Combinatorial pigmentation mutants yield transparent adult zebrafish
The characteristic adult pigmentation pattern of the zebrafish consists of three distinct classes of pigment cells arranged in stripes (): black melanophores, reflective iridophores, and yellow xanthophores(Rawls, et al, 2001
). The nacre
mutant () has a complete lack of melanocytes due to a mutation in the gene encoding the mitfa
gene.(Lister, et al, 1999
). The roy orbison
) zebrafish () is a spontaneous mutant, and has a complete lack of iridophores, uniformly pigmented eyes, sparse melanocytes, and a translucency of the skin. The gene responsible for this mutant phenotype is currently unknown.
Figure 1 Zebrafish pigmentation mutants exhibit defects in specific neural crest derived populations. A wild-type zebrafish (1A) demonstrates alternating patterns of deeply pigmented stripes comprised largely of melanocytes. The interstripe regions, devoid of (more ...)
Fish that are doubly mutant for nacre
are shown in . This line, which we have named casper
for its ghost like appearance, demonstrates a complete lack of all melanocytes and iridophores in both embryogenesis and adulthood. Most strikingly, the body of the fish is almost entirely transparent. In female adults, individual eggs are easily observable, as seen in . We compared the optical transparency of the casper
line to several commonly used zebrafish pigmentation mutants, including golden, albino, rose
, and panther
. Organs that can be seen using standard stereomicroscopy in casper
include the heart, aorta, intestinal tube, liver and gallbladder. Brain tissue is visible to a small extent. None of these organs are easily visible in any of the other pigment mutants studied (Supplementary Figure 1
), other than the heart and intestinal tube in rose
. Examination of the cardiac region of the casper
mutant (Supplementary Video 1
) shows that ventricular contraction can be observed, making this mutant useful for in vivo
analysis of genetic perturbations in cardiac function. The casper
mutant is entirely viable, with incrossed adults producing large numbers of viable offspring at expected mendelian ratios and no heterozygous phenotype.
Ocular and skin transparency is due to loss of iridophore crystals
fish were sectioned and stained with hematoxylin and eosin, as well as Masson’s trichrome to identify collagenous fibers. The gross morphology of the casper
eye is normal, with maintenance of the scleral-corneal border and normal pigmented retinal epithelial cells. However, there is a complete absence of the scleral iridophore layer (, arrow), which is demonstrated by the loss of refractive crystalline plates(Clothier, et al, 1987
) referred to as schemochromes. In wild-type animals, the pigmented retinal epithelium is normally obscured by the reflective iridophores; their absence in roy
mutants exposes the black cells across the entire surface of the eye.
Figure 2 Transverse section of wild-type (2A) and casper (roy;nacre) eyes (2B) demonstrates that the uniformly pigmented eye in the mutant line is primarily due to a loss of reflective iridophores in the sclera (arrow shows iridophores in wild-type fish), which (more ...)
The iridophores of the skin are a dermal-hypodermal structure which are normally intimately connected to the surrounding xanthophores and lie just above the trunk skeletal muscle(Le Guellec, et al, 2004
). While wild-type fish (, arrow) show a substantial layer of these reflective cells, the casper
mutant has a complete absence of this layer, while the remaining skin structures are relatively intact, including epidermal scales and dermal collagen fibers as demonstrated by Masson’s trichrome stain (data not shown). These data suggest that the relative transparency of the casper
mutant is due to a combination of melanocyte loss (which normally absorbs incident light and protects subdermal structures) and iridophore loss (which normally reflect incident light away from the internal organs).
The transparent casper mutant reveals timing and homing of hematopoietic stem cells to the marrow compartment
Prior work from our laboratory has demonstrated that the transplantable hematopoietic stem/progenitor (HSPC) marrow population resides within the adult kidney, and can be isolated using flow cytometric analysis of forward and side scatter(Traver, et al, 2003
). We isolated whole kidney marrow from beta-actin:GFP
transgenic fish (which labels all cell types except red blood cells) and performed intra-cardiac (ventricular) transplantation of 100,000 whole kidney marrow cells along with 200,000 carrier red blood cells into a recipient casper
mutant that had previously been irradiated with 25Gy. At 4 hours post transplant, a pool of blood surrounds the cardiac chambers, which likely represents extravasated blood in the pericardial sac. Within 2 weeks (, left), a GFP labeled population of cells is seen within the region of the zebrafish kidney as well as in the gill vasculature. By 4 weeks, a significant increase in the size and distribution of this GFP labeled population can be easily visualized, and is now largely confined to the anatomical area of the kidney of the fish (, right). For comparison, a wild-type fish transplanted in an identical manner is shown, demonstrating that essentially no GFP can normally be seen in an opaque adult. These data suggest that the transparent casper
mutant allows for ready visualization of hematopoietic progenitor engraftment after a sublethal dose of 25Gy, consistent with short-term hematopoietic engraftment.
Figure 3 Whole kidney marrow from beta actin:GFP labeled donors was transplanted into either wild-type (n=6) or casper (n=6) irradiated recipients via intra-cardiac (IC) injection of 100,000 cells. Fish were imaged from 4 hours until 5 weeks post transplant at (more ...)
