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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

Association of lamb1a expression and function with key regeneration effector pathways.(A) Longitudinal sections of 2 dpa fin regenerates stained for lamb1a by ISH, indicating sparse expression in fgf20a mutants (dob). Scale bars, 50 μm. (B) RT-qPCR analysis indicating depleted levels of lamb1a RNA in fgf20a mutants (left). When Fgf signaling is blocked by induced expression of a dominant–negative Fgf receptor for just 6 hours at 4 dpa, lamb1a levels drop by nearly 50%. qPCR results were normalized to rpl13a and to the basal expression of lamb1a at 0 hpa. (n = 4; mean ± SEM; Student’s t -test, ***P < 0.001, **P < 0.01). (C) Whole-mount images of fin regenerates stained by alizarin red staining for calcium deposition, after treatment of sde1 animals with DMSO or FK506. Scale bars, 0.5 mm. (D) Measurement of the length of alizarin red-positive domains at 7 dpa (n = 9 and 10; mean ± SEM; Student’s t -test, ***P < 0.001). (E) Antibody staining for Laminin protein in vehicle- or FK506-treated sde1 fin regenerates at 7 dpa. Laminin, red; DAPI, blue. (F) Antibody co-staining for aPKC (red) and P63 (green) expression in vehicle or FK506-treated sde1 fin regenerates at 7 dpa. FK506 treatment partially rescued bone regeneration in sde1 mutants, with no detectable impact on Laminin localization or basal epithelial cell polarity. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. White arrows indicate basal cell nuclei. Red arrows indicate plane of amputation.
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pgen.1005437.g007: Association of lamb1a expression and function with key regeneration effector pathways.(A) Longitudinal sections of 2 dpa fin regenerates stained for lamb1a by ISH, indicating sparse expression in fgf20a mutants (dob). Scale bars, 50 μm. (B) RT-qPCR analysis indicating depleted levels of lamb1a RNA in fgf20a mutants (left). When Fgf signaling is blocked by induced expression of a dominant–negative Fgf receptor for just 6 hours at 4 dpa, lamb1a levels drop by nearly 50%. qPCR results were normalized to rpl13a and to the basal expression of lamb1a at 0 hpa. (n = 4; mean ± SEM; Student’s t -test, ***P < 0.001, **P < 0.01). (C) Whole-mount images of fin regenerates stained by alizarin red staining for calcium deposition, after treatment of sde1 animals with DMSO or FK506. Scale bars, 0.5 mm. (D) Measurement of the length of alizarin red-positive domains at 7 dpa (n = 9 and 10; mean ± SEM; Student’s t -test, ***P < 0.001). (E) Antibody staining for Laminin protein in vehicle- or FK506-treated sde1 fin regenerates at 7 dpa. Laminin, red; DAPI, blue. (F) Antibody co-staining for aPKC (red) and P63 (green) expression in vehicle or FK506-treated sde1 fin regenerates at 7 dpa. FK506 treatment partially rescued bone regeneration in sde1 mutants, with no detectable impact on Laminin localization or basal epithelial cell polarity. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. White arrows indicate basal cell nuclei. Red arrows indicate plane of amputation.

Mentions: Like lamb1a, the Fgf ligand gene fgf20a is induced during zebrafish fin regeneration. Moreover, fgf20a mutants are also defective in lef1 induction and maturation of the regeneration epidermis [20]. Fgf signaling has been implicated in control of Laminin production in the context of embryoid bodies [47]. Because of these links, we investigated possible expression associations between lamb1a and fgf20a. We found that the induction of fgf20a was not affected in sde1 regenerates (S6A Fig). By contrast, lamb1a induction was severely disrupted in fgf20a mutants at 2 dpa (Fig 7A and 7B), at which point Laminin protein was detectable at very low levels along the epithelial-mesenchymal boundary (S6B Fig). To determine whether Fgf signaling actively sustains lamb1a expression during regeneration, we employed a transgenic line Tg(hsp70:dnfgfr1-EGFP)pd1 that drives a dominant negative Fgfr1 cassette from a heat-shock-inducible promoter [48]. A single heat-shock treatment at 4 dpa to transiently attenuate Fgf signaling during regeneration was sufficient to reduce lamb1a expression by 48% within 6 hours (Fig 7B), suggesting direct control of lamb1a at the transcriptional level by Fgf signaling. By contrast, 24 hours treatment from 3 to 4 dpa with either the Igf receptor antagonist NVP-AEW541 or the Igf signaling agonist NBI-31772 did not significant alter lamb1a expression (S6C Fig). Similarly, the induction of lamb1a was not affected in sde1 regenerates at 2 dpa at the restrictive temperature, as assayed by qPCR (S6D Fig). These results implicate fgf20a upstream of lamb1a in activation of morphogenesis of the regeneration epidermis.


