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Plenary Paper
HEMATOPOIESIS AND STEM CELLS
Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl
David Lopez,1,2 Li Lin,1,2 James R. Monaghan,3 Christopher R. Cogle,1,4 Frank J. Bova,5 Malcolm Maden,6
and Edward W. Scott1,2
1
Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL; 2Department of Molecular Genetics and Microbiology,
University of Florida, Gainesville, FL; 3Department of Biology, Northeastern University, Boston, MA; 4Division of Hematology and Oncology, Department of
Medicine, University of Florida, Gainesville, FL; 5Department of Neurosurgery, University of Florida, Gainesville, FL; and 6Department of Biology, University
of Florida, Gainesville, FL
Hematopoietic stem cell (HSC)-derived cells are involved in wound healing responses
throughout the body. Unfortunately for mammals, wound repair typically results in scarring
and nonfunctional reparation. Among vertebrates, none display such an extensive ability for
• Establishing HSC
adult regeneration as urodele amphibians, including 1 of the more popular models: the
transplantation and assay
axolotl. However, a lack of knowledge of axolotl hematopoiesis hinders the use of this animal
methods for the axolotl.
tissue regeneration. We
• Axolotl sites of hematopoiesis for the study of hematopoietic cells in scar-free wound healing and
used white and cytomegalovirus:green fluorescent protein1 transgenic white axolotl
are the spleen and liver.
strains to map sites of hematopoiesis and develop hematopoietic cell transplant methodology. We also established a fluorescence-activated cell sorter enrichment technique for major blood lineages and colony-forming
unit assays for hematopoietic progenitors. The liver and spleen are both active sites of hematopoiesis in adult axolotls and contain
transplantable HSCs capable of long-term multilineage blood reconstitution. As in zebrafish, use of the white axolotl mutant allows
direct visualization of homing, engraftment, and hematopoiesis in real time. Donor-derived hematopoiesis occurred for >2 years in recipients
generating stable hematopoietic chimeras. Organ segregation, made possible by embryonic microsurgeries wherein halves of 2 differently
colored embryos were joined, indicate that the spleen is the definitive site of adult hematopoiesis. (Blood. 2014;124(8):1232-1241)
Key Points
Introduction
In mammals, the ability to regenerate limbs and organs is progressively lost during ontogeny and correlates closely with maturation
of immune competence. Research in scar-free healing, primarily
observed in model systems with dysfunctional neutrophils and macrophages, has led to the hypothesis that the immune system dictates the
balance between scarring and regeneration.1,2 Unfortunately, presently
available genetic models of vertebrate wound healing, such as the
African spiny mouse (Acomys), tend to lack significant regenerative
abilities.3 Thus, although the role of hematopoietic stem cell (HSC)derived blood cells in wound healing via inflammation and paracrine
regulation is well understood during fibrotic healing, the same cannot
be said for a scar-free regenerative response.4,5
Among vertebrates, urodele amphibians, such as the axolotl
(Ambystoma mexicanum), display a unique and extensive ability for
adult regeneration. Axolotls can replace a wide variety of tissues and
complex structures including muscle, cartilage, skin, spinal cord, brain,
heart, jaw, and limbs.6 The axolotl’s nearest relatives, anuran frogs and
reptiles, lose the ability to reform tissues after metamorphosis or show
limited tail regeneration. This suggests that adult regeneration may be
an acquired trait in axolotls that might be inducible in mammals if the
required genetic pathways are mapped. Recent advances in production of transgenic axolotls,6-10 complete mapping of the axolotl transcriptome,11 and production of gene expression arrays12-16 finally
allow molecular mapping of regeneration pathways. Additionally, given
the extensive conservation of synteny between axolotls and humans, this
amphibian may be a powerful genetic model to study hematopoietic cell
function in scar-free wound healing and tissue regeneration.17
However, 1 major challenge in using axolotls is a limited knowledge of their hematopoiesis. Axolotls produce similar blood lineages as
mammals with the exception of persistent orthochromatic normoblasts
in adults.18,19 Yet to be defined are fundamental questions such as
sites of hematopoiesis and the HSC niche. Therefore, in this study,
we define the primary and secondary sites of hematopoiesis in both
larval and adult axolotls. Furthermore, we developed the classic
tools of hematology to study axolotl hematopoiesis including hematopoietic cell transplantations (HCTs), colony-forming unit
(CFU) assays, and methods to map axolotl hematopoiesis. Finally,
we have taken advantage of axolotl embryo manipulations to
generate stable hematopoietic chimeras (green fluorescent protein
[GFP]1 blood in a white recipient) in nonirradiated models with
directly visible immune cells similar to the zebrafish.
Submitted September 17, 2013; accepted April 25, 2014. Prepublished online
as Blood First Edition paper, May 6, 2014; DOI 10.1182/blood-2013-09526970.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
Materials and methods
Axolotls
White mutant (d/d), GFP1 , or nucCherryRed1 cytomegalovirus (CMV):
b-actin promoter-driven transgenic axolotls and embryos were purchased
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
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© 2014 by The American Society of Hematology
BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
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AXOLOTL HEMATOPOIESIS AND TRANSPLANTATION
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from the Ambystoma Genetic Stock Center (AGSC) or bred in-house from
AGSC founder animals. Animals were staged as previously described20 and
maintained in Holtfreter’s solution.21 This study was approved under the
University of Florida Institutional Animal Care and Use Committee protocol
201202645.
Irradiation
Adult axolotls were anesthetized by submersion in 0.1% tricainemethanesulfonate (Sigma-Aldrich), pH 7.4, and a 900- to 1000-cGy dose
of irradiation22 was given in a 137Cs source irradiator with or without
lead shielding used to protect the gills, or a 1000- to 1200-cGy dose of
g-irradiation was specifically targeted to the spleen and liver region via
a six Mev linear accelerator.
Cell collection
As a general note, axolotl cells require a different osmotic pressure than
mammalian cells, thus requiring standard buffers/media be diluted to 75%
normal strength with water prior to use. Axolotl cells were maintained in
either 0.603 L-15 media, 5% fetal bovine serum (FBS), 13 penicillin/
streptomycin, and 13 insulin-transferrin or 0.753 phosphate-buffered
saline (PBS) (referred to as axolotl PBS [APBS]). Cells were kept at 18°C to
20°C in ambient air. Axolotls were anesthetized, tissues were collected, and
single cell suspensions were made by maceration in APBS and 5% FBS.
Axolotl blood was collected into 10 mM EDTA in APBS to prevent
coagulation. Ficoll-Paque Plus (GE) density gradients were performed as
described elsewhere23 for lymphoblastic “buffy” coat and erythrocyte
enrichment.
Flow cytometry
Single-cell suspensions of liver and spleen cells from GFP1 axolotls in APBS
with 5% FBS were stained with 7-amino-actinomycin D (BD Biosciences) to
exclude dead cells and debris. Analysis and sorting was accomplished on
a fluorescence-activated cell sorter (FACS) Aria flow cytometer (BD Biosciences) based on 7-amino-actinomycin D exclusion, GFP fluorescence, side
scatter (SSC), and forward scatter (FSC) for cell size and granularity.
Histology
Tissues were dissected and fixed in 4% paraformaldehyde at 4°C, washed with
PBS, and transferred to 20% sucrose in PBS for 3 days at 4°C. Tissues were
embedded in Optimal Cutting Temperature Compound (Sakura Finctek) and
stored at 220°C. Sections were cut to a thickness of 5 mm and stained with
hematoxylin and eosin (H&E). Transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed using the In Situ
Cell Death Detection Kit, TMR red (Roche), in accordance with the manufacturer’s instructions. Cytospins were made with 5 3 104 to 1 3 105 cells.
Samples were spun at 800 rpm for 3 minutes onto glass slides. Prepared
slides were stained with Protocol HEMA 3 stain set (Fisher Scientific),
Wright-Giemsa, Alcian blue, or a-naphthyl butyrate esterase. Antibodies
were mouse monoclonal F4/80 IgG (Serotec; MCAP497) 1:50, rabbit
polyclonals CD79a IgG (Abcam; AB5691) 1:100, and CD2 IgG (AB37212;
Abcam) 1:50. Microscopy of tissue sections and cytospins was performed
on a Leica DM5500B microscope using a Hamamatsu digital camera model
C7780.
Phagocyte assay
pHrodo Red Escherichia coli BioParticles (Molecular Probes) were mixed
with whole blood and incubated at room temperature for 1 to 3 hours.
Erythrocytes were separated by Ficoll-Paque density gradient, and all other
fractions were cytospun onto glass slides for visualization.
Figure 1. HPC activity from axolotl spleen and liver. (A) Representative view
(310) of spleen CFU at 10 days. (B) Magnification (340) of a large CFU-E colony.
(C) Magnification (340) of a non-CFU-E colony. (D) Quantitation of CFUs in the liver
and spleen reveals that the spleen contains more than twice as many CFUs as the
liver (12 253 vs 5825, P 5 .0009; N 5 3). Graphs show mean 6 standard error of the
mean of $3 independent experiments. (E) The proportion of CFU-E to other CFU
colonies (P 5 .4). CFU assays were imaged on a Leica DMIRB inverted microscope
using a SPOT Flex digital camera.
were performed using borosilicate glass capillary needles (1-mm outer diameter,
no filament; World Precision Instruments) made using a micropipette puller.
Procedures were done via micromanipulator and a screw-actuated air/oil
microinjector; 1 3 104 to 5 3 105 cells were injected intracardially into tricaine
anesthetized animals. Axolotls were imaged on a Leica MZ16FA microscope
using a Hamamatsu digital camera model C7780 and Volocity Imaging
software (Perkin Elmer).
Fused 2-color chimeras
Embryos at stages 14 to 20 were dejelled and washed in fresh 100% Steinberg’s
solution24 with a pH of 7.4 containing 1% antibiotic-antimycotic and 0.0025%
gentamycin (25 mg/L). Under a dissecting microscope, embryos of each color
were cut transversely in half with microsurgical scissors, and the anterior end of
one was matched with the posterior end of the other. Paired halves were moved
into depressions made in agar with neural folds touching. Embryos were left
undisturbed for 96 hours at 20°C, transferred into fresh 100% Steinberg’s
solution for another 7 days, and then moved to 40% Holtfreter’s solution.
CFU assays
Axolotl cellular responses to mammalian colony stimulating factors (CSFs)
are unknown. Therefore, we produced axolotl pokeweed mitogen-stimulated
spleen cell-conditioned media (PWM-SCM) to serve as a source of axolotl
CSFs for CFU assays. Axolotl spleen cells were suspended in 60% L-15
media, 10% PBS, penicillin/streptomycin, insulin-transferrin-selenium, and
1% pokeweed mitogen solution (1 mg/mL), pH 6.4, at a cell concentration of
1 3 106 to 2 3 106 cells/mL, and allowed to condition medium for 7 days at
18°C. Conditioned media were collected by centrifugation, filtered through
a 0.45-mm filter, and stored at 280°C.
Single-cell suspensions of spleen and liver cells were suspended at a final
concentration of 5 3 104 cells/mL in 3 mL of 2% methylcellulose (Methocel),
50% PWM-SCM, and 0.603 L-15 media, pH 6.4, in 35-mm Petri plates.
Human erythropoietin was added at 1 U/mL. Cultures were incubated up to
5 weeks at 18°C in ambient air.
Polymerase chain reaction
Hematopoietic cell transplantation
Lethally irradiated (950 cGy)22 white adult animals were anesthetized and
received a minimum of 1 3 104 whole spleen or liver cells intravenously
through a 26-gauge needle in a maximum volume of 300 mL. Microinjections
into nonirradiated embryos (stages 25 to hatching) and larvae (3 months old)
Genomic DNA was isolated from whole blood or Ficoll-Paque Plus density
gradient-purified erythrocyte fractions and prepared as described elsewhere.23 Primers were designed to amplify a 173-bp region within the GFP
gene. The forward primer was AAGTTCATCTGCACCACCG, and the
reverse primer was TCCTTGAAGAAGATGGTGCG. Reactions (25 mL)
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LOPEZ et al
BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
Figure 2. Identification of major hematopoietic cell
lineages from the liver and spleen by FACS lightscatter characteristics, fluorescence, and staining.
FACS and H&E staining are presented for (A) axolotl
blood, (B) liver, and (C) spleen. The CMV:GFP transgene expresses in all tissue types, but not in every cell.
Depending on cell lineage, GFP expression can vary
from 10% to 90% of individual cells. This is similar to
the variable transgene expression seen in the equivalent transgenic mouse.31 All erythrocytes are GFP2
(red). Mature myeloid cell enriched populations are
GFP1SSChi (blue). Lymphocyte enriched populations
are GFP1SSClo (blue). The liver contains almost even
numbers of myeloid cells and lymphoblasts. The spleen
contains more lymphoblasts than myeloid cells. Black
cells are excluded debris. (D) Cytospins followed by
Wright-Giemsa, Alcian blue, a-naphthyl acetate esterase, and myeloperoxidase staining were performed to
confirm cell morphology and characteristics. Scale bar:
20 mm (applies to the 8 individual cells stained with
Wright-Giemsa). Blood composition was 3.7 6 2.5%
lymphoblastic cells, 1.3 6 0.5% mature myeloid cells,
and 95 6 1.6% erythrocytes. Following removal of
adipocytes, the liver was composed of 3.7 6 0.6%
lymphoblastic cells, 5.3 6 1.5% mature myeloid cells,
and 68.5 6 10.2% erythrocytes (N 5 3; Figure 1A). In
comparison, the spleen contained 31.3 6 12% lymphoblastic cells, 5 6 0.8% mature myeloid cells, and
60.7 6 15.5% erythrocytes (N 5 3).
were prepared with the addition of 4% dimethylsulfoxide, and the thermal
cycler was programed as described elsewhere.25
Statistical analysis
Microsoft Excel 2013 was used to perform 2-tailed Student t tests. P , .05
was considered significant.
Results
Defining sites of hematopoiesis in the axolotl
Previous studies of the axolotl immune system noted that the liver
and spleen both contain significant numbers of hematopoietic cells,
whereas the bone marrow appears to be nonhematopoietic.22,26,27 To
confirm which organs in adult axolotls contain hematopoietic progenitor cells (HPCs), we developed an axolotl-specific CFU assay
based on early mouse CFU assays using PWM-SCM as a source
of CSFs. We tested the addition of a variety of mammalian growth
factors routinely used for serum-free CFU assays. Only erythropoietin (EPO) induced formation of additional colonies. Axolotl cells
are notoriously difficult to grow in tissue culture. To date, only a
single cell line (AL-1 fibroblasts)28 has been established from axolotls.
Primary cultures of axolotl cells usually fail after only a few passages.
Colonies generated from axolotl (N 5 5 each organ) spleen and liver
cells in response to axolotl PWM-SCM/EPO were similar in morphology to classically defined erythroid burst-forming unit and erythroid
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AXOLOTL HEMATOPOIESIS AND TRANSPLANTATION
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Figure 3. Organs and donor derived GFP1 hematopoietic cells can be visualized directly through the skin in the axolotl. (A) The underside of a wild-type white larval
axolotl. The internal organs can be seen clearly through the skin. (B) The head of an irradiated white adult axolotl 1 month after HCT. The iris of the eyes (arrows) are
autofluorescent but the nodes are regions of highly concentrated GFP1 donor-derived cells. (C) GFP channel with lighting to visualize GFP1 cells and non-GFP vasculature.
Two GFP1 blood cells (white arrowheads) are flowing through vasculature in the tail of a transplanted axolotl in this frame. The orange is white light diffracting off of non-GFP
tissues. (D) The foot of a transplanted axolotl with a patch of GFP1 HSPC-derived cells in the skin. Dashed line indicates a previous amputation site (transverse cut) and the
section used for E. Dense recruitment of donor-derived cells suggests the potential involvement of blood cells in limb regeneration. (E) DIC image (320) of the foot sectioned
in D. Superior aspect of the inferior portion of the foot. Blue, DAPI staining of cell nuclei; green, donor-derived GFP1 cells. Scale bar: 100 mm. (F) Dissecting microscope view
of static donor-derived cells in the skin.
CFU (CFU-E) (Figure 1A-B). Given how little is known about
hematopoiesis/hematopoietic development in axolotls, we use the
term CFU-E to describe all erythroid-only colonies in this study. The
second major colony type present resembled granulocyte, erythrocyte,
monocyte, megakaryocyte CFU (CFU-GEMM) colonies (Figure 1C)
containing a mixture of myeloid, megakaryocyte, and erythroid
lineage cells. After culture initiation, colonies were first visible by
4 days and peaked at 10 days. Axolotl spleens generated twice the
number of total colonies than livers (12 253 vs 5825, P 5 .0009;
Figure 1D). Axolotl spleen and liver generated similar proportions of CFU-E (70% vs 62%, P 5 .4) and CFU-GEMM (30% vs
38%, P 5 .4) colonies (Figure 1E). We also tested cells from all
other major organs including bone marrow, thymus, and kidneys for
CFU activity and found none. Therefore, we concluded that spleen and
liver are the active sites of hematopoiesis in the adult axolotl.
One limitation to hematologic analysis in axolotl is a lack of
cell-specific antibodies that work for flow cytometry. For transplant studies, we use GFP1 animals as HSC donors, thereby permitting detection of erythroid, myeloid, and lymphoblastic cells
via a combination of FSC, SSC, and GFP. Analysis of peripheral
blood, spleen, and liver confirmed that these organs contain multiple
hematopoietic lineages (Figure 2).22 Spleens are mostly composed of
hematopoietic cells, whereas livers have a thin outer layer of predominantly hematopoietic cells with rarer hematopoietic cells
found in the inner hepatocyte dominated layers. Erythrocytes,
mature myeloid cells, and lymphoblasts were sorted based on
FSC/SSC/GFP properties, and their enriched populations were
confirmed by cytospin, H&E, and Wright-Giemsa staining (Figure
2A-D). The lymphoblastic population is likely to contain not
only B and T lymphocytes, but monocytes and HPCs depending on tissue source. The mature myeloid cell population
contained Alcian blue-positive mast cells, a-naphthyl butyrate
esterase-expressing macrophages, and myeloperoxidase-positive
neutrophils18,29-33 (Figure 2D).
Ablative transplants in the axolotl
Next we developed HCT protocols for axolotl to test for HSC activity
and to define its primary niche(s) during axolotl development. Initial
transplants adapted our murine ablative bone marrow transplant
protocol to the axolotl. White axolotl mutants were used as recipients
(Figure 3A) to allow for noninvasive visualization of internal tissues
and organs as in zebrafish, but with the added benefits of increased
size and visualization persisting throughout adulthood. This trait
facilitated monitoring of donor GFP1 cell engraftment after transplant. Cohorts of adult white axolotls received 950-cGy whole body
irradiation, with lead shielding of the head to protect the gills from
damage, followed by intravenous transplant of test GFP1 donor
cell populations. We tested all tissues/organs to avoid bias. Standard
transplants received 5 3 106 cells from test organs, or all cells from
smaller organs into a single recipient if the yield was lower (ie, bone
marrow flushing yielded 1 3 105 cells per donor). From adult donors,
only recipients of liver or spleen cells showed a progressive reconstitution with GFP1 blood easily visualized in white recipients
(Figure 3B-F). Increasing numbers of GFP1 leukocytes could be
seen flowing through blood vessels (Figure 3C; supplemental Videos
1 and 2, available on the Blood Web site) that stabilized after 5 to 6
months. Accumulation of GFP1 cells was visible in what appears to
be a lymph-like network beneath the skin (Figure 3B). When adult
spleen/liver was used as a donor source, excellent initial reconstitution was observed, but the majority of recipients (.75%) would
develop symptoms consistent with graft-versus-host disease (GVHD)
by 2 to 5 months after transplant (supplemental Figure 1). Previous
studies on axolotl immune function suggested that larvae from 3 to
7 months of age still have immature immune systems.34-36 Therefore,
we used larvae in this age range as donors for transplantation. These
transplants showed a reduced incident of GVHD (,25%) and robust
GFP1 hematopoietic reconstitution. We now use 3- to 7-month-old
donor tissue for transplant into gill-shielded, whole body irradiated
recipients as current “best practice” for ablative HCT experiments.
We have observed up to 20% (1% GFP1 cells in peripheral blood vs
5% average for donors) chimerism of donor cells within the first
3 months. Ficoll-Paque density centrifugation was used to enrich
for lymphoblastic cells (buffy coat). Transplantation of 104 GFP1
lymphoblastic spleen cells resulted in equivalent levels of donor cell
engraftment as 107 unfractionated GFP1 spleen cells, a roughly 1000fold enrichment. We concluded that in adult axolotls, both liver and
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LOPEZ et al
BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
spleen contained HSC populations that are enriched in the lymphoblastic population.
We also used a 6-Mev linear accelerator to provide 1000 cGy of
irradiation targeted specifically to the spleen and liver. These cohorts
reconstituted well with adult GFP1 spleen and/or liver cells. An
example from these cohorts is seen in Figure 3D-E. This animal
happened to have a front limb eaten by its tankmates. This limb
regenerated with participation of large numbers of donor-derived
GFP1 cells during tissue remodeling/regeneration, which is a
result consistent with a recent study showing macrophages are
required for axolotl limb regeneration.29 Targeted irradiation of
liver and spleen without transplant resulted in hematopoietic failure/
anemia necessitating euthanasia by 6 weeks, demonstrating that they
are the primary hematopoietic organs.
We confirmed the effects of irradiation on liver and spleen by
TUNEL staining. Spleens showed a gradual increase in apoptotic
cells until nearly every cell appeared TUNEL1 at 7 weeks (supplemental Figure 2A,D,G). The number of apoptotic cells in liver
seemed to remain relatively constant and mostly in the periphery
(supplemental Figure 2B,E,H). The tail (composed mainly of muscle)
served as a radioresistant control and had a small increase in apoptotic
cells after irradiation that continued unchanged from 3 to 7 weeks
(supplemental Figure 2C,F,I).
Representative histology of HCT recipients (N 5 10 animals)
shows a distinct peripheral layer in liver composed primarily of
lymphoblastic cells (Figure 4A) that was ablated entirely within
1 month of irradiation, whereas parenchymal liver cells showed
minimal signs of ablation (Figure 4C). Following irradiation, spleens
demonstrated a decrease in lymphoblasts and erythrocytes, but no
distinct regions or cell types were differentially affected (Figure 4B,D).
Tissues examined at 3 weeks after irradiation and transplant showed
widespread engraftment in spleen and liver (Figure 4E-H). Interestingly, engraftment in liver appeared to concentrate in a peripheral hematopoietic layer (Figure 4E,G). Engraftment in spleen
was evenly distributed throughout the organ (Figure 4F,H). In
6-week-old livers, GFP1 donor cell engraftment was considerably
less than seen in spleen without major preference for the periphery
(Figure 4I,K). Engraftment in spleen remained extensive (Figure 4J,L).
Overall, there were a noticeably higher proportion of GFP1 cells engrafted in spleen than in liver (Figure 4M-N) at 6 weeks after transplant.
Collectively, these results indicate that both liver and spleen
contain transplantable HSCs and are active sites of hematopoiesis.
Nonablative in utero/larval transplants prevent GVHD
Irradiative conditioning has been shown to impair regeneration of the
axolotl.37,38 To avoid irradiation, and generate stable blood chimeras,
2 microsurgical techniques in embryonic and larval axolotls were
used: parabiosis and intracardiac microinjection HCT. Parabiosis
allows anatomical joining of 2 embryos resulting in a shared
Figure 4. Effects of ablative HCT on the liver and spleen. Characteristic histology
analysis of irradiated and or transplanted axolotl. The examples shown are from
recipients of both spleen and liver cells, but the results are similar in spleen or
liver only transplants. No other donor tissue transplant resulted in GFP1 donor cell
engraftment. (A,C) H&E staining (340) of the liver. Irradiation destroys the peripheral
hematopoietic layer of the liver demarcated by the red line and “H”. The black line
marks where the hematopoietic layer ends and hepatocytes begin. (B,D) H&E
staining (340) of the spleen. The tissue displays some degradation after irradiation.
(E,G) H&E staining (320) of the liver with matching direct GFP 3 weeks after
irradiation and transplant. The hematopoietic peripheral layer is still present but
partially ablated and shows more engraftment by donor cells than the rest of the liver.
Figure 4 (continued) (F,H) H&E staining (320) of the spleen with matching direct
GFP 3 weeks after irradiation and transplant. Donor cells engraft all through the
organ without preference as seen in the liver. (I,K) H&E staining (320) of the liver
with matching direct GFP 6 weeks after irradiation and transplant. The hematopoietic
layer has been ablated and donor cells show poor engraftment without preference for
the periphery. (J,L) H&E staining (320) of the spleen with matching direct GFP 6
weeks after irradiation and transplant. There is no change in engraftment as seen in
the liver. (M-N) Direct GFP section (35) of the liver and spleen at 4 weeks after
irradiation and transplant. Donor cells show some preference for engraftment near
the periphery in the liver but none in the spleen and there is substantially more
engraftment in the spleen.
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circulation. The procedure requires 1 surgery to fuse a GFP1 embryo
with a white mutant embryo side by side (supplemental Figure 3A-D),
and then another 24 hours later to denervate the GFP1 embryo.
Denervation prevents injury as the 2 axolotls develop together. It is
then possible to track GFP1 blood in the white axolotl (supplemental
Figure 3E-F). This method proved useful for short-term studies, but
difficulties with rearing parabiotic axolotls to adulthood compelled
us to explore further options.
HCT normally requires chemical or irradiative conditioning, and
GVHD is expected in unmatched donor-recipient pairs. One exception across a number of species is HCT in the embryonic stages of
development. Thus, we sought to induce tolerance by intracardiac
microinjection (N 5 25) of GFP1 hematopoietic cells into recipient
embryos and larvae prior to immune system maturation (up to 3
months of age). In our experiments, 18 of 25 (72%) recipients of
whole adult liver or spleen cells showed persistence of GFP1 blood
for the life of the animal with varying levels of chimerism (4.0-8.0%
in blood). GFP1 cells were directly visualized homing and engrafting in the larval liver (Figure 5A,C) and spleen (Figure 5B,D) as
the animals developed irrespective of cell origin. GVHD was never
observed in animals that underwent the procedure from stage 36 to
3 months of age. In transplants of 3-month-old larvae, donor engraftment persisted for .2 years in 9 of 10 recipients of $1 3 104
whole adult spleen or liver cells (Figure 5E-H) with blood chimerism
levels at 4% to 8%. Fewer axolotls showed engraftment, and blood
chimerism was undetectable beyond 2 months when less than 1 3 104
spleen cells were injected. In comparison with irradiated chimeras,
microinjected axolotls showed fewer GFP1 donor cells in the liver’s
peripheral hematopoietic layer (Figure 5E,G). However, spleens
showed greater engraftment than livers as previously observed in
irradiated chimeras (Figure 5G-H). Together, these results provide
conclusive evidence that both liver and spleen contain HSCs capable
of long-term durable engraftment.
First transplantable HSC arises in the embryonic axolotl liver
To determine at which point in embryonic development transplantable HSCs can be isolated, hematopoietic cells were collected from
GFP1 axolotls throughout development. Cells from blood islands
(developing at stages 32-38),31 larval livers (developing at stage 39),
and larval spleens (developing after hatching) were used as graft
sources. No animals receiving blood island cell transplants showed
evidence of donor hematopoiesis. However, all animals receiving
larval liver cell transplant or larval spleen cell transplant exhibited
donor engraftment (data not shown). These results imply that a
transplantable HSC first develops in the axolotl liver at stage 39 prior
to the formation of the spleen. On formation, the spleen also contains
transplantable HSC in the larvae, which is a condition retained
through adulthood.
Spleen supports long-term HSC self-renewal in adult axolotl
Because both livers and spleens in embryos and adult axolotls contain transplantable HSCs, our next goal was to determine if one
or both organs provide a microenvironment capable of supporting
long-term HSC (LT-HSC) self-renewal. By bisecting GFP1
embryos and fusing the cephalic portion with the caudal portion
of either white or nucCherryRed1 embryos (Figure 6A-D), spleens
would develop either the same or a different color than livers
facilitating identification of the long-term HSC niche. Nine of 9
(100%) animals showed evidence of GFP1 hematopoiesis for the
first 5 months in the juvenile stage of axolotl development
Figure 5. Donor HSCs engraft in the liver and spleen in microinjected embryos
and larvae to create stable chimeras. (A) Anesthetized larva 1 month after microinjection transplant with the liver visible through the skin (white box). (B) Anesthetized
3-month-old larvae 4 days after microinjection transplant with the spleen visible
through the skin (white box). (C-D) In vivo GFP channel view of the magnified liver
in A and spleen in B shows engraftment of a large number of GFP1 donor cells. (E-F)
H&E staining (340) of the liver and spleen from a 2-year-old microinjected
chimera. (G) Section (340) displaying donor GFP1 cell engraftment in the liver in E.
No preference for the peripheral hematopoietic layer is visible. (H) Section (340)
displaying greater donor GFP1 cell engraftment in the spleen in F than in the liver.
(Figure 6E), but as the animals developed into adulthood (.5
months), GFP1 hematopoiesis gradually disappeared by 9 months
in some axolotls. In these adult animals, GFP1 cells first
disappeared from vasculature and then from skin (Figure 6F). On
examination of internal organs, the liver and other organs were
found to be GFP1 , whereas the spleen was white (Figure 6G-H;
supplemental Figure 4). GFP1 cells are therefore derived from the
liver, implying that liver supports hematopoietic progenitor activity
but not LT-HSC self-renewal.
When cephalic GFP1 bisections (containing liver) were fused
with nucCherryRed1 bisections (containing spleen), we were able to
confirm our previous observations (Figure 6I-J). In larval stages,
there were relatively equal numbers of GFP1 and Red1 cells in circulation, but as the animals continued to develop, Red1 blood predominated. The density of HSC-derived cells with each fluorescent
protein fixed in the skin provides confirmation (Figure 6K-L; supplemental Figure 5). Long-term adult blood color always matches
spleen color. Thus, even though liver contains transplantable
LT-HSCs and contributes to blood production in adult axolotls, the
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BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
LOPEZ et al
primary microenvironment that supports LT-HSC self-renewal in
the adult axolotl is spleen.
Axolotl HCT yields long-term multilineage engraftment
Our next goal was to demonstrate that HSC transplant in axolotls
results in long-term multilineage engraftment. Unfortunately, almost
no lineage-specific antibodies are available for axolotl hematopoietic
cells. Figure 7A and supplemental Figure 6 show FACS analysis of
peripheral blood demonstrating reconstitution with both GFPLow
and GFPHigh populations in recipient animals. As in mice, the CMV:
b-actin promoter driven GFP transgene is expressed at variable levels
even within a given lineage.39 We have multiple animals that now have
GFP1 donor engraftment .2 years after transplant, thus showing
long-term repopulation. Figure 7B shows that long-term engraftment remains clearly visible in the skin of recipient animals with
a GFP1/nucCherryRed1 fusion chimera as a control.
We next wanted to show multilineage reconstitution in transplant
recipients. We and others have extensively screened extant commercially available antibodies for ones that will cross-react with
axolotl blood cells. To date, F4/80 cross-reacts with mature axolotl
macrophages, CD79a (Mb-1) cross-reacts with B lymphocytes, and
CD2 with T cells (see Materials and methods for details). However,
none of these cross-reactive antibodies work well for flow cytometry.
Therefore, we used Ficoll gradient sedimentation of peripheral blood/
spleen/liver to separate erythroid populations from leukocytes,
followed by cytospins and functional assays or immunohistochemical staining for lineage markers and GFP expression. Functional
characterization of blood cells by a fluorescent E coli phagocytic
assay confirms that donor-derived GFP1 phagocytic myeloid cells are
present in both nonablative microinjected chimeras and irradiated
chimeras along with abundant nonphagocytic GFP1 lymphocytes
(Figure 7C). Immunohistochemistry for mature macrophage marker
F4/80 (Figure 7D), B-lymphocyte marker CD79a (mb-1) (Figure 7E),
and T-lymphocyte marker CD2 (Figure 7F) in red fluorescence combined with GFP immunostaining in green fluorescence clearly
demonstrates reconstitution of multiple white blood cell lineages.
Peripheral blood shows the presence of both donor-GFPLow and
recipient non-GFP erythrocytes (Figure 7G). Polymerase chain
reaction (PCR)-based genotyping of Ficoll-purified erythrocyte blood
cell fractions verifies the presence of the GFP gene in erythrocytes
from GFP donors, GFP1 /white fusion chimeras, and irradiated
chimeras (Figure 7H). Collectively, the data demonstrate long-term
multilineage reconstitution following axolotl HCT.
Discussion
Figure 6. Embryonically fused chimeras reveal that the spleen is the adult and
long-term hematopoietic organ. Embryonic fusions were created by surgical
juxtapositioning of the anterior and posterior halves from GFP1 embryos and either
white or nucCherryRed1 embryos. Depending on exact positioning of the split, the
liver and spleen can be derived from the same or differing halves yielding the same
or differing colors of blood cell produced by each organ. (A) Half GFP1, half white
wild-type axolotl with a GFP1 liver. (B) Half GFP1, half nucCherryRed1 larva. (C)
Underside of the larva in B displaying how various tissues and organs are composed
of genetically different cells. (D) Forelimb with GFP1 skin and muscle and Red1
bone. (E) GFP1 hematopoietic cells in the GFP 2 tail of a 4-month-old fused
chimera. (F) The GFP2 tail of a 9-month-old fused chimera that previously had
GFP1 blood. (G-H) The liver and spleen of a GFP1 cephalic half and white tail fused
chimera with no visible GFP1 blood in circulation. The liver is GFP1 but the spleen is
GFP- and has no GFP1 blood cells. (I-J) GFP1 and Red1 larva with a Red1 spleen
and a GFP1 liver visible through the skin. (K) nucCherryRed1 spleen-derived blood
cells visualized in the GFP1 Red2 skin of a green/red fusion animal at 7 months of
Salamanders are an order of amphibia comprised of .650 extant
species. Among vertebrates, salamanders exhibit an unparalleled
ability for adult regeneration. The axolotl offers numerous advantages
over other highly regenerative species. Unlike other urodeles, axolotls
Figure 6 (continued) age. (L) GFP1 liver-derived blood cells visualized in the
GFP2 Red1 skin of the same fusion animal at the same time point as in K. In these
fusions, the liver-derived contribution slowly diminishes over time. See Figure 7
and supplemental Figures 5 and 6. The FACS plots shown in Figure 7 and
supplemental Figure 6 for the GFP/white fusion are of the peripheral blood in the
same animal but taken several months apart. The FACS plot in supplemental
Figure 6 was taken earlier and displays a greater percentage of GFP1 cells than
the plot in Figure 7, again supporting the results observed in the pictures.
