Regeneration and reprogramming compared

Abstract

Background: Dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. Dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is over expressed in mature cell types.

Results: We hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. We analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. We found that some of the pluripotency associated factors (oct4/pou5f1, sox2, c-myc, klf4, tert, sall4, zic3, dppa2/4 and fut1, a homologue of ssea1) were expressed before and during regeneration and that at least two of these factors (oct4, sox2) were also required for normal fin regeneration in the zebrafish. However these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.

Conclusions: By comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. However, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. We therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes.

Figure 1

qPCR of a pluripotent cell type compared with cells of the regeneration blastema. A) Relative expression of pluripotency associated genes in Xenopus cap cells compared to limb blastema (st52) at different time points during regeneration. In red is relative expression of animal cap cells, in green the relative expression of st52 limb blastemas at 0 dpa, 1 dpa, 3 dpa and 5 dpa. (note: the results for oct25, oct60 and oct79 expression was combined as oct4 expression). B) Relative expression of pluripotency associated genes in Xenopus cap cells and st52 tail blastema cells. C) Relative expression of pluripotency associated genes in Xenopus cap cells and regenerative incompetent pseudo blastemas of st57 limbs at 0 dpa, 1 dpa, 3 dpa, and 5 dpa. Asterisks indicate significant (P < 0.05) upregulation of expression compared to 0 dpa. D) Relative gene expression for pluripotency associated markers in zebrafish non-regenerating (0 hpa), regenerating (6 hpa to 72 hpa) fin and embryonic cells in oblong stage. E) Relative expression of blastema markers in cap cells and regenerating limbs. Asterisks indicate significant (P < 0.05) upregulation of gene expression compared to the previous time point. F) Relative expression of blastema markers in zebrafish non-regenerating (0 hpa), regenerating (6 hpa to 72 hpa) fin and embryonic cells in oblong stage. For each gene, expression was normalized to highest expressing tissue. Error bars indicate Standard deviation (SD). (For statistical differences see Additional file 1).

Figure 2

In situ hybridization of c-myc, sall4 and sox2 in developing and regenerating limbs and tails. A-A"') Expression of c-myc in developing st52 limbs (A) and amputated limbs at 0 dpa, 1 dpa and 3 dpa (A'-A"'). c-myc expression is diffuse and very weak in the developing limb bud and the blastema. B-B"') Expression of sall4 in developing st52 (B) and regenerating limbs at 0 dpa, 1 dpa, 3 dpa (B'-B"'). sall4 is expressed diffusely in the distal mesenchyme of the developing limb bud and after amputation in the whole of the blastema. C-C"') Expression of sox2 in developing (C) and regenerating (C'-C"') limbs. sox2 is expressed proximal to the blastema in the posterior mesenchyme. D) c-myc mRNA is expressed in the entire regeneration bud of a 3 dpa tail and is much stronger than in limbs. E) sall4 mRNA expression in 3 dpa tail is mainly seen in the distal tip of the regenerating tail. F) sox2 mRNA expression in 3 dpa tail is confined to the neural tube. (dpa = days post amputation). Thin black line indicates amputation plane

Figure 3

Cell cycle analysis. (A) Cell survival after blastomere and blastemal cell recovery at different time points (0 h, 48 h and 4 d). (B) Representation of cells in G0/G1 (blue) and G2/M (red) cell cycle phases. Results are expressed as percentages. FACS histogram of non-regenerating (C) and 4 dpa regenerating fin blastema cells (D). Cells in G0/G1 are included in R9 and G2/M in area R10.

Figure 4

Morpholino injection into zebrafish caudal fins. A-D) Bright field and fluorescence picture of a dextran (A) msxb MO (B) pou MO (C) and sox2 MO (D) injected and electroporated fin: injections were done into the dorsal half of a 3 dpa blastema and photographed 24 h later. E, F) Time course for pou MO (E) and sox2 MO (F) injected and electroporated fins at 3 dpa, 2 dpa, 1 dpa and 0 dpa. Pictures were taken 24 h after injection. G-I) Percentage of dorsal versus ventral fin outgrowth (in green) and average inhibition of dorsal versus ventral fin (in red) of fins which were injected 3 dpa (G, H) and on day 0 to 3 pa as indicated (I). (see Method for exact calculation) (Asterisk indicate significant difference (P < 0.5) compared to dextran or pou 5-mis MO control or for 1 dpa injected fins between pou MO and sox2 MO injected fins). J) Measurement of apoptosis and cell proliferation in pou MO injected fins. There is no significant difference between dorsal and ventral fins in apoptosis as measured by TUNEL while there is a significant reduction of cell divisions in the dorsal fin as measured by pH3 staining (P ≤ 0.05). Black lines indicate blastema size on day of injection. Error bars indicate standard error.