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Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain.

Miner JH, Cunningham J, Sanes JR - J. Cell Biol. (1998)

Bottom Line: Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development.Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate.Our results identify new roles for laminins and BLs in diverse developmental processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Renal Division, St. Louis, Missouri, USA.

ABSTRACT
Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are alpha/beta/gamma heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin alpha5 chain is required during embryogenesis. The alpha5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose alpha5 retain or acquire other alpha chains. Embryos lacking laminin alpha5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are alpha5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.

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Related in: MedlinePlus

Targeted mutagenesis of the Lama5 gene. (A) The targeting vector deleted exons encoding 113 amino acids and replaced them with an in frame lacZ cDNA and the PGK neo selectable marker. N, NcoI; X, XbaI. (B) Southern analysis of  genomic DNA from E13.5 embryos demonstrates the existence  of the three expected genotypes, confirming that targeting was  successful and that homozygous mutants were alive at this age.  The probe, shown in A, was from outside the short arm of the targeting vector. (C and D) Presence of laminin α5 protein in control (C) but not mutant (D) distal limb ectodermal BL at E13.5,  detected immunohistochemically with antisera to epitopes outside of the targeted region. Bar, 50 μm.
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Figure 2: Targeted mutagenesis of the Lama5 gene. (A) The targeting vector deleted exons encoding 113 amino acids and replaced them with an in frame lacZ cDNA and the PGK neo selectable marker. N, NcoI; X, XbaI. (B) Southern analysis of genomic DNA from E13.5 embryos demonstrates the existence of the three expected genotypes, confirming that targeting was successful and that homozygous mutants were alive at this age. The probe, shown in A, was from outside the short arm of the targeting vector. (C and D) Presence of laminin α5 protein in control (C) but not mutant (D) distal limb ectodermal BL at E13.5, detected immunohistochemically with antisera to epitopes outside of the targeted region. Bar, 50 μm.

Mentions: A λ clone containing exons encoding parts of domains VI, V, and IVb of laminin α5 was obtained by screening a 129sv mouse genomic library (Stratagene, La Jolla, CA) with the 5′ 900 bp of the cDNA previously described by Miner et al. (1995). To construct a targeting vector, two consecutive XbaI fragments totaling 3.5 kb and encoding 113 amino acids (129– 241 in Miner et al., 1995) were replaced with an in frame lacZ cDNA and a PGK neo cassette. The neo cassette was derived from the vector pPNT, which also supplied PGK HSV-tk for negative selection (Tybulewicz et al., 1991; see Fig. 2 A). The mutated chromosomal segment was transferred to R1 ES cells by electroporation, and transfectants were selected with G418 (400 μg/ml) and FIAU (0.2 μM). Approximately 450 clones were screened by PCR, and a single homologous recombinant clone was obtained. Cells from this clone were injected into C57BL/6J blastocysts by standard methods. Three chimeras transmitted the mutation to their offspring. Homozygotes derived from each of these founders exhibited the same phenotypes, and all phenotypes segregated with the mutation, even after five generations of crossing to C57BL/6J mice. We never detected lacZ activity in heterozygotes or homozygotes, presumably because the signal sequence of the laminin α5 protein forces the catalytic domain of the lacZ protein into the endoplasmic reticulum, where it is inactive.


Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin alpha5 chain.

Miner JH, Cunningham J, Sanes JR - J. Cell Biol. (1998)

Targeted mutagenesis of the Lama5 gene. (A) The targeting vector deleted exons encoding 113 amino acids and replaced them with an in frame lacZ cDNA and the PGK neo selectable marker. N, NcoI; X, XbaI. (B) Southern analysis of  genomic DNA from E13.5 embryos demonstrates the existence  of the three expected genotypes, confirming that targeting was  successful and that homozygous mutants were alive at this age.  The probe, shown in A, was from outside the short arm of the targeting vector. (C and D) Presence of laminin α5 protein in control (C) but not mutant (D) distal limb ectodermal BL at E13.5,  detected immunohistochemically with antisera to epitopes outside of the targeted region. Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132973&req=5

Figure 2: Targeted mutagenesis of the Lama5 gene. (A) The targeting vector deleted exons encoding 113 amino acids and replaced them with an in frame lacZ cDNA and the PGK neo selectable marker. N, NcoI; X, XbaI. (B) Southern analysis of genomic DNA from E13.5 embryos demonstrates the existence of the three expected genotypes, confirming that targeting was successful and that homozygous mutants were alive at this age. The probe, shown in A, was from outside the short arm of the targeting vector. (C and D) Presence of laminin α5 protein in control (C) but not mutant (D) distal limb ectodermal BL at E13.5, detected immunohistochemically with antisera to epitopes outside of the targeted region. Bar, 50 μm.
Mentions: A λ clone containing exons encoding parts of domains VI, V, and IVb of laminin α5 was obtained by screening a 129sv mouse genomic library (Stratagene, La Jolla, CA) with the 5′ 900 bp of the cDNA previously described by Miner et al. (1995). To construct a targeting vector, two consecutive XbaI fragments totaling 3.5 kb and encoding 113 amino acids (129– 241 in Miner et al., 1995) were replaced with an in frame lacZ cDNA and a PGK neo cassette. The neo cassette was derived from the vector pPNT, which also supplied PGK HSV-tk for negative selection (Tybulewicz et al., 1991; see Fig. 2 A). The mutated chromosomal segment was transferred to R1 ES cells by electroporation, and transfectants were selected with G418 (400 μg/ml) and FIAU (0.2 μM). Approximately 450 clones were screened by PCR, and a single homologous recombinant clone was obtained. Cells from this clone were injected into C57BL/6J blastocysts by standard methods. Three chimeras transmitted the mutation to their offspring. Homozygotes derived from each of these founders exhibited the same phenotypes, and all phenotypes segregated with the mutation, even after five generations of crossing to C57BL/6J mice. We never detected lacZ activity in heterozygotes or homozygotes, presumably because the signal sequence of the laminin α5 protein forces the catalytic domain of the lacZ protein into the endoplasmic reticulum, where it is inactive.

Bottom Line: Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development.Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate.Our results identify new roles for laminins and BLs in diverse developmental processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Renal Division, St. Louis, Missouri, USA.

ABSTRACT
Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are alpha/beta/gamma heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin alpha5 chain is required during embryogenesis. The alpha5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose alpha5 retain or acquire other alpha chains. Embryos lacking laminin alpha5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are alpha5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin alpha chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.

Show MeSH
Related in: MedlinePlus