Evaluation of presumptive caudal fin blastema cells as candidate donors in intraspecies zebrafish ( Danio rerio ) chimaeras

The blastema is a regenerative tissue with remarkable pluripotency. The aim of this work done on zebrafish (Danio rerio) was to define technical procedures required for obtaining and integrating blastema cells into embryos at the mid blastula transition stage (MBT) and the effect on survival, as well as the capacity to produce pigmented chimaeras. Wild type blastema cells were injected into gold type MBT embryos (E). Wild MBT blastomere cells were also injected into gold type MBT embryos as a control (C1). A second control group, C2, was not subjected to any manipulation. Survival was evaluated at 24, 48 and 72 h after performing the chimaerism, and the rate of adult chimaeras evaluated. The results showed significant differences in embryo survival between the E and C1 groups in embryo survival at 24 and 48 h postchimaerism (24 h: E-83.49% vs C1-54.8%, p < 0.05; 48 h: E-98.83% vs C1-85.13%, p < 0.05). There was no significant difference, at any time, between E and C2. The results at 72 h for E and the controls (E-89.41%; C1-84.12% and C2-92.55%) indicate that insertion of blastema cells does not have a negative effect on embryo development. The results in adults (E: 0 chimaeras from 7 specimens; C1: 5 chimaeras from 17 specimens) suggest that the dedifferentiation grade of the blastema cells may not be enough to generate germ-line chimaeras, but their condition of potentially dedifferentiating cells may be an advantage when using them as donor nuclei in somatic cloning by nuclear transplant. Additional key words: biodiversity, chimaerism, embryo, zebrafish.

The cell type used to carry out a biodiversity preservation strategy is important.Until now, current cryopreservation techniques have not enabled the preservation of embryos from species with meroblastic development, such as fish.However, it is possible to store embryonic cells (Cardona-Costa and García-Ximénez, 2007) which can give rise to germ-line chimaeras (Lin et al., 1992), although this involves destroying the donor embryo of the lineage to be preserved.
During caudal fin regeneration in fish, a regenerative tissue appears called blastema that grows atop each fin ray, between the wound epidermis and the amputation surface of the stump, which can regenerate the fin structure (Poss et al., 2003).It is not clear whether blastema formation involves cellular dedifferentiation, the activation of quiescent cells, or both processes, but its pluripotency is remarkable (Akimenko et al., 2003;Poss et al., 2003).
If blastema cells are pluripotent embryo stem cells (ESCs), they would be ideal candidates for use as donor cells in germ-line chimaerism with the aim of preserving biodiversity of these animal species, as these undifferentiated cells can be obtained from adult specimens without causing any permanent damages to them.For this reason, it would be interesting to determine the potency of blastema cells and to investigate if it is possible to integrate them in the germ-line of chimera specimens, which has not been reported to date.
The aim of this work on zebrafish (Danio rerio), was to test the capacity of blastema cells to colonize and integrate into chimaeras, defining the technical procedures required and the effect on embryo and larval survival, as well as the capacity to produce pigmented chimaeras.
Adult zebrafish kept in a 6:3 proportion (females: males), and fed granular food supplemented with recently defrosted hen egg yolk and shrimp meat (Simao et al., 2007), where used to obtain dechorionated embryos at the mid-blastula transition (MBT) stage (1,000 cells), the optimal stage to carry out chimaerism (Lin et al., 1992).All chemicals and the culture media were from Sigma-Aldrich.
After washing with tap water on nylon mesh, embryos were selected under a microscope and washed again with tap water.Embryos in a suitable development stage and perfectly clean were kept in dechlorinated and decalcified tap water (Westerfield, 2003).No bleach treatment was applied, but sterilized media and materials (pipettes) and aseptic conditions were used.
Dechorionation was carried out by pronase treatment (1.5 mg mL -1 in H10), being H10 Hanks buffered salt solution (HBSS) diluted 10% in distilled water, v/v) followed by immersion twice in H10 with an osmolarity of 35 mOsm.Damaged embryos were discarded and only intact embryos were used in the experiments.
To remove the yolk, sterile hypodermic needles were used to puncture the yolk sac, and the embryo was then placed on a liquid surface where the yolk was removed immediately from the embryo by surface tension.The intact blastoderms were placed in HBSS without Ca 2+ or Mg 2+ .The blastoderm was disaggregated into blastomeres using a stretch and fire polished Pasteur pipette with an approximately inner diameter of 1/5 the size of a zebrafish blastoderm.
The blastema cells were from adult wild type zebrafish.Animals were anaesthetized with a clove oil solution (100 µL in conventional tap water; Grush et al., 2004) and the caudal fins were amputated.After 4 days at 28.5ºC these fish were again anaesthetized and the distal region, where blastema was growing, was cut and kept in H10.The tissue was cleaned with a 0.2% bleach solution for 2 min, then washed twice in H10 and finally incubated for 30 min in HBSS without Ca 2+ or Mg 2+ .After this, the blastema region was isolated from the rest of the tissue using a scalpel, under a microscope.To dissociate blastema cells, three 100 µL drops of different incubation media were placed on a Petri dish, one of Trypsin-EDTA and two of L-15 media with 20% bovine foetal serum.The three drops were covered with mineral oil.The whole blastema was first incubated in the Trypsin-EDTA drop for 5 min and the cells were dissociated using a pulled Pasteur pipette.Treatment was stopped adding 10 µL of serum to the drop, next cells were washed twice in L-15 medium by transferring them to the other two drops, and finally cell viability was checked using 0.4% trypan blue.
Around 50-100 presumptive blastema cells were injected per recipient embryo into the marginal zone, where primordial germ cells (PGCs) are localized in MBT embryos The micromanipulation was carried out in H10 medium.Both blastema and MBT cells, were kept in HBSS without Ca 2+ or Mg 2+ .The outer diameter of the injection micropipette was 30 µm for blastema cells and 50 µm for MBT cells.Micromanipulation was carried out using a Leitz micromanipulator inverted microscope (Nikon ECLIPSE TE200).After performing the chimaerism, survival of remaining cells was tested using the trypan blue test.
One experimental and two control groups were established.In the experimental group (E) wild type blastema cells were injected into gold type MBT embryos.To evaluate the effect of the cell type on embryo and larval survival, wild type embryonic cells at the MBT stage were also injected into gold type MBT embryos (first control group, C 1 ).The aim was to evaluate the technical efficiency and the effect of chimaerism on survival, the second control group (C 2 ) was not subjected to any manipulation and embryos were kept in H10 for the same period of time as those that were micromanipulated.At least 100 embryos were manipulated, both in the experimental group (E) and in the control groups in the different sessions.
Survival was evaluated at 24, 48 and 72 h after performing the chimaerism, and the rate of adult chimaeras was evaluated after about three months.
Results were analysed using the c 2 test (Statgraphics Plus 4.0).When a single degree of freedom was involved, the Yates correction for continuity was performed.
During growth and elongation of the blastema, this transitory structure generates a cell proliferating-differentiation balance (Nechiporuk and Keating, 2002).This characteristic is important when selecting donor cells for transplanting because the regenerative outgrowth has different kinetic, morphological and molecular qualities in the days after amputation.In this work, the blastema used in our chimaerism experiments was recovered at 4 d post-amputation.At this stage, the transition between blastema formation and blastema outgrowth begins and the blastema precursor cells have just undergone the dedifferentiation process and have a high proliferation rate (Poleo et al., 2001), so most cells retrieved will be undifferentiated.
In our results, cells from the blastema differed in their morphology to cells from a non-amputated fin with regard to size and lobopodia signs (Harvey, 1983), typical movements of undifferentiating cells.The blastema cells tolerated both manipulation and treatments (bleach, trypsin, etc.) to isolate them, as shown by the viability tests after each session of chimaerism.
