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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

Forward genetic screen for signaling defects in the fin regeneration epidermis.(A) Whole-mount images of wild-type and (chc1-9) mutant regenerates at 7 days post amputation (dpa). Red dashed lines indicate plane of amputation. Scale bars, 1 mm. (B) Whole-mount RNA ISH of lef1 expression in wild-type and mutant regenerates at 1 dpa. chc1, chc3, and chc4 mutant families show reduced or undetectable lef1 expression when compared to their respective heterozygous siblings. (C, D) Longitudinal sections of 2 dpa fin regenerates assessed by RNA ISH, showing reduced lef1 and shha in chc1 mutant regenerates. (E) Whole-mount images of sde1 (formerly, chc1) fin regenerates at 4 dpa. Scale bars, 0.5 mm. (F) Longitudinal sections of 3 dpa fin regenerates show impaired patterning of osteoblasts in sde1 (chc1) mutants, assessed by Zns-5 antibody staining (green). DAPI, blue. Scale bars, 50 μm (unless otherwise indicated). Red arrows indicate plane of amputation.
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pgen.1005437.g001: Forward genetic screen for signaling defects in the fin regeneration epidermis.(A) Whole-mount images of wild-type and (chc1-9) mutant regenerates at 7 days post amputation (dpa). Red dashed lines indicate plane of amputation. Scale bars, 1 mm. (B) Whole-mount RNA ISH of lef1 expression in wild-type and mutant regenerates at 1 dpa. chc1, chc3, and chc4 mutant families show reduced or undetectable lef1 expression when compared to their respective heterozygous siblings. (C, D) Longitudinal sections of 2 dpa fin regenerates assessed by RNA ISH, showing reduced lef1 and shha in chc1 mutant regenerates. (E) Whole-mount images of sde1 (formerly, chc1) fin regenerates at 4 dpa. Scale bars, 0.5 mm. (F) Longitudinal sections of 3 dpa fin regenerates show impaired patterning of osteoblasts in sde1 (chc1) mutants, assessed by Zns-5 antibody staining (green). DAPI, blue. Scale bars, 50 μm (unless otherwise indicated). Red arrows indicate plane of amputation.

Mentions: Previous genetic screens for mutants in zebrafish fin regeneration involved parthenogenesis of F1 generation females [1, 28]. This approach saves considerable time and animal facility space, as progeny with homozygous ENU-induced mutations can be screened in the F2 generation. Yet, it also limits animal survival and access to chromosomal regions far from centromeres [29]. For this study, we conducted a three-generation screen, in which we raised 423 F3 families from 108 F2 generation crosses to adulthood at a temperature of 25°C. To identify temperature-sensitive mutants that can be used for toggling gene function, we shifted these adults to 33°C after amputating ~50% of the caudal fins, and then assessed regeneration 7 days later. After several rounds of outcrossing to identify stable phenotypes and dilute unlinked ENU mutations from the genetic background, we found 9 families (chc1-9) with temperature-sensitive defects in fin regeneration inherited as a single recessive determinant (Fig 1A).


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)

Forward genetic screen for signaling defects in the fin regeneration epidermis.(A) Whole-mount images of wild-type and (chc1-9) mutant regenerates at 7 days post amputation (dpa). Red dashed lines indicate plane of amputation. Scale bars, 1 mm. (B) Whole-mount RNA ISH of lef1 expression in wild-type and mutant regenerates at 1 dpa. chc1, chc3, and chc4 mutant families show reduced or undetectable lef1 expression when compared to their respective heterozygous siblings. (C, D) Longitudinal sections of 2 dpa fin regenerates assessed by RNA ISH, showing reduced lef1 and shha in chc1 mutant regenerates. (E) Whole-mount images of sde1 (formerly, chc1) fin regenerates at 4 dpa. Scale bars, 0.5 mm. (F) Longitudinal sections of 3 dpa fin regenerates show impaired patterning of osteoblasts in sde1 (chc1) mutants, assessed by Zns-5 antibody staining (green). DAPI, blue. Scale bars, 50 μm (unless otherwise indicated). Red arrows indicate plane of amputation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4549328&req=5

pgen.1005437.g001: Forward genetic screen for signaling defects in the fin regeneration epidermis.(A) Whole-mount images of wild-type and (chc1-9) mutant regenerates at 7 days post amputation (dpa). Red dashed lines indicate plane of amputation. Scale bars, 1 mm. (B) Whole-mount RNA ISH of lef1 expression in wild-type and mutant regenerates at 1 dpa. chc1, chc3, and chc4 mutant families show reduced or undetectable lef1 expression when compared to their respective heterozygous siblings. (C, D) Longitudinal sections of 2 dpa fin regenerates assessed by RNA ISH, showing reduced lef1 and shha in chc1 mutant regenerates. (E) Whole-mount images of sde1 (formerly, chc1) fin regenerates at 4 dpa. Scale bars, 0.5 mm. (F) Longitudinal sections of 3 dpa fin regenerates show impaired patterning of osteoblasts in sde1 (chc1) mutants, assessed by Zns-5 antibody staining (green). DAPI, blue. Scale bars, 50 μm (unless otherwise indicated). Red arrows indicate plane of amputation.
Mentions: Previous genetic screens for mutants in zebrafish fin regeneration involved parthenogenesis of F1 generation females [1, 28]. This approach saves considerable time and animal facility space, as progeny with homozygous ENU-induced mutations can be screened in the F2 generation. Yet, it also limits animal survival and access to chromosomal regions far from centromeres [29]. For this study, we conducted a three-generation screen, in which we raised 423 F3 families from 108 F2 generation crosses to adulthood at a temperature of 25°C. To identify temperature-sensitive mutants that can be used for toggling gene function, we shifted these adults to 33°C after amputating ~50% of the caudal fins, and then assessed regeneration 7 days later. After several rounds of outcrossing to identify stable phenotypes and dilute unlinked ENU mutations from the genetic background, we found 9 families (chc1-9) with temperature-sensitive defects in fin regeneration inherited as a single recessive determinant (Fig 1A).

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