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)

Association of lamb1a expression and function with key regeneration effector pathways.(A) Longitudinal sections of 2 dpa fin regenerates stained for lamb1a by ISH, indicating sparse expression in fgf20a mutants (dob). Scale bars, 50 μm. (B) RT-qPCR analysis indicating depleted levels of lamb1a RNA in fgf20a mutants (left). When Fgf signaling is blocked by induced expression of a dominant–negative Fgf receptor for just 6 hours at 4 dpa, lamb1a levels drop by nearly 50%. qPCR results were normalized to rpl13a and to the basal expression of lamb1a at 0 hpa. (n = 4; mean ± SEM; Student’s t -test, ***P < 0.001, **P < 0.01). (C) Whole-mount images of fin regenerates stained by alizarin red staining for calcium deposition, after treatment of sde1 animals with DMSO or FK506. Scale bars, 0.5 mm. (D) Measurement of the length of alizarin red-positive domains at 7 dpa (n = 9 and 10; mean ± SEM; Student’s t -test, ***P < 0.001). (E) Antibody staining for Laminin protein in vehicle- or FK506-treated sde1 fin regenerates at 7 dpa. Laminin, red; DAPI, blue. (F) Antibody co-staining for aPKC (red) and P63 (green) expression in vehicle or FK506-treated sde1 fin regenerates at 7 dpa. FK506 treatment partially rescued bone regeneration in sde1 mutants, with no detectable impact on Laminin localization or basal epithelial cell polarity. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. White arrows indicate basal cell nuclei. Red arrows indicate plane of amputation.
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pgen.1005437.g007: Association of lamb1a expression and function with key regeneration effector pathways.(A) Longitudinal sections of 2 dpa fin regenerates stained for lamb1a by ISH, indicating sparse expression in fgf20a mutants (dob). Scale bars, 50 μm. (B) RT-qPCR analysis indicating depleted levels of lamb1a RNA in fgf20a mutants (left). When Fgf signaling is blocked by induced expression of a dominant–negative Fgf receptor for just 6 hours at 4 dpa, lamb1a levels drop by nearly 50%. qPCR results were normalized to rpl13a and to the basal expression of lamb1a at 0 hpa. (n = 4; mean ± SEM; Student’s t -test, ***P < 0.001, **P < 0.01). (C) Whole-mount images of fin regenerates stained by alizarin red staining for calcium deposition, after treatment of sde1 animals with DMSO or FK506. Scale bars, 0.5 mm. (D) Measurement of the length of alizarin red-positive domains at 7 dpa (n = 9 and 10; mean ± SEM; Student’s t -test, ***P < 0.001). (E) Antibody staining for Laminin protein in vehicle- or FK506-treated sde1 fin regenerates at 7 dpa. Laminin, red; DAPI, blue. (F) Antibody co-staining for aPKC (red) and P63 (green) expression in vehicle or FK506-treated sde1 fin regenerates at 7 dpa. FK506 treatment partially rescued bone regeneration in sde1 mutants, with no detectable impact on Laminin localization or basal epithelial cell polarity. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. White arrows indicate basal cell nuclei. Red arrows indicate plane of amputation.
Mentions: Like lamb1a, the Fgf ligand gene fgf20a is induced during zebrafish fin regeneration. Moreover, fgf20a mutants are also defective in lef1 induction and maturation of the regeneration epidermis [20]. Fgf signaling has been implicated in control of Laminin production in the context of embryoid bodies [47]. Because of these links, we investigated possible expression associations between lamb1a and fgf20a. We found that the induction of fgf20a was not affected in sde1 regenerates (S6A Fig). By contrast, lamb1a induction was severely disrupted in fgf20a mutants at 2 dpa (Fig 7A and 7B), at which point Laminin protein was detectable at very low levels along the epithelial-mesenchymal boundary (S6B Fig). To determine whether Fgf signaling actively sustains lamb1a expression during regeneration, we employed a transgenic line Tg(hsp70:dnfgfr1-EGFP)pd1 that drives a dominant negative Fgfr1 cassette from a heat-shock-inducible promoter [48]. A single heat-shock treatment at 4 dpa to transiently attenuate Fgf signaling during regeneration was sufficient to reduce lamb1a expression by 48% within 6 hours (Fig 7B), suggesting direct control of lamb1a at the transcriptional level by Fgf signaling. By contrast, 24 hours treatment from 3 to 4 dpa with either the Igf receptor antagonist NVP-AEW541 or the Igf signaling agonist NBI-31772 did not significant alter lamb1a expression (S6C Fig). Similarly, the induction of lamb1a was not affected in sde1 regenerates at 2 dpa at the restrictive temperature, as assayed by qPCR (S6D Fig). These results implicate fgf20a upstream of lamb1a in activation of morphogenesis of the regeneration epidermis.

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