With regard to embryo viability, survival of embryos that supported the manipulation process, were evaluated at 24, 48 and 72 h after performing the chimaerism (103 from 134 in E; 135 from 202 in the control group 1, C 1 ).The technical efficiency of embryos that survived manipulation was 76.86% in blastema cells and 66.83% in blastomeres at the MBT stage.Embryo survival is shown in Table 1.Statistical analysis of the results showed significant differences (p > 0.05) between the E and C 1 groups in embryo survival at 24 and 48 h postchimaerism.This means that insertion of blastema cells involves less immediate damage on embryo survival than insertion of blastomeres at the MBT stage.The cause of the increase in deaths at 24 h, in the control group C 1 may have been due to pipette size (30 µm for blastema cells vs 50 µm for MBT cells) because mechanical damage caused by the injection process are higher as micropipette size increases.There were also significant differences (p < 0.05) between the two control groups, C 1 and C 2 , at 24 and 48 h.However, at no time was there a significant difference between group E and group C 2 .Another factor to take into account in embryo viability is the number of cells injected.Although blastema cells are larger than somatic cells (fibroblasts), they are much smaller than blastomere cells (Figure 1).This means that mechanical damage caused by injection of MBT blastomeres is greater than the damage caused by injection of the same number of blastema cells.The survival at 72 h, in E and the controls (C 1 and C 2 ) (  (Lee et al., 2005;Piliszek et al., 2007).The results of this work suggest that insertion of blastema cells does not have a negative effect on embryo development for at least three days post fertilization.
Although the process of cell dedifferentiation explains the formation of the blastema after a limb lesion in aquatic urodele amphibians (Straube et al., 2004;Brockes and Kumar, 2005;Straube and Tanaka, 2006), in teleost fishes there are no molecular markers available to prove the cellular reprogramming before forming the blastema.However, several cytological (Becerra et al., 1996) and cell signalling studies (Santamaría et al., 1996;Poleo et al., 2001;Nechiporuk and Keating, 2002;Santos-Ruiz et al., 2002) suggest that dedifferentiation of mesenchymal cells would be a possible mechanism of blastema formation.If this were true, their integration into a chimera specimen would be possible, even in the germ line.
Somatic chimaerism begins to show at 48 h postfecundation, because this is the stage at which the first melanocytes differentiate (Rawls et al., 2001).As skin pigmentation chimaerism (Figure 2) is a good marker for detecting germ-line contributions from transplanted cells in zebrafish (Lin et al., 1992), the results from adults in this work (E: 0 chimaeras from 7 specimens; C 1 : 5 chimaeras from 17 specimens; data not shown) suggest that blastema cells are not totipotent, which precludes their use as donor cells in strategies of biodiversity preservation via chimaerism.Although the dedifferentiation grade of the blastema cells may not be enough to generate germ-line chimaeras, their condition of potentially dedifferentiating cells may be an advantage   previous conclusion that mechanical damage in the manipulation process causes the differences observed.Thus, although blastema cells have molecular qualities and expression patterns that differ from the blastomeres at the MBT stage, embryo survival is higher in the former.It is worth noting that zebrafish embryos generates a favourable microenvironment for keeping somatic cells in an undifferentiated state and human metastatic cells can survive, divide, and even migrate and integra-when using them as donor nuclei in somatic cloning by nuclear transplant, increasing technical efficiency in fish.

Table 1
) supports the Columns with different superscripts are statistically different (Statgraphics 4.0). 1 Experimental group (E): wild type blastema cells injected into gold type MBT embryos. 2 Control group 1 (C 1 ): wild type MBT cells injected into gold type MBT embryos. 3Control group 2 (C 2 ): embryos not subjected to manipulation.

Table 1 .
Embryo survival at different stages te into zebrafish embryos