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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.


Prrx-1 expression in blastema slices.Fold change in Prrx-1 levels after seven days of culture under different culture conditions. The value for “Uninjured Skin” was determined for samples of skin that had not been cultured. The value for “Pre-culture Slice” was determined for blastema slices that had not been cultured. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances. The number of biological replicates for the conditions tested were as follows: Pre-culture slice: N = 5, Basal medium: N = 7, 5%FBS: N = 5, Pre-conditioned DRG: N = 5, 100ng/mL BMP: N = 3. Each biological replicate consisted of four technical replicates. Asterisk (*) = P<0.05.
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pone.0123186.g004: Prrx-1 expression in blastema slices.Fold change in Prrx-1 levels after seven days of culture under different culture conditions. The value for “Uninjured Skin” was determined for samples of skin that had not been cultured. The value for “Pre-culture Slice” was determined for blastema slices that had not been cultured. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances. The number of biological replicates for the conditions tested were as follows: Pre-culture slice: N = 5, Basal medium: N = 7, 5%FBS: N = 5, Pre-conditioned DRG: N = 5, 100ng/mL BMP: N = 3. Each biological replicate consisted of four technical replicates. Asterisk (*) = P<0.05.

Mentions: Expression of Prrx-1 (Paired-related Homeobox 1) in OSC blastema mesenchymal cells was maintained and increased in vitro (Fig 4). Prrx-1 is a transcription factor that is expressed at high levels in developing and regenerating axolotl limb mesenchymal cells [41], but is expressed at low levels that are not detected by in situ hybridization in uninjured skin. Expression of Prrx-1 in the blastema is regulated by interactions between the wound epithelium and the nerve [41], and thus it is a marker for regenerating blastema cells [3,41,46]. As reported previously [47], Prrx-1 expression was detected at low levels by qPCR in uninjured skin (Fig 4). Expression in blastema sections prior to being culture was significantly upregulated an average of 6-fold, which is comparable to the increased level of expression in vivo when comparing blastemas to uninjured skin [3]. Blastema slices cultured in basal medium for 7 days expressed Prrx-1 at a 23-fold higher level relative to uninjured skin (about a 4-fold increase during the culture period. Since Prrx-1 expression is restricted to the distal tip of blastemas in vivo, this increase in expression in vitro could be a result of increased numbers of cells in the slices being induced to express this gene rather than an increase in the level of expression in the distal blastema cells. Better understanding the mechanisms of Prrx-1 expression during regeneration is a goal of future experiments using OSC. Taken together, these data indicate that blastema slices cultured in basal media are viable and maintain expression of a gene that is characteristic of regenerating 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)

Prrx-1 expression in blastema slices.Fold change in Prrx-1 levels after seven days of culture under different culture conditions. The value for “Uninjured Skin” was determined for samples of skin that had not been cultured. The value for “Pre-culture Slice” was determined for blastema slices that had not been cultured. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances. The number of biological replicates for the conditions tested were as follows: Pre-culture slice: N = 5, Basal medium: N = 7, 5%FBS: N = 5, Pre-conditioned DRG: N = 5, 100ng/mL BMP: N = 3. Each biological replicate consisted of four technical replicates. Asterisk (*) = P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414535&req=5

pone.0123186.g004: Prrx-1 expression in blastema slices.Fold change in Prrx-1 levels after seven days of culture under different culture conditions. The value for “Uninjured Skin” was determined for samples of skin that had not been cultured. The value for “Pre-culture Slice” was determined for blastema slices that had not been cultured. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances. The number of biological replicates for the conditions tested were as follows: Pre-culture slice: N = 5, Basal medium: N = 7, 5%FBS: N = 5, Pre-conditioned DRG: N = 5, 100ng/mL BMP: N = 3. Each biological replicate consisted of four technical replicates. Asterisk (*) = P<0.05.
Mentions: Expression of Prrx-1 (Paired-related Homeobox 1) in OSC blastema mesenchymal cells was maintained and increased in vitro (Fig 4). Prrx-1 is a transcription factor that is expressed at high levels in developing and regenerating axolotl limb mesenchymal cells [41], but is expressed at low levels that are not detected by in situ hybridization in uninjured skin. Expression of Prrx-1 in the blastema is regulated by interactions between the wound epithelium and the nerve [41], and thus it is a marker for regenerating blastema cells [3,41,46]. As reported previously [47], Prrx-1 expression was detected at low levels by qPCR in uninjured skin (Fig 4). Expression in blastema sections prior to being culture was significantly upregulated an average of 6-fold, which is comparable to the increased level of expression in vivo when comparing blastemas to uninjured skin [3]. Blastema slices cultured in basal medium for 7 days expressed Prrx-1 at a 23-fold higher level relative to uninjured skin (about a 4-fold increase during the culture period. Since Prrx-1 expression is restricted to the distal tip of blastemas in vivo, this increase in expression in vitro could be a result of increased numbers of cells in the slices being induced to express this gene rather than an increase in the level of expression in the distal blastema cells. Better understanding the mechanisms of Prrx-1 expression during regeneration is a goal of future experiments using OSC. Taken together, these data indicate that blastema slices cultured in basal media are viable and maintain expression of a gene that is characteristic of regenerating 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.