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Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration.

Chen CH, Merriman AF, Savage J, Willer J, Wahlig T, Katsanis N, Yin VP, Poss KD - PLoS Genet. (2015)

Bottom Line: Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins.These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone.By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Howard Hughes Medical Institute, Duke University School of Medicine, Durham, North Carolina, United States of America.

ABSTRACT
The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.

No MeSH data available.


Related in: MedlinePlus

sde1 encodes a laminin beta1 paralog.(A) Analysis of genomic homozygosity in sde1 mutants. (B) Log likelihood analysis using SNPtrack. (C) High-resolution mapping using linked SNP markers. Three and five recombinants were found for SNP32559436 and SNP32681121, respectively, on each side of a 121 kb region. After genotyping 453 adult animals, no recombinants were identified for a novel SNP at position 32605161. (D) Sanger sequencing readouts from wild-type and sde1 cDNA. SNP32605161 is within the coding region of the gene laminin beta 1a (lamb1a), causing a leucine to proline change. (E) Amino acid alignment across distant species. Red star marks the location of the leucine. Differential gray scale indicates level of conservation across listed species. (F) Cartoon depicting major structural domains in Lamb1a. Blue and red arrows indicate the locations of the sde1 mutation, along with two previously identified alleles gupm189 and s804. Lam NT, Laminin N-terminal domain; Blue hexagons, Laminin-type epidermal growth factor-like domain; CC, uncharacterized coiled-coil domain. (G) sde1 embryos incubated at 31°C have shortened trunks. Representative embryos from an sde1 x sde1/+ cross. Embryos were transferred to 31°C at 3 hours post-fertilization (hpf). Images were acquired at 28 hpf. Approximately 48% of embryos (32 out of 67) showed phenotypes representative of the gupm189 mutation after the temperature shift, consistent with expected Mendelian ratio. The phenotype and the ratio were consistent across three independent crosses. (H) Complementation tests showing both gupm189 and s804 alleles fail to complement the 7 dpa regeneration defects of the sde1 mutation in adult animals, yielding expected ratios (~50%; n = 65 and 30). Red dashed lines indicate plane of amputation. Scale bars, 1 mm.
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pgen.1005437.g003: sde1 encodes a laminin beta1 paralog.(A) Analysis of genomic homozygosity in sde1 mutants. (B) Log likelihood analysis using SNPtrack. (C) High-resolution mapping using linked SNP markers. Three and five recombinants were found for SNP32559436 and SNP32681121, respectively, on each side of a 121 kb region. After genotyping 453 adult animals, no recombinants were identified for a novel SNP at position 32605161. (D) Sanger sequencing readouts from wild-type and sde1 cDNA. SNP32605161 is within the coding region of the gene laminin beta 1a (lamb1a), causing a leucine to proline change. (E) Amino acid alignment across distant species. Red star marks the location of the leucine. Differential gray scale indicates level of conservation across listed species. (F) Cartoon depicting major structural domains in Lamb1a. Blue and red arrows indicate the locations of the sde1 mutation, along with two previously identified alleles gupm189 and s804. Lam NT, Laminin N-terminal domain; Blue hexagons, Laminin-type epidermal growth factor-like domain; CC, uncharacterized coiled-coil domain. (G) sde1 embryos incubated at 31°C have shortened trunks. Representative embryos from an sde1 x sde1/+ cross. Embryos were transferred to 31°C at 3 hours post-fertilization (hpf). Images were acquired at 28 hpf. Approximately 48% of embryos (32 out of 67) showed phenotypes representative of the gupm189 mutation after the temperature shift, consistent with expected Mendelian ratio. The phenotype and the ratio were consistent across three independent crosses. (H) Complementation tests showing both gupm189 and s804 alleles fail to complement the 7 dpa regeneration defects of the sde1 mutation in adult animals, yielding expected ratios (~50%; n = 65 and 30). Red dashed lines indicate plane of amputation. Scale bars, 1 mm.

Mentions: To identify the gene that is disrupted in sde1 mutants, we performed whole-exome sequencing of clutchmate DNA from an sde1 x sde1/+ cross (see Materials and Methods) [36]. Sequencing data were analyzed using the web-based mapping tool SNPtrack, which facilitates linkage analysis based on single nucleotide polymorphisms (SNPs) and regions of homozygosity [37]. Primary analysis from SNPtrack revealed a single 6.2 Mb peak on chromosome 25 containing sde1 (Fig 3A and 3B). We genotyped 453 adults from several sde1 × sde1/+ mapping crosses for polymorphic SNPs in this region. From this linkage analysis, we identified 3 closely linked SNPs that flanked a 121 kb region containing two genes: laminin beta 1a (lamb1a) and laminin beta 4b (lamb4b) (Fig 3C). By filtering against a SNP database established in the Poss lab (see Materials and Methods) and cDNA sequencing, we identified one novel non-synonymous mutation (T to C) in the coding region of the gene lamb1a causing a lysine to proline change at position 46 (Fig 3D). No coding mutations were identified in lamb4b. Zebrafish have two unlinked paralogs encoding laminin beta 1, lamb1a and lamb1b, emerging from a partial genome duplication event estimated at 350 million years ago [38]. Sequence analysis revealed that lysine 46 is conserved between these two zebrafish paralogs, and also among laminin beta 1 genes in other vertebrate species like human and Xenopus laevis (Fig 3E). Additionally, the candidate sde1 mutation is located in a highly conserved Laminin N-terminal (Lam NT) domain (Fig 3F), which has been demonstrated to mediate interactions with other Laminin members [39, 40]. Thus, genetic mapping associates sde1 with a mutation in a conserved residue of the laminin beta 1 paralog, lamb1a.


Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration.

Chen CH, Merriman AF, Savage J, Willer J, Wahlig T, Katsanis N, Yin VP, Poss KD - PLoS Genet. (2015)

sde1 encodes a laminin beta1 paralog.(A) Analysis of genomic homozygosity in sde1 mutants. (B) Log likelihood analysis using SNPtrack. (C) High-resolution mapping using linked SNP markers. Three and five recombinants were found for SNP32559436 and SNP32681121, respectively, on each side of a 121 kb region. After genotyping 453 adult animals, no recombinants were identified for a novel SNP at position 32605161. (D) Sanger sequencing readouts from wild-type and sde1 cDNA. SNP32605161 is within the coding region of the gene laminin beta 1a (lamb1a), causing a leucine to proline change. (E) Amino acid alignment across distant species. Red star marks the location of the leucine. Differential gray scale indicates level of conservation across listed species. (F) Cartoon depicting major structural domains in Lamb1a. Blue and red arrows indicate the locations of the sde1 mutation, along with two previously identified alleles gupm189 and s804. Lam NT, Laminin N-terminal domain; Blue hexagons, Laminin-type epidermal growth factor-like domain; CC, uncharacterized coiled-coil domain. (G) sde1 embryos incubated at 31°C have shortened trunks. Representative embryos from an sde1 x sde1/+ cross. Embryos were transferred to 31°C at 3 hours post-fertilization (hpf). Images were acquired at 28 hpf. Approximately 48% of embryos (32 out of 67) showed phenotypes representative of the gupm189 mutation after the temperature shift, consistent with expected Mendelian ratio. The phenotype and the ratio were consistent across three independent crosses. (H) Complementation tests showing both gupm189 and s804 alleles fail to complement the 7 dpa regeneration defects of the sde1 mutation in adult animals, yielding expected ratios (~50%; n = 65 and 30). Red dashed lines indicate plane of amputation. Scale bars, 1 mm.
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pgen.1005437.g003: sde1 encodes a laminin beta1 paralog.(A) Analysis of genomic homozygosity in sde1 mutants. (B) Log likelihood analysis using SNPtrack. (C) High-resolution mapping using linked SNP markers. Three and five recombinants were found for SNP32559436 and SNP32681121, respectively, on each side of a 121 kb region. After genotyping 453 adult animals, no recombinants were identified for a novel SNP at position 32605161. (D) Sanger sequencing readouts from wild-type and sde1 cDNA. SNP32605161 is within the coding region of the gene laminin beta 1a (lamb1a), causing a leucine to proline change. (E) Amino acid alignment across distant species. Red star marks the location of the leucine. Differential gray scale indicates level of conservation across listed species. (F) Cartoon depicting major structural domains in Lamb1a. Blue and red arrows indicate the locations of the sde1 mutation, along with two previously identified alleles gupm189 and s804. Lam NT, Laminin N-terminal domain; Blue hexagons, Laminin-type epidermal growth factor-like domain; CC, uncharacterized coiled-coil domain. (G) sde1 embryos incubated at 31°C have shortened trunks. Representative embryos from an sde1 x sde1/+ cross. Embryos were transferred to 31°C at 3 hours post-fertilization (hpf). Images were acquired at 28 hpf. Approximately 48% of embryos (32 out of 67) showed phenotypes representative of the gupm189 mutation after the temperature shift, consistent with expected Mendelian ratio. The phenotype and the ratio were consistent across three independent crosses. (H) Complementation tests showing both gupm189 and s804 alleles fail to complement the 7 dpa regeneration defects of the sde1 mutation in adult animals, yielding expected ratios (~50%; n = 65 and 30). Red dashed lines indicate plane of amputation. Scale bars, 1 mm.
Mentions: To identify the gene that is disrupted in sde1 mutants, we performed whole-exome sequencing of clutchmate DNA from an sde1 x sde1/+ cross (see Materials and Methods) [36]. Sequencing data were analyzed using the web-based mapping tool SNPtrack, which facilitates linkage analysis based on single nucleotide polymorphisms (SNPs) and regions of homozygosity [37]. Primary analysis from SNPtrack revealed a single 6.2 Mb peak on chromosome 25 containing sde1 (Fig 3A and 3B). We genotyped 453 adults from several sde1 × sde1/+ mapping crosses for polymorphic SNPs in this region. From this linkage analysis, we identified 3 closely linked SNPs that flanked a 121 kb region containing two genes: laminin beta 1a (lamb1a) and laminin beta 4b (lamb4b) (Fig 3C). By filtering against a SNP database established in the Poss lab (see Materials and Methods) and cDNA sequencing, we identified one novel non-synonymous mutation (T to C) in the coding region of the gene lamb1a causing a lysine to proline change at position 46 (Fig 3D). No coding mutations were identified in lamb4b. Zebrafish have two unlinked paralogs encoding laminin beta 1, lamb1a and lamb1b, emerging from a partial genome duplication event estimated at 350 million years ago [38]. Sequence analysis revealed that lysine 46 is conserved between these two zebrafish paralogs, and also among laminin beta 1 genes in other vertebrate species like human and Xenopus laevis (Fig 3E). Additionally, the candidate sde1 mutation is located in a highly conserved Laminin N-terminal (Lam NT) domain (Fig 3F), which has been demonstrated to mediate interactions with other Laminin members [39, 40]. Thus, genetic mapping associates sde1 with a mutation in a conserved residue of the laminin beta 1 paralog, lamb1a.

Bottom Line: Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins.These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone.By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Howard Hughes Medical Institute, Duke University School of Medicine, Durham, North Carolina, United States of America.

ABSTRACT
The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.

No MeSH data available.


Related in: MedlinePlus