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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.


Related in: MedlinePlus

Nuclear P-Smad 1/5/8 fluorescence in response to BMP2.Nuclear Phospho-Smad 1/5/8 staining in blastema mesenchymal cells in blastema slices cultured in the presence or absence of exogenous human BMP2 in amounts as indicated. The corrected nuclear fluorescence was calculated in order to normalize the intensity of nuclear staining for variation in the area of the nuclei being analyzed as well as for the background fluorescence [40]. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances.
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pone.0123186.g006: Nuclear P-Smad 1/5/8 fluorescence in response to BMP2.Nuclear Phospho-Smad 1/5/8 staining in blastema mesenchymal cells in blastema slices cultured in the presence or absence of exogenous human BMP2 in amounts as indicated. The corrected nuclear fluorescence was calculated in order to normalize the intensity of nuclear staining for variation in the area of the nuclei being analyzed as well as for the background fluorescence [40]. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances.

Mentions: To test whether cultured blastemas cell respond to exogenous BMP2, we added recombinant human BMP2 to the basal medium of OSC blastema cells at concentrations that span our best estimate for the in vivo concentration of BMP2 [49–53]. We first determined that the axolotl blastema cells responded to the addition of non-axolotl BMP2 by quantifying the changes in the level of phospho-Smad 1/5/8 (p-Smad 1/5/8) immunofluorescence within the nucleus of OSC blastema cells (Fig 6). Treatment with BMP2 at all three concentrations tested (1–100 ng/ml) induced a significant increase (nearly double at 100 ng/ml) in the amount of nuclear-localized p-Smad 1/5/8 relative to basal medium, indicating that human BMP2 activated the canonical BMP signaling pathway in 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)

Nuclear P-Smad 1/5/8 fluorescence in response to BMP2.Nuclear Phospho-Smad 1/5/8 staining in blastema mesenchymal cells in blastema slices cultured in the presence or absence of exogenous human BMP2 in amounts as indicated. The corrected nuclear fluorescence was calculated in order to normalize the intensity of nuclear staining for variation in the area of the nuclei being analyzed as well as for the background fluorescence [40]. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123186.g006: Nuclear P-Smad 1/5/8 fluorescence in response to BMP2.Nuclear Phospho-Smad 1/5/8 staining in blastema mesenchymal cells in blastema slices cultured in the presence or absence of exogenous human BMP2 in amounts as indicated. The corrected nuclear fluorescence was calculated in order to normalize the intensity of nuclear staining for variation in the area of the nuclei being analyzed as well as for the background fluorescence [40]. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances.
Mentions: To test whether cultured blastemas cell respond to exogenous BMP2, we added recombinant human BMP2 to the basal medium of OSC blastema cells at concentrations that span our best estimate for the in vivo concentration of BMP2 [49–53]. We first determined that the axolotl blastema cells responded to the addition of non-axolotl BMP2 by quantifying the changes in the level of phospho-Smad 1/5/8 (p-Smad 1/5/8) immunofluorescence within the nucleus of OSC blastema cells (Fig 6). Treatment with BMP2 at all three concentrations tested (1–100 ng/ml) induced a significant increase (nearly double at 100 ng/ml) in the amount of nuclear-localized p-Smad 1/5/8 relative to basal medium, indicating that human BMP2 activated the canonical BMP signaling pathway in 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.


Related in: MedlinePlus