Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis

Probably not a substantial claim: may not pass S3 (load-bearing).

Bioluminescence has met with only limited success in the zebrafish system.

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

• cites Creton et al., 1997 sourceNot checked — no full text of the source
• cites Kaneko and Cahill, 2005 sourceDisputed

  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.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Prior work from the authors' laboratory demonstrated that the zebrafish is a useful model for understanding the genetics of hematopoietic stem/progenitor cells and tumor stem cell biology.

“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”

• cites Burns et al., 2005 sourceSupported
• cites Traver et al., 2004aNot matched

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

• cites Langenau et al., 2007 sourceSupported

Classified as probably not a substantial claim.

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.

“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”

• cites Rawls et al., 2001 sourceSupported

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

In vivo imaging is a critical counterpart to studying molecular processes in cell culture systems.

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

• cites Koo et al., 2006 sourceSupported

May combine two claims.

MRI 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.

“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”

• cites Lee et al., 2007 sourceNot checked — no full text of the source
• cites Lyons, 2005 sourcePartly 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)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Bioluminescence has been utilized to detect engraftment and hematopoietