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

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.

Fig 1. Organotypic slice culture model for axolotl blastemas.

(A1) The limb of an axolotl (Ambystoma mexicanum) was amputated and allowed to regenerate a medium-late bud stage. (A2) The blastema was surgically removed from the animal, (A3) was sectioned using a vibratome (A3) and cultured (A4, B, C). Depending on the size of the blastema, a typical blastema yielded 3–4 slices that appeared similar when cultured (B and C). A region of pooled blood cells in the apical region of adjacent slices is indicated by the arrows in (B and C). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.

Fig 2. Organotypic blastema slices survive and proliferate in vitro.

(A) Hematoxylin and eosin stained section of a blastema slice that had been cultured for 10 days in the presence of 5% FBS. Regions of pre-cartilage condensations are evident (arrows). The blastema epithelium has begun to migrate over the proximal cut end. (B) EdU labeling of a blastema slice after 10 days in vitro. EdU positive nuclei (green) are present in the mesenchyme but absent from the epithelium. (C) A bright field image of a blastema slice (upper right) and DRG (lower left) after 10 days of co-culture. (D) Immunofluorescence of the blastema slice/DRG co-culture illustrated in (C). Proliferating cells in the slices (green) are restricted to the mesenchyme, nuclei are stained with DAPI (blue), and neurofilaments are labeled with RT97 (red). The proximal (P) to distal (D) orientation of the sections is indicated. Scale bars = 500μm.

Fig 3. Cell death in blastema slices after culture for 3, 5, and 7 days.

The percentage of TUNEL positive cells was not significantly different between treatments at any of the time points, or between the three time points. Error bars represent one Standard Deviation of the Mean, and P-values were determined by t-test with 2 tails assuming unequal variances. The number of biological replicates for 3, 5, and 7 days were as follows: Basal medium: 5, 5, and 3, 5%FBS: 4, 4, and 5, DRG co-culture: 3, 3, and 3.

Fig 4. 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.

Fig 5. Labeling index and growth fraction in blastema slices.

A) Labeling index of blastema mesenchymal cells in blastema slices over a period of seven days in culture under different culture conditions as indicated. Labeling period was for five hours. The value for “in vivo” was determined by injecting animals with BrdU two hours prior to collecting the blastema for sectioning and counting of labeled cells without culture. 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 in vivo: N = 22. The number of biological replicates for the following conditions on 3,5, and 7 days were as follows: Basal medium: N = 6, 8, and 9. 5%FBS: N = 7, 7, and 7. DRG co-culture: N = 3, 5, and 6. Pre-conditioned DRG: N = 5, 5, and 4. Asterisk (*) = P<0.05 when compared to the LI in basal medium at equivalent time point. B) Growth Fraction of blastema mesenchymal cells in blastema slices after five days of culture under different culture conditions as indicated. Labeling period was for 80 hours. Error bars represent S.E.M., and P-values were determined by t-test with 2 tails assuming unequal variances. N = 4. Asterisk (*) = P<0.04.

Fig 6. 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.

Fig 7. 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.

Fig 8. Labeling index in response to exogenous BMP2 and LDN193189.

A) Labeling index of blastema mesenchymal cells in blastema slices over a period of five days in culture in the presence or absence of exogenous human BMP2 in amounts as indicated. The number of biological replicates = 4. B) Labeling index of blastema mesenchymal cells in blastema slices over a period of 5 days cultured in 5%FBS and in the presence or absence of LDN193189 in amounts as indicated. The number of biological replicates = 3. Error bars in both A and B represent S.E.M. and P-values were determined by t-test with 2 tails assuming unequal variances.