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Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

Lehrberg J, Gardiner DM - PLoS ONE (2015)

Bottom Line: Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo.Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves.Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

No MeSH data available.


Proliferation in response to BMP2.Immunofluorescence showing EdU labeling of blastema slices originating from the same blastema and cultured in either basal medium or 100ng/mL BMP2. EdU positive proliferating cells are green, nuclei are stained with DAPI and are blue. The proximal (P) to distal (D) orientation of the sections is indicated. Scale bar = 1mm.
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pone.0123186.g007: Proliferation in response to BMP2.Immunofluorescence showing EdU labeling of blastema slices originating from the same blastema and cultured in either basal medium or 100ng/mL BMP2. EdU positive proliferating cells are green, nuclei are stained with DAPI and are blue. The proximal (P) to distal (D) orientation of the sections is indicated. Scale bar = 1mm.

Mentions: OSC blastema mesenchymal cells responded to increased BMP2 signaling by increasing expression of Prrx-1 to the level observed in response to 5% FBS (Fig 4). In addition, the rate of proliferation increased significantly (double that for cultures in basal medium) at all three concentrations of BMP2 that were tested (Figs 7 and 8A). The average labeling indices for all three concentrations were comparable to each other and to in vivo rates of proliferation [13,28–30]. Since the mean LI did not change in response to increasing dose, and the variability (standard error) was considerable smaller at the lowest dose tested, we concluded that 1 ng/ml BMP2 was an appropriate dose for experiments with OSC axolotl blastema cells.


Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

Lehrberg J, Gardiner DM - PLoS ONE (2015)

Proliferation in response to BMP2.Immunofluorescence showing EdU labeling of blastema slices originating from the same blastema and cultured in either basal medium or 100ng/mL BMP2. EdU positive proliferating cells are green, nuclei are stained with DAPI and are blue. The proximal (P) to distal (D) orientation of the sections is indicated. Scale bar = 1mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414535&req=5

pone.0123186.g007: Proliferation in response to BMP2.Immunofluorescence showing EdU labeling of blastema slices originating from the same blastema and cultured in either basal medium or 100ng/mL BMP2. EdU positive proliferating cells are green, nuclei are stained with DAPI and are blue. The proximal (P) to distal (D) orientation of the sections is indicated. Scale bar = 1mm.
Mentions: OSC blastema mesenchymal cells responded to increased BMP2 signaling by increasing expression of Prrx-1 to the level observed in response to 5% FBS (Fig 4). In addition, the rate of proliferation increased significantly (double that for cultures in basal medium) at all three concentrations of BMP2 that were tested (Figs 7 and 8A). The average labeling indices for all three concentrations were comparable to each other and to in vivo rates of proliferation [13,28–30]. Since the mean LI did not change in response to increasing dose, and the variability (standard error) was considerable smaller at the lowest dose tested, we concluded that 1 ng/ml BMP2 was an appropriate dose for experiments with OSC axolotl blastema cells.

Bottom Line: Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo.Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves.Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, United States of America.

ABSTRACT
We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

No MeSH data available.