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BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
AXOLOTL HEMATOPOIESIS AND TRANSPLANTATION
1239
Figure 7. Multilineage potential. (A) FACS blood
plots of the white wild-type (WT), GFP donor, spleen
microinjections (MI), GFP/white 2-color, and ablative
liver and spleen HCT. (B) Representative images of
GFP1 donor-derived immune cell types in the skin of
2-color, spleen microinjected HCT, and ablative spleen
and liver HCT. (C) Phagocytic and nonphagocytic cells
isolated from spleen or liver microinjected animals,
and ablative HCT chimeras. (D) Mature macrophages
isolated from microinjected and ablative HCT animals
identified by F4/80 staining. (E) B cells isolated from
microinjected and ablative HCT animals. Green particles are fluorescent E coli used in the phagocytic
assay. (F) T cells isolated from microinjected and
ablative HCT axolotls. (G) GFP immunostaining shows
various levels of GFP expression across blood cells.
Low levels of GFP expression can be detected in
nucleated donor-derived erythrocytes (large white
arrow). No GFP expression is detected in recipient
erythrocytes (small white arrow). (H) PCR-based
genotyping. Lane 1, GFP donor whole blood; lane 2,
GFP donor red blood cell fraction; lane 3, spleen microinjected chimera; lane 4, irradiated and spleen transplanted chimera with no GVHD; lane 5, irradiated and
transplanted chimera with GVHD; lane 6, GFP1 cephalic
half and white tail chimera; lane 7, white wild-type axolotl;
lane 8, water control. Generuler low range DNA ladder
(25-700 bp) confirms the expected 173-bp amplified
band of the GFP gene.
are easily bred in captivity, with a single female producing hundreds
of eggs per spawning. Eggs are 3 mm in diameter, which allows
unique embryonic manipulations.40 The axolotl’s immune system
has a great deal of homology with that of higher vertebrates. All major
immunologic components are present,1,18,19 and hematopoiesis undergoes similar lineage differentiation as mammals with the exception
of erythropoiesis in which circulating erythrocytes often retain nuclei
in axolotls.18,19 Axolotl innate immunity is composed of antimicrobial peptides, neutrophils, macrophages, complement, and oxidant
protective factors.1 Their adaptive immune system has T and B lymphocytes and 3 classes of antibodies (immunoglobulin [Ig]M, IgY, and
IgX).41 Also, immune signaling pathways such as nuclear factor kB
have been detected, along with major histocompatibility complexes
I and II.1
Axolotls are 1 of a few salamander species that is an obligate
neotene. Neoteny is a form of paedomorphyism where juvenile traits
are retained by adults. Axolotl reach sexual maturity without undergoing metamorphosis to a more terrestrial form; hence, they retain
many larval traits and live in water. The immune system of axolotl
also exhibits neoteny. Like mammalian neonates, axolotl do not
immunize well with soluble antigens and have a reduced ability to
mount a memory (secondary) immune response, and graft rejection is
a chronic event taking 3 to 8 weeks.42,43 The axolotl hematopoietic
system is known to undergo a gradual cryptic metamorphosis wherein
B cells, T cells, and erythrocytes express more major histocompatibility complex II, and there is a juvenile to adult hemoglobin switch,
which is believed to be the end of establishing immune tolerance in
amphibians.34-36 Axolotls induced with exogenous thyroid hormone
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BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
LOPEZ et al
undergo metamorphosis to an adult terrestrial phenotype. Some aspects of their immune system have also been reported to mature
following these changes, and the result is an improved response to
soluble antigens, production of secondary immune responses, and
accelerated graft rejection.18,34,44 Yet, like all salamanders, mature
axolotls retain full regenerative capabilities. This represents a
unique model to facilitate discrimination between developmental
vs regenerative-related changes in the axolotl hematopoietic system.
Now, the necessary genetic tools to map regenerative pathways
are becoming available for the axolotl, thus allowing comparative
studies between scarring and regeneration.6,11-16 Regeneration being
a complex process involving precisely coordinated interplay between
various cell types, of which the HSC and its progeny are proving
themselves integral.29,45 Here we developed basic axolotl CFU
assays that were used to map the liver and spleen as sites of active
hematopoiesis in adult axolotls. We also developed both ablative and
nonablative HCTs, yielding long-term multilineage engraftment of
fluorescent protein-tagged blood cells in white/translucent axolotls.
By directly manipulating axolotl embryos and tracking tagged cells,
we were able to prove that spleen, and not liver, contains the selfrenewing HSC niche in adult axolotls. Although the adult liver
remains a secondary site of hematopoiesis, containing both CFU
generating progenitors and transplantable HSCs, it is not capable of
supporting HSC self-renewal. These findings fit well with a recent
study of TdT gene expression in axolotls that shows TdT gene
shutdown occurs in liver prior to spleen during axolotl maturation.27
HCT was also used to demonstrate that transplantable HSCs first
arise in axolotl larval livers at stage 39 of development. In the absence
of available donor markers in the axolotl, these interpretations are
based on expression of CMV:b-actin driven transgenes expressing
fluorescent proteins. Expression from theses transgenes varies from
low to high within and across specific lineages as seen in Figure 7. Our
interpretations are based on GFPHigh expression levels, which will
under-report overall levels of engraftment. CMV-based transgenes
can also experience gene silencing, which would also result in underreporting of donor engraftment in axolotl transplants.46-49
Axolotls are outbred animals, and initial adult donor transplants
had significant problems with apparent GVHD complications. Cryptic
metamorphosis of the axolotl immune system is usually complete by 6
to 7 months,34 providing a window of opportunity to isolate donor cells
with immature immunity that greatly reduced incidence of GVHD in
adult recipients. Concerns with diminishing the axolotl’s regenerative
capacity through irradiative conditioning were eliminated by performing the equivalent of nonablative in utero transplants into embryonic
and juvenile stage axolotls prior to immune system maturation to
induce graft tolerance. Resultant animals never exhibited apparent
GVHD during long-term multilineage engraftment, albeit at slightly
reduced levels of overall engraftment compared with ablative transplants. With a stable engraftment efficiency of .90%, this technique
has an excellent success rate in comparison with in utero or intracardiac
microinjection transplants performed in other species.50-53
We recently demonstrated that axolotls completely regenerate
dermal lesions, including all skin structures, without scar formation,
whereas control mice exhibit classic mammalian scarring.30 Combining the in vivo and in vitro hematopoietic tools described will
help elucidate the HSC’s contribution to vertebrate regeneration in
the axolotl.
