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The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR - J. Cell Biol. (1997)

Bottom Line: Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes.Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains.Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

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In situ hybridization of laminin α chain probes to P1 kidney sections. α1 was observed in primitive structures in the cortex and  in some tubules (a). α2 was absent from the cortical structures but distributed diffusely in the interior (b). α4 was detected primarily in  clusters at the cortex (vesicle and comma stage nephrons) but also diffusely in the medulla (d). α5 was mainly in the collecting ducts, but  there were also grain clusters in the inner cortex and in the medulla (e). α3 was absent (c), and a sense control was negative (f). These  patterns suggest that the developmental transitions demonstrated immunohistochemically in Figs. 7 and 8 reflect, in part, developmental  transitions in α chain gene expression. The cortical surface of the kidney is outlined in white. Insets in a–e show higher power views of  the cortex. Bars: (f) 0.5 mm; (a, inset) 0.1 mm.
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Figure 9: In situ hybridization of laminin α chain probes to P1 kidney sections. α1 was observed in primitive structures in the cortex and in some tubules (a). α2 was absent from the cortical structures but distributed diffusely in the interior (b). α4 was detected primarily in clusters at the cortex (vesicle and comma stage nephrons) but also diffusely in the medulla (d). α5 was mainly in the collecting ducts, but there were also grain clusters in the inner cortex and in the medulla (e). α3 was absent (c), and a sense control was negative (f). These patterns suggest that the developmental transitions demonstrated immunohistochemically in Figs. 7 and 8 reflect, in part, developmental transitions in α chain gene expression. The cortical surface of the kidney is outlined in white. Insets in a–e show higher power views of the cortex. Bars: (f) 0.5 mm; (a, inset) 0.1 mm.

Mentions: To determine whether the isoform transitions detected at the protein level reflected regulation of gene expression, we performed in situ hybridizations on P1 kidney sections using probes for the α1–5 chains. As noted above, nephrons at all stages of development are present in a corticomedullary gradient in neonates, with the most primitive just beneath the cortical surface and the most mature at deeper levels. Laminin α4 transcripts were clustered at the cortex, as expected from its early appearance in vesicular BL (Fig. 9 d). Laminin α1 transcripts were detected in cortical and subcortical clusters, consistent with the expression of this chain in late vesicle, comma, and S-shaped stages (Fig. 9 a). Segments of tubules were also α1 positive, and lower level expression (above background) was found throughout the kidney. Low levels of laminin α5 RNA were present in the superficial layer of the cortex, consistent with the later appearance of this chain in the developing nephron. Occasional clusters that were observed are likely to be the tips of the ureteric buds that have BLs rich in α5 (Fig. 9 e). Deep in the medulla, laminin α5 RNA was abundant in the collecting ducts, which are derived from the α5-positive ureteric bud. α5 labeling was not abundant in all structures that contained the protein (e.g., capillary loop stage glomeruli), suggesting that, in some cases, α5 RNA is unstable or simply present at low levels but translated efficiently. Laminin α2 RNA was concentrated in the deep cortical and medullary portions of the kidney (Fig. 9 b), consistent with its localization in a subset of tubules (Fig. 6 b). Laminin α3 RNA was not detectable within the renal cortex but was found at P15 in the papilla (data not shown), consistent with its protein localization in adult kidney (Fig. 6 c).


The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform.

Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR - J. Cell Biol. (1997)

In situ hybridization of laminin α chain probes to P1 kidney sections. α1 was observed in primitive structures in the cortex and  in some tubules (a). α2 was absent from the cortical structures but distributed diffusely in the interior (b). α4 was detected primarily in  clusters at the cortex (vesicle and comma stage nephrons) but also diffusely in the medulla (d). α5 was mainly in the collecting ducts, but  there were also grain clusters in the inner cortex and in the medulla (e). α3 was absent (c), and a sense control was negative (f). These  patterns suggest that the developmental transitions demonstrated immunohistochemically in Figs. 7 and 8 reflect, in part, developmental  transitions in α chain gene expression. The cortical surface of the kidney is outlined in white. Insets in a–e show higher power views of  the cortex. Bars: (f) 0.5 mm; (a, inset) 0.1 mm.
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Figure 9: In situ hybridization of laminin α chain probes to P1 kidney sections. α1 was observed in primitive structures in the cortex and in some tubules (a). α2 was absent from the cortical structures but distributed diffusely in the interior (b). α4 was detected primarily in clusters at the cortex (vesicle and comma stage nephrons) but also diffusely in the medulla (d). α5 was mainly in the collecting ducts, but there were also grain clusters in the inner cortex and in the medulla (e). α3 was absent (c), and a sense control was negative (f). These patterns suggest that the developmental transitions demonstrated immunohistochemically in Figs. 7 and 8 reflect, in part, developmental transitions in α chain gene expression. The cortical surface of the kidney is outlined in white. Insets in a–e show higher power views of the cortex. Bars: (f) 0.5 mm; (a, inset) 0.1 mm.
Mentions: To determine whether the isoform transitions detected at the protein level reflected regulation of gene expression, we performed in situ hybridizations on P1 kidney sections using probes for the α1–5 chains. As noted above, nephrons at all stages of development are present in a corticomedullary gradient in neonates, with the most primitive just beneath the cortical surface and the most mature at deeper levels. Laminin α4 transcripts were clustered at the cortex, as expected from its early appearance in vesicular BL (Fig. 9 d). Laminin α1 transcripts were detected in cortical and subcortical clusters, consistent with the expression of this chain in late vesicle, comma, and S-shaped stages (Fig. 9 a). Segments of tubules were also α1 positive, and lower level expression (above background) was found throughout the kidney. Low levels of laminin α5 RNA were present in the superficial layer of the cortex, consistent with the later appearance of this chain in the developing nephron. Occasional clusters that were observed are likely to be the tips of the ureteric buds that have BLs rich in α5 (Fig. 9 e). Deep in the medulla, laminin α5 RNA was abundant in the collecting ducts, which are derived from the α5-positive ureteric bud. α5 labeling was not abundant in all structures that contained the protein (e.g., capillary loop stage glomeruli), suggesting that, in some cases, α5 RNA is unstable or simply present at low levels but translated efficiently. Laminin α2 RNA was concentrated in the deep cortical and medullary portions of the kidney (Fig. 9 b), consistent with its localization in a subset of tubules (Fig. 6 b). Laminin α3 RNA was not detectable within the renal cortex but was found at P15 in the papilla (data not shown), consistent with its protein localization in adult kidney (Fig. 6 c).

Bottom Line: Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes.Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains.Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
Laminin trimers composed of alpha, beta, and gamma chains are major components of basal laminae (BLs) throughout the body. To date, three alpha chains (alpha1-3) have been shown to assemble into at least seven heterotrimers (called laminins 1-7). Genes encoding two additional alpha chains (alpha4 and alpha5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant alpha4 and alpha5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that alpha4 and alpha5 assemble into four novel laminin heterotrimers (laminins 8-11: alpha4beta1gamma1, alpha4beta2gamma1, alpha5beta1gamma1, and alpha5beta2gamma1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of alpha1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, alpha4 and alpha5 exhibited the broadest patterns of expression, while expression of alpha1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the alpha chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one alpha chain, all alpha chains were present in multiple BLs, and some BLs contained two or three alpha chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different alpha chains and two different beta chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five alpha chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length alpha3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Show MeSH
Related in: MedlinePlus