient mice (Fig. 1 d; not shown) show the absence of NrCAM in the mutants.


Targeted ablation of NrCAM or ankyrin-B results in disorganized lens fibers leading to cataract formation.

Moré MI, Kirsch FP, Rathjen FG - J. Cell Biol. (2001)

NrCAM gene disruption. (a) Map of the targeting vector, and of part of the genomic locus before and after homologous recombination with the targeting vector. The position of exons 5, 6, and 8 as well as the exon numbering was adopted from the human NrCAM locus (Dry et al., 2001). neo, PGKneopA cassette; tk, MC1TKpA cassette; H, HindIII; K, KpnI; S, SacI; P, PauI; B, BamHI; N, NheI; Sp, SpeI. Only relevant restriction sites are shown. The positions of the external Southern blot probe, as well as the PCR primers, are indicated. (b) Southern blot of DNA from tail biopsies of wild-type, heterozygous, and homozygous mice, using the restriction enzymes BamHI and KpnI. (c) PCR of genomic DNA using the primers Mimo71 and Moré1 (c), and the primers neoL1 and Moré1. (d) Immunoblot blot analysis using whole brain from adult wild-type and NrCAM−/− mice using polyclonal antibody 463 against NrCAM. The NrCAM band is completely absent in the mutants. Additional bands are due to nonspecific binding activity of the antibody. To demonstrate equal loading, a parallel blot was analyzed with polyclonal antibodies to L1. Molecular weight markers are indicated on the left. Immunohistological studies using neural tissue as well as immunoblots of brain and lens tissue using a polyclonal antibody provided by M. Grumet also reveal that NrCAM is absent (not shown).
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Related In: Results  -  Collection

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fig1: NrCAM gene disruption. (a) Map of the targeting vector, and of part of the genomic locus before and after homologous recombination with the targeting vector. The position of exons 5, 6, and 8 as well as the exon numbering was adopted from the human NrCAM locus (Dry et al., 2001). neo, PGKneopA cassette; tk, MC1TKpA cassette; H, HindIII; K, KpnI; S, SacI; P, PauI; B, BamHI; N, NheI; Sp, SpeI. Only relevant restriction sites are shown. The positions of the external Southern blot probe, as well as the PCR primers, are indicated. (b) Southern blot of DNA from tail biopsies of wild-type, heterozygous, and homozygous mice, using the restriction enzymes BamHI and KpnI. (c) PCR of genomic DNA using the primers Mimo71 and Moré1 (c), and the primers neoL1 and Moré1. (d) Immunoblot blot analysis using whole brain from adult wild-type and NrCAM−/− mice using polyclonal antibody 463 against NrCAM. The NrCAM band is completely absent in the mutants. Additional bands are due to nonspecific binding activity of the antibody. To demonstrate equal loading, a parallel blot was analyzed with polyclonal antibodies to L1. Molecular weight markers are indicated on the left. Immunohistological studies using neural tissue as well as immunoblots of brain and lens tissue using a polyclonal antibody provided by M. Grumet also reveal that NrCAM is absent (not shown).
Mentions: To study the function of NrCAM in an in vivo situation, we generated a mouse strain deficient for this gene. To do this, we first characterized the NrCAM locus surrounding the ATG using a mouse embryonic stem (ES) cell BAC clone. A long and a short arm was subcloned from the BAC clone to construct the targeting vector to destroy the start codon within exon 4 (Fig. 1 a). Electroporated ES cells were screened for homologous recombination and used to create chimeric mice via blastocyst injection. The correct integration of the targeting vector and a deletion of the ATG-containing exon was confirmed by Southern hybridization as shown in Fig. 1 b and using the PauI-NheI fragment (region deleted in the mutant) as probe (not shown). Alternatively, the replacement of the ATG-containing exon by the neo cassette in the mutant mice was shown by PCR (Fig. 1 c). To detect NrCAM at the protein level, we generated polyclonal antibodies against a glutathione S-transferase fusion protein of the FNIII domains 1–4 of NrCAM. Immunohistochemical studies and an immunoblot from brains of both wild-type and NrCAM-deficient mice (Fig. 1 d; not shown) show the absence of NrCAM in the mutants.

Bottom Line: The NgCAM-related cell adhesion molecule (NrCAM) is an immunoglobulin superfamily member of the L1 subgroup that interacts intracellularly with ankyrins.The disorganization of fiber cells becomes histologically distinct during late embryonic development and includes abnormalities of the cytoskeleton and of connexin50-containing gap junctions.Also, these studies provide genetic evidence of an interaction between NrCAM and ankyrin-B.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany.

ABSTRACT
The NgCAM-related cell adhesion molecule (NrCAM) is an immunoglobulin superfamily member of the L1 subgroup that interacts intracellularly with ankyrins. We reveal that the absence of NrCAM causes the formation of mature cataracts in the mouse, whereas significant pathfinding errors of commissural axons at the midline of the spinal cord or of proprioceptive axon collaterals are not detected. Cataracts, the most common cause of visual impairment, are generated in NrCAM-deficient mice by a disorganization of lens fibers, followed by cellular disintegration and accumulation of cellular debris. The disorganization of fiber cells becomes histologically distinct during late embryonic development and includes abnormalities of the cytoskeleton and of connexin50-containing gap junctions. Furthermore, analysis of lenses of ankyrin-B mutant mice also reveals a disorganization of lens fibers at postnatal day 1, indistinguishable from that generated by the absence of NrCAM, indicating that NrCAM and ankyrin-B are required to maintain contact between lens fiber cells. Also, these studies provide genetic evidence of an interaction between NrCAM and ankyrin-B.

Show MeSH
Related in: MedlinePlus