Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis

In the paper

“In addition, bioluminescence has met with only limited success in the zebrafish system (Creton et al., 1997; Kaneko and Cahill, 2005”

What the sources say

• ?Not checked — no full text of the sourcecites Creton et al., 1997 source
• Disputed

  The Discussion (page unavailable) describes the authors’ bioluminescent transgenic zebrafish as a high-throughput system for studying circadian gene expression in whole animals, contrary to the broad claim of only limited success. It notes a narrower limitation: embryos were unsuitable for circadian studies with these lines, whereas older larvae produced robust rhythms. (LLM, conf=0.82)

  Checked by a reasoning model — may be wrong.

  cites Kaneko and Cahill, 2005 sourceExperimental — accuracy not yet measured

In the paper

“Prior work from our laboratory has demonstrated that the zebrafish is a useful model for understanding the genetics of both HSPC (Burns et al., 2005; Traver et al., 2004a”

What the sources say

• Supported

  The citation is attached to the HSPC portion of the citing sentence. In the section on Mind bomb and HSC specification, the paper uses zebrafish mutants to investigate genes controlling hematopoietic stem and progenitor cells, supporting zebrafish as a model for that purpose. The supplied sentence does not attach this citation to tumor stem cell biology. (LLM, conf=0.91)

  cites Burns et al., 2005 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Traver et al., 2004a
• Supported

  The section ‘Identifying the cancer stem cell in zERMS’ reports that zebrafish experiments isolated a cancer stem cell and helped identify pathways relevant to its self-renewal. This supports the tumor-stem-cell part attributed to this paper; the printed sentence cites Burns and Traver for HSPCs. (LLM, conf=0.91)

  cites Langenau et al., 2007 source

In the paper

“The characteristic adult pigmentation pattern of the zebrafish consists of three distinct classes of pigment cells arranged in stripes (Figure 1A): black melanophores, reflective iridophores, and yellow xanthophores”

What the sources say

• Supported

  The “Zebrafish Melanocyte Development” section names yellow xanthophores, reflective iridophores, and black melanocytes, noting that melanocytes are also called melanophores. “Simple Models of Organogenesis” discusses all three cell types in relation to adult body stripes. (LLM, conf=0.91)

  cites Rawls et al., 2001 source

In the paper

“In vivo imaging is a critical counterpart to studying molecular processes in cell culture systems (Koo et al., 2006”

What the sources say

• Supported

  The abstract (p. 1) calls in vivo molecular imaging an “essential bridge between in vitro data and their translation into clinical applications.” The Introduction (p. 1) also describes its use in studying molecular and cellular mechanisms in animal models. Together, these support the claim’s characterization of in vivo imaging as a critical counterpart to in vitro study. (LLM, conf=0.89)

  cites Koo et al., 2006 source

In the paper

“MRI is a useful technique for visualizing transplanted cells in vivo and has a spatial resolution of 25–50 μm but requires a significant amount of magnetic dye treatment of transplanted cells, has limited sensitivity, and requires considerably complex and expensive equipment (Lee et al., 2007; Lyons, 2005”

What the sources say

• ?Not checked — no full text of the sourcecites Lee et al., 2007 source
• Partly supported

  Table 1 (Conclusions; page not provided) supports MRI's high spatial resolution and low sensitivity, but gives a resolution of 25–100 μm, not 25–50 μm. The MRI imaging section describes contrast probes, not a requirement for substantial magnetic-dye treatment of transplanted cells. The paper notes that equipment costs vary between modalities but does not specifically describe MRI equipment as complex and expensive. (LLM, conf=0.90)

  cites Lyons, 2005 sourceExperimental — accuracy not yet measured

In the paper

“Bioluminescence has been utilized to detect engraftment and hematopoietic reconstitution after transplantation of luciferase-positive HSC populations (Welsh and Kay, 2005; Cao et al., 2006”

What the sources say

• ?Not checked — no full text of the sourcecites Welsh and Kay, 2005 source
• Partly supported

  The Results (page not provided) report using bioluminescence to assess engraftment after transplantation of luciferase-expressing embryonic stem cells into rat hearts. The source does not report transplantation of hematopoietic stem cell (HSC) populations or detection of hematopoietic reconstitution. (LLM, conf=0.88)

  cites Cao et al., 2006 sourceExperimental — accuracy not yet measured

In the paper

“Another concern is the dose of radiation in both CT and PET that would need to be delivered to the fish to achieve adequate resolution, which may have independent biological effects (Paulus et al., 2000; Pickhardt et al., 2005”

What the sources say

• Partly supported

  The Radiation Dose section calls radiation exposure an important concern in microCT studies and compares the estimated dose in mice with published lethal-dose values; Table 1 reports organ doses for high-resolution and screening scans. This supports a concern about CT radiation, but the paper does not establish the dose needed for adequate resolution in fish or address biological effects from PET imaging. (LLM, conf=0.82)

  cites Paulus et al., 2000 sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion says radiation from microCT scanning could potentially affect tumor growth and interfere with serial monitoring in mice. Materials and Methods says the authors chose parameters that provided adequate resolution while minimizing radiation dose. The paper does not establish this concern for PET or fish. (LLM, conf=0.88)

  cites Pickhardt et al., 2005 sourceExperimental — accuracy not yet measured

In the paper

“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”