Acknowledgments
The authors thank Dr Ying Li for assistance and knowledge of
cytochemical stains; Neal Benson and Dr Koji Hosaka for technical assistance with flow cytometry; Dr Gregory Marshall for
providing technical help and critically reviewing the manuscript;
Drs Ashley Seifert, Seung-bum Kim, Anitha Shenoy, Huiming
Xia, Liya Pi, and Mr Gary Brown for technical help; Anna K. Rodgers
and Dr William Slayton for editorial assistance; Dr David Fuller
for project guidance; and the Ambystoma Genetic Stock Center
(AGSC) for providing axolotls.
Ambystoma Genetic Stock Center is supported by National
Science Foundation grant NSF-DBI-0951484.
This work was funded by National Institutes of Health, National
Institute of Neurological Disorders and Stroke grant RC2 NS069480,
National Institute of Diabetes and Digestive and Kidney Diseases
grant T32 DK 074367, and National Heart, Lung, and Blood Institute
grant HL70758.
Authorship
Contribution: D.L. designed experiments, performed research, collected and analyzed data, and wrote the manuscript; L.L. assisted
with research and carrying out experiments; J.R.M. provided technical assistance and performed experiments; C.R.C. helped with
interpreting data and editing the manuscript; F.J.B. contributed his
expertise and use of the Varian Clinac 600/C linear accelerator; M.M.
aided in the design of the experiments; and E.W.S. was responsible
for conception and design, data analysis and interpretation, and
redaction of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Edward W. Scott, University of Florida, Department of Molecular Genetics, Box 100201, 1600 SW Archer Rd,
Gainesville, FL 32610-0201; e-mail: escott@ufl.edu.
References
1. Harty M, Neff AW, King MW, Mescher AL.
Regeneration or scarring: an immunologic
perspective. Dev Dyn. 2003;226(2):
268-279.
4. Fathke C, Wilson L, Hutter J, et al. Contribution
of bone marrow-derived cells to skin: collagen
deposition and wound repair. Stem Cells. 2004;
22(5):812-822.
2. Martin P, Souza DD, Martin J, et al. Wound
healing in the PU.1 null mouse—tissue repair is
not dependent on inflammatory cells. Curr Biol.
2003;13(13):1122-1128.
5. Gurtner GC, Werner S, Barrandon Y, Longaker
MT. Wound repair and regeneration. Nature.
2008;453(7193):314-321.
3. Seifert AW, Kiama SG, Seifert MG, Goheen JR,
Palmer TM, Maden M. Skin shedding
and tissue regeneration in African spiny
mice (Acomys). Nature. 2012;489(7417):
561-565.
6. Sobkow L, Epperlein HH, Herklotz S, Straube
WL, Tanaka EM. A germline GFP transgenic
axolotl and its use to track cell fate: dual origin
of the fin mesenchyme during development and
the fate of blood cells during regeneration. Dev
Biol. 2006;290(2):386-397.
7. Whited JL, Lehoczky JA, Tabin CJ. Inducible
genetic system for the axolotl. Proc Natl Acad Sci
USA. 2012;109(34):13662-13667.
8. Khattak S, Murawala P, Andreas H, et al.
Optimized axolotl (Ambystoma mexicanum)
husbandry, breeding, metamorphosis,
transgenesis and tamoxifen-mediated
recombination. Nat Protoc. 2014;9(3):529-540.
9. Sandoval-Guzmán T, Wang H, Khattak S, et al.
Fundamental differences in dedifferentiation and
stem cell recruitment during skeletal muscle
regeneration in two salamander species. Cell
Stem Cell. 2014;14(2):174-187.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8
AXOLOTL HEMATOPOIESIS AND TRANSPLANTATION
10. Khattak S, Schuez M, Richter T, et al. Germline
Transgenic Methods for Tracking Cells and
Testing Gene Function during Regeneration in
the Axolotl. Stem Cell Rev. 2013;1(1):90-103.
25. Meeker ND, Hutchinson SA, Ho L, Trede NS.
Method for isolation of PCR-ready genomic DNA
from zebrafish tissues. Biotechniques. 2007;
43(5):610-614, 612, 614.
11. SGP/Sal-Site. 2014. Available at: http://
www.ambystoma.org/. Accessed May 29, 2012.
26. Durand C, Charlemagne J, Fellah JS. RAG
expression is restricted to the first year of life in
the Mexican axolotl. Immunogenetics. 2000;
51(8-9):681-687.
12. Wu C-H, Tsai M-H, Ho C-C, Chen C-Y, Lee H-S.
De novo transcriptome sequencing of axolotl
blastema for identification of differentially
expressed genes during limb regeneration.
BMC Genomics. 2013;14(1):434.
13. Habermann B, Bebin A-G, Herklotz S, et al. An
Ambystoma mexicanum EST sequencing project:
analysis of 17,352 expressed sequence tags from
embryonic and regenerating blastema cDNA
libraries. Genome Biol. 2004;5(9):R67.
14. Monaghan JR, Epp LG, Putta S, et al. Microarray
and cDNA sequence analysis of transcription
during nerve-dependent limb regeneration. BMC
Biol. 2009;7:1.
15. Monaghan JR, Athippozhy A, Seifert AW, et al.
Gene expression patterns specific to the
regenerating limb of the Mexican axolotl. Biol
Open. 2012;1(10):937-948.
16. Campbell LJ, Suárez-Castillo EC, Ortiz-Zuazaga
H, Knapp D, Tanaka EM, Crews CM. Gene
expression profile of the regeneration epithelium
during axolotl limb regeneration. Dev Dyn. 2011;
240(7):1826-1840.
17. Voss SR, Kump DK, Putta S, et al. Origin of
amphibian and avian chromosomes by fission,
fusion, and retention of ancestral chromosomes.
Genome Res. 2011;21(8):1306-1312.
18. Ussing AP, Rosenkilde P. Effect of induced
metamorphosis on the immune system of the
axolotl, Ambystoma mexicanum. Gen Comp
Endocrinol. 1995;97(3):308-319.
19. Charlemagne J. Aspects morphologiques de la
différenciation des éléments sanguins chez
l’Axolotl,Ambystoma mexicanum Shaw. Zeitschrift
Zellforsch Mikroskopische Anat. 1971;123(2):
224-239.
20. Bordzilovskaya NP, Dettlaff TA, Duhon ST,
Malacinski GM. Developmental-Stage Series of
Axolotl Embryos. In: Armstrong JB, Malacinski
GM, eds. Developmental Biology of the Axolotl.
New York, NY: Oxford University Press; 1989:
201-219.
27. Golub R, André S, Hassanin A, Affaticati P,
Larijani M, Fellah JS. Early expression of two TdT
isoforms in the hematopoietic system of the
Mexican axolotl. Implications for the evolutionary
origin of the N-nucleotide addition.
