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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

lamb1a induction defines cell polarity and signaling competence in basal cells of the regeneration epidermis.(A) Antibody co-staining for aPKC (red) and P63 (green; an epithelial maker for all basal and some suprabasal epithelial cells) in longitudinal sections of sde1/+ and sde1 fin regenerates at 2 dpa. (B) Antibody staining for Laminin in fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating induced mislocalization. Laminin, red; DAPI, blue. White dashed boxes indicate areas of enlarged view. (C) Antibody co-staining for aPKC (red) and P63 (green) in longitudinal sections of sde1/+ and sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating loss of basal cell polarity. iii: distal regenerated tissue (sde1/+); iv: distal regenerated tissue (sde1). White arrows indicate basal cell nuclei. (D) Antibody co-staining for phosphorylated Igf1r (red) and P63 (green) in longitudinal sections of 5 dpa sde1/+ and sde1 fin regenerates, after a temperature shift from 25°C to 33°C at 4 dpa. The basal localization of p-Igf1r is enriched in basal epithelial cells in sde1 mutants. (E) shha RNA expression is reduced in sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.
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pgen.1005437.g006: lamb1a induction defines cell polarity and signaling competence in basal cells of the regeneration epidermis.(A) Antibody co-staining for aPKC (red) and P63 (green; an epithelial maker for all basal and some suprabasal epithelial cells) in longitudinal sections of sde1/+ and sde1 fin regenerates at 2 dpa. (B) Antibody staining for Laminin in fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating induced mislocalization. Laminin, red; DAPI, blue. White dashed boxes indicate areas of enlarged view. (C) Antibody co-staining for aPKC (red) and P63 (green) in longitudinal sections of sde1/+ and sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating loss of basal cell polarity. iii: distal regenerated tissue (sde1/+); iv: distal regenerated tissue (sde1). White arrows indicate basal cell nuclei. (D) Antibody co-staining for phosphorylated Igf1r (red) and P63 (green) in longitudinal sections of 5 dpa sde1/+ and sde1 fin regenerates, after a temperature shift from 25°C to 33°C at 4 dpa. The basal localization of p-Igf1r is enriched in basal epithelial cells in sde1 mutants. (E) shha RNA expression is reduced in sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.

Mentions: Laminin is widely studied as a component of the basement membrane; thus, its induction in the basal epithelial layer strongly suggested a role in creating this structure. We examined Laminin presence in 2 dpa regenerates of sde1 mutants at 33°C, and found it ectopically localized to all basal epithelial cell regions including the apical and lateral portions (Fig 4D), indicate of intracellular residence. This result supports the idea that Lamb1a interaction with other Laminin members through the N-terminal domain may be important for secretion of Laminin complexes [39, 40]. Laminin was also aberrantly localized in sde1 juvenile fins maintained at 33°C for 14 days (Fig 5D). To examine the polarity of the basal epithelial layer at 2 dpa, we used an antibody raised against atypical Protein kinase C (aPKC), a well-characterized apical marker that is essential to maintain epithelial polarity in many systems, including nematodes, flies, and mammalian cells [43]. Whereas aPKC was localized to apical and lateral regions of basal epithelial cells by 2 dpa in wild-type fins, sde1 regenerates accumulated aPKC expression in basal regions of this cell layer (Fig 6A). This finding is consistent with in vitro [44, 45] and in vivo [46] functional studies indicating Laminin as a polarizing cue for epithelial cells.


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)

lamb1a induction defines cell polarity and signaling competence in basal cells of the regeneration epidermis.(A) Antibody co-staining for aPKC (red) and P63 (green; an epithelial maker for all basal and some suprabasal epithelial cells) in longitudinal sections of sde1/+ and sde1 fin regenerates at 2 dpa. (B) Antibody staining for Laminin in fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating induced mislocalization. Laminin, red; DAPI, blue. White dashed boxes indicate areas of enlarged view. (C) Antibody co-staining for aPKC (red) and P63 (green) in longitudinal sections of sde1/+ and sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating loss of basal cell polarity. iii: distal regenerated tissue (sde1/+); iv: distal regenerated tissue (sde1). White arrows indicate basal cell nuclei. (D) Antibody co-staining for phosphorylated Igf1r (red) and P63 (green) in longitudinal sections of 5 dpa sde1/+ and sde1 fin regenerates, after a temperature shift from 25°C to 33°C at 4 dpa. The basal localization of p-Igf1r is enriched in basal epithelial cells in sde1 mutants. (E) shha RNA expression is reduced in sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4549328&req=5

pgen.1005437.g006: lamb1a induction defines cell polarity and signaling competence in basal cells of the regeneration epidermis.(A) Antibody co-staining for aPKC (red) and P63 (green; an epithelial maker for all basal and some suprabasal epithelial cells) in longitudinal sections of sde1/+ and sde1 fin regenerates at 2 dpa. (B) Antibody staining for Laminin in fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating induced mislocalization. Laminin, red; DAPI, blue. White dashed boxes indicate areas of enlarged view. (C) Antibody co-staining for aPKC (red) and P63 (green) in longitudinal sections of sde1/+ and sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa, indicating loss of basal cell polarity. iii: distal regenerated tissue (sde1/+); iv: distal regenerated tissue (sde1). White arrows indicate basal cell nuclei. (D) Antibody co-staining for phosphorylated Igf1r (red) and P63 (green) in longitudinal sections of 5 dpa sde1/+ and sde1 fin regenerates, after a temperature shift from 25°C to 33°C at 4 dpa. The basal localization of p-Igf1r is enriched in basal epithelial cells in sde1 mutants. (E) shha RNA expression is reduced in sde1 fin regenerates at 4 dpa after a temperature shift from 25°C to 33°C at 3 dpa. Scale bars, 50 μm. White dashed boxes indicate areas of enlarged view. Red arrows indicate plane of amputation.
Mentions: Laminin is widely studied as a component of the basement membrane; thus, its induction in the basal epithelial layer strongly suggested a role in creating this structure. We examined Laminin presence in 2 dpa regenerates of sde1 mutants at 33°C, and found it ectopically localized to all basal epithelial cell regions including the apical and lateral portions (Fig 4D), indicate of intracellular residence. This result supports the idea that Lamb1a interaction with other Laminin members through the N-terminal domain may be important for secretion of Laminin complexes [39, 40]. Laminin was also aberrantly localized in sde1 juvenile fins maintained at 33°C for 14 days (Fig 5D). To examine the polarity of the basal epithelial layer at 2 dpa, we used an antibody raised against atypical Protein kinase C (aPKC), a well-characterized apical marker that is essential to maintain epithelial polarity in many systems, including nematodes, flies, and mammalian cells [43]. Whereas aPKC was localized to apical and lateral regions of basal epithelial cells by 2 dpa in wild-type fins, sde1 regenerates accumulated aPKC expression in basal regions of this cell layer (Fig 6A). This finding is consistent with in vitro [44, 45] and in vivo [46] functional studies indicating Laminin as a polarizing cue for epithelial cells.

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