What the sources say

• Partly supported

  In “An overview of the current knowledge of fish skin” (page unavailable), the paper places iridophores and xanthophores in the hypodermis, between the dermis and underlying muscle. It does not say that iridophores are normally intimately connected to xanthophores, or specifically describe iridophores as a dermal-hypodermal structure. (LLM, conf=0.84)

  cites Le Guellec et al., 2004 sourceExperimental — accuracy not yet measured

In the paper

“However, there is a complete absence of the scleral iridophore layer (Figure 2A, arrow), which is demonstrated by the loss of refractive crystalline plates (Clothier and Lythgoe, 1987”

What the sources say

• ?Not checked — no full text of the sourcecites Clothier and Lythgoe, 1987 source

In the paper

“The gross morphology of the casper eye is normal, with maintenance of the scleral-corneal border and normal pigmented retinal epithelial cells.”

In the paper

“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.”

In the paper

“To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells.”

In the paper

“The roy orbison (roy) zebrafish (Figure 1C) is a spontaneous mutant and has a complete lack of iridophores, uniformly pigmented eyes, sparse melanocytes, and a translucency of the skin.”

In the paper

“The casper mutant is entirely viable, with incrossed adults producing large numbers of viable offspring at expected mendelian ratios and no heterozygous phenotype.”

In the paper

“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 posttransplant, and many of these cells are GFP positive (Figure 3B, top).”

In the paper

“The pattern of engraftment at 5 days in the intracardiac recipients of NRAS-GFP cells (Figure 4A, bottom) largely reflects local deposition of tumor cells along the transplantation tract, although some cells can be seen migrating dorsally.”

In the paper

“In a similarly transplanted wild-type fish, no visible tumors are seen until 2 to 3 weeks posttransplant, and metastatic cells are not detectable.”

In the paper

“As shown in Figure 4C, 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.”

In the paper

“At 4 hr posttransplant, a pool of blood surrounds the cardiac chambers, which likely represents extravasated blood in the pericardial sac.”

In the paper

“All of the hematopoietic cells are strongly GFP-positive by IHC (Figure 3C, right, brown staining), whereas none of the kidney tubule cells stain for GFP.”

In the paper

“The inset of Figure 4A (bottom) demonstrates that the tumor cells (taken at 10 days posttransplant) continue to be strongly GFP positive and indicates that they could be localized even in the absence of black pigmentation.”

In the paper

“Mediolateral and ventral views of this tumor allow for three-dimensional calculation of tumor volume (in this case 1496 mm3).”

In the paper

“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.”

In the paper

“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 (Figure 3B, right).”

In the paper

“Real-time microscopy of cardiac contraction in the casper mutant. Distinct phases of systole and diastole can be examined by light microscopy.”

In the paper

“While wild-type fish (Figure 2B, 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).”

In the paper

“Between 5 and 28 days posttransplant, 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 the paper

“The development of a robust, fertile, and easily maintained transparent adult zebrafish provides an important adult complement to the studies that are achievable in the transparent zebrafish embryo.”

In the paper

“The ability to calculate 3D 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.”

In the paper

“Because nonmelanized but fluorescently labeled tumor cells can be visualized in the same manner, this tumor transplant model should be amenable to virtually any transplantable tumor.”

In the paper

“These data suggest that the transparent casper mutant allows for ready visualization of hematopoietic progenitor engraftment after a sublethal dose of 25 Gy, consistent with short-term hematopoietic engraftment.”

In the paper

“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”

What the sources say

• ?Not checked — no full text of the sourcecites Traver et al., 2003 source

In the paper

“Our data suggests that the casper fish will serve as a useful platform for researchers interested in processes such as stem cell engraftment and homing.”

In the paper

“Based on the scatter profile, multilineage engraftment has occurred, which indicates that the engrafted cells represent a primitive hematopoietic population.”

In the paper

“One limitation of this line is that it requires that both mutations (nacre and roy) be present in the homozygous state, which will require at least two breeding cycles to take advantage of currently available transgenic lines.”

In the paper

“Although similar to the “see through” medaka strain (Wakamatsu et al., 2001”

What the sources say

• ?Not checked — no full text of the sourcecites Wakamatsu et al., 2001 source

In the paper

“Finally, imaging of very deep tissues may require multiphoton microscopy, which is currently experimental in the zebrafish system.”

In the paper

“Because the adult zebrafish is well suited to genetic and small molecule based screens, the casper fish will allow for detailed in vivo study of stem and cancer biology in a vertebrate organism with great relevance to human biology.”

In the paper

“In addition, because the mutation in roy is currently unknown (but under active investigation), it is possible that this gene defect may affect some unknown aspects of tumor or stem cell biology, although it clearly does not affect survival.”

In the paper

“PET imaging, which can detect relatively small numbers of tumor cells, requires radioactive labeling of cell populations, is only moderately sensitive, and necessitates small animal PET scanners, which are not broadly available (Beckmann et al., 2007; Tai et al., 2005”

What the sources say

• ?Not checked — no full text of the sourcecites Beckmann et al., 2007 source
• ?Not checked — no full text of the sourcecites Tai et al., 2005

In the paper

“Although bioluminescence had high sensitivity in this study, it had limited spatial resolution (on the order of 1–10 mm), required several minutes of imaging per animal, showed detection of light from deep tissues is limited, and requires intravenous administration of substrate.”