The kidneys were dissected from the 4 week post transplant fish (both casper and wild-type) and analyzed for their forward/side scatter profile as well as GFP. Although no GFP can be visualized with a fluorescent stereoscope in the wild-type, a normal distribution of hematopoietic cells is seen at 4 weeks post transplant, and many of these cells are GFP positive (, top). Based on the scatter profile, multilineage engraftment has occurred, which indicates that the engrafted cells represent a primitive hematopoietic population. The kidney from the casper mutant shows a similar reconstitution of hematopoietic lineages which are also GFP positive, to the same degree (, bottom). These data demonstrate that the anatomic localization of GFP positive cells in the casper mutant is indeed indicative of marrow reconstitution.
Recipient fish at 4 weeks post transplant were fixed and processed for H&E and anti-GFP IHC. Kidney sections shows large numbers of hematopoietic cells (H)surrounding the tubules (T) and glomeruli (G) of the kidney (, left), consistent with previous observations(Traver, et al, 2004b
). All of the hematopoietic cells are strongly GFP-positive by IHC (, right - brown staining), whereas none of the kidney tubule cells stain for GFP. This confirms the proper homing of the GFP-labeled marrow cells back to the kidney marrow of the recipient casper
Because one of the major advantages of an optically clear adult animal is the ability to achieve single-cell resolution in a live animal, we next used confocal microscopy on adult zebrafish that had been transplanted with beta-actin GFP HSPCs 4 weeks earlier. Imaging of a transplanted wild-type recipient yielded no discernable GFP signal due to the opacity of the normal skin. In contrast, the casper mutant allowed for identification of cells both singly and in clusters () at a resolution of approximately 5 um. The maximal depth of field achieved with a Zeiss confocal microscope was 88um from the surface of the skin.
To ensure that the casper mutant will be generally applicable to stem cell biologists, we measured survival after HSPC transplant in wild-type and casper recipients. As shown in , although both groups show an initial procedural mortality, there is no significant difference in survival between the groups, indicating that casper will function similarly to wild-type animals in transplant studies.
The casper mutant allows for rapid identification of transplanted tumor cells
We next assessed whether the transparent casper zebrafish could be used to analyze in vivo tumor engraftment and migration after transplantation. Stable transgenic zebrafish carrying either the mutated human B-raf or N-ras-GFP fusion oncogene under the control of the melanocyte specific mitf promoter were crossed to p53-/- fish. These fish reliably develop highly aggressive melanoma in 4-12 months. A tumor from an adult fish was disaggregated into a single cell suspension and 200,000 cells were transplanted either into the ventral peritoneum (IP) or via intra-ventricular (IC) injection into an irradiated casper recipient. Within 5 days post IP transplantation of the NRAS-GFP melanoma (, top), a large mass of deeply pigmented tumor cells could be visualized within the peritoneum. In addition, a small number of cells have localized to the dorsal epidermal scales (, top, circled). The pattern of engraftment at 5 days in the intra-cardiac recipients of NRAS-GFP cells (, bottom) largely reflects local deposition of tumor cells along the transplantation tract, although some cells can be seen migrating dorsally. The inset of (bottom) demonstrates that the tumor cells (taken at 10 days post transplant) continue to be strongly GFP positive and indicates that they could be localized even in the absence of black pigmentation. In , a large mass is seen 14 days after IP transplantation of 200,000 BRAF;p53 melanoma. Mediolateral and ventral views of this tumor allow for 3-dimensional calculation of tumor volume (in this case 1496mm3). The ability to calculate 3-D tumor volume and whole body distribution of tumor cells provides an important quantitative analysis of engraftment after small numbers of tumor cells, and offers the potential for monitoring in vivo effects of therapeutically useful molecules.
Figure 4 mitf-NRAS-GFP;p53-/-(4A) driven melanoma cells were transplanted into casper adults into either the peritoneum (IP, n=6) or via intracardiac injection (IC, n=5) at a dose of 200,000 cells. By 5 days post IP transplant of NRAS-GFP cells (4A, top), a large (more ...)
We repeatedly imaged a single recipient fish over a period of 4 weeks using standard stereomicroscopy in order to quantify tumor growth over time. As shown in , this individual fish is fully viable over this period of time, and the tumor volume can be assessed at each time point without sacrificing the animal. Because non-melanized, but fluorescently labeled tumor cells can be visualized in the same manner, this tumor transplant model should be amenable to virtually any transplantable tumor.
Finally, to explore the utility of casper as a tool in understanding metastatic progression, we examined each fish that had been transplanted with BRAF;p53 melanoma cells for evidence of tumor dissemination. Between 5 and 28 days post transplant, 9/24 (37.5%) of all transplant recipients had evidence of distant metastasis, with the peak of initial dissemination occurring between 5 and 10 days. In , two representative fish are shown, with the initial site of implantation demonstrated by the arrow. In the left-hand figure, very widespread dissemination is seen throughout the animal, both as single cells as well as in clusters of cells. In the right-hand figure, a small cluster of individual cells (see inset) have disseminated far away from the implantation site and have become embedded in the dorsal skin. At least one of these cells has the stellate appearance of a migratory melanoma cell. Because there are no pigmented cells between the primary mass and this single dorsal cell, this supports the concept that these cells represent dissemination from the primary transplant. In a similarly transplanted wild-type fish, no visible tumors are seen until 2-3 weeks post transplant, and metastatic cells are not detectable. Although the mechanism of such early and widespread dissemination is unclear at this point, it may represent the accumulation of complex genetic changes in the BRAF;p53 donor cells, since the donor tumors had been present for well over a month. In the future, we plan on studying the factors that determine the capacity for transplanted cells to undergo early dissemination, as well as the mechanism of the “switch” between disseminated micrometastatic disease and bulk macrometastases.