Immunogenetics. 2004;56(3):204-213.
28. Lévesque M, Gatien S, Finnson K, et al.
Transforming growth factor: beta signaling is
essential for limb regeneration in axolotls. PLoS
ONE. 2007;2(11):e1227.
29. Godwin JW, Pinto R. Rosenthal NA.
Macrophages are required for adult salamander
limb regeneration. Proc Natl Acad Sci USA. 2013;
110(23):9415-9420.
30. Seifert AW, Monaghan JR, Voss SR, Maden M.
Skin regeneration in adult axolotls: a blueprint for
scar-free healing in vertebrates. PLoS ONE.
2012;7(4):e32875.
31. Durand C, Kerfourn F, Charlemagne J, Fellah JS.
Identification and expression of Helios, a member
of the Ikaros family, in the Mexican axolotl:
implications for the embryonic origin of
lymphocyte progenitors. Eur J Immunol. 2002;
32(6):1748-1752.
32. Charlemagne J. Aspects morphologiques de la
différenciation des éléments sanguins chez
l’axolotl, Ambystoma mexicanum Shaw. Z
Zellforsch Mikrosk Anat. 1972;123(2):224-239.
33. Kappers JA, Ten Kate IB, De Bruyn HJ. On
mast cells in the choroid plexus of the axolotl
(Ambystoma mex.). Z Zellforsch Mikrosk Anat.
1958;48(6):617-634.
34. Völk H, Charlemagne J, Tournefier A, et al.
Wide tissue distribution of axolotl class II
molecules occurs independently of thyroxin.
Immunogenetics. 1998;47(5):339-349.
35. Ducibella T. The occurrence of biochemical
metamorphic events without anatomical
metamorphosis in the axolotl. Dev Biol. 1974;
38(1):175-186.
21. Armstrong JB, Malacinski GM. Developmental
Biology of the Axolotl. New York: Oxford
University Press; 1989.
36. Tompkins R. Genic control of axolotl
metamorphosis. Am Zool. 1978;18(2):313-319.
22. Brunst VV. The effect of total-body x-irradiation on
the adult axolotl (Siredon mexicanum). Radiat
Res. 1958;8(1):32-45.
37. Brunst VV. The effects of partial irradiation on tail
regeneration in adult Siredon mexicanum. Am J
Roentgenol Radium Ther Nucl Med. 1969;105(1):
196-206.
23. Higuchi R. Simple and rapid preparation of
samples for PCR. In: Erlich HA, ed. PCR
Technology: Principles and Applications for DNA
Amplification. New York, NY: Stockton Press;
1989:31-38.
24. Zackson SL, Steinberg MS. Cranial neural crest
cells exhibit directed migration on the pronephric
duct pathway: further evidence for an in vivo
adhesion gradient. Dev Biol. 1986;117(2):
342-353.
38. Brunst VV. Effects of ionizing radiation on the
development of amphibians. Q Rev Biol. 1965;
40(1):1-67.
39. Okabe M, Ikawa M, Kominami K, Nakanishi T,
Nishimune Y. ‘Green mice’ as a source of
ubiquitous green cells. FEBS Lett. 1997;407(3):
313-319.
40. Voss SR, Epperlein HH, Tanaka EM. Ambystoma
mexicanum, the Axolotl: A Versatile Amphibian
1241
Model for Regeneration, Development, and
Evolution Studies. Cold Spring Harb. Protoc.
2009;2009(8):pdb.emo128.
41. Schaerlinger B, Frippiat J-P. IgX antibodies in the
urodele amphibian Ambystoma mexicanum. Dev
Comp Immunol. 2008;32(8):908-915.
42. André S, Kerfourn F, Affaticati P, Guerci A,
Ravassard P, Fellah JS. Highly restricted diversity
of TCR delta chains of the amphibian Mexican
axolotl (Ambystoma mexicanum) in peripheral
tissues. Eur J Immunol. 2007;37(6):1621-1633.
43. Tournefier A, Laurens V, Chapusot C, et al.
Structure of MHC class I and class II cDNAs and
possible immunodeficiency linked to class II
expression in the Mexican axolotl. Immunol Rev.
1998;166:259-277.
44. Tahan AM, Jurd RD. Effect of temperature
on transplantation immunity in Ambystoma
mexicanum. Dev Comp Immunol. 1978;2(3):
435-442.
45. Jones JE, Corwin JT. Replacement of lateral
line sensory organs during tail regeneration in
salamanders: identification of progenitor cells and
analysis of leukocyte activity. J Neurosci. 1993;
13(3):1022-1034.
46. Brooks AR, Harkins RN, Wang P, Qian HS,
Liu P, Rubanyi GM. Transcriptional silencing is
associated with extensive methylation of the CMV
promoter following adenoviral gene delivery to
muscle. J Gene Med. 2004;6(4):395-404.
47. Löser P, Jennings GS, Strauss M, Sandig V.
Reactivation of the previously silenced
cytomegalovirus major immediate-early promoter
in the mouse liver: involvement of NFkappaB.
J Virol. 1998;72(1):180-190.
48. Kong Q, Wu M, Huan Y, et al. Transgene
expression is associated with copy number
and cytomegalovirus promoter methylation in
transgenic pigs. PLoS ONE. 2009;4(8):e6679.
49. Morrissey D, Collins SA, Rajenderan S, Casey G,
O’Sullivan GC, Tangney M. Plasmid transgene
expression in vivo: promoter and tissue variables. In:
Martin F, ed. Gene Therapy - Tools and Potential
Applications. InTech. 2013. Available at http://www.
intechopen.com/books/gene-therapy-tools-andpotential-applications/plasmid-transgeneexpression-in-vivo-promoter-and-tissue-variables.
50. Traver D, Paw BH, Poss KD, Penberthy WT, Lin
S, Zon LI. Transplantation and in vivo imaging of
multilineage engraftment in zebrafish bloodless
mutants. Nat Immunol. 2003;4(12):1238-1246.
51. Chen X, Gong XL, Katsumata M, Zeng YT, Huang
SZ, Zeng F. Hematopoietic stem cell engraftment
by early-stage in utero transplantation in a mouse
model. Exp Mol Pathol. 2009;87(3):173-177.
52. Zanjani ED, Flake AW, Rice H, Hedrick M,
Tavassoli M. Long-term repopulating ability of
xenogeneic transplanted human fetal liver
hematopoietic stem cells in sheep. J Clin Invest.
1994;93(3):1051-1055.
53. Zeng F, Chen M, Katsumata M, et al. Identification
and characterization of engrafted human cells
in human/goat xenogeneic transplantation
chimerism. DNA Cell Biol. 2005;24(7):403-409.
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2014 124: 1232-1241
doi:10.1182/blood-2013-09-526970 originally published
online May 6, 2014
Mapping hematopoiesis in a fully regenerative vertebrate: the axolotl
David Lopez, Li Lin, James R. Monaghan, Christopher R. Cogle, Frank J. Bova, Malcolm Maden and
Edward W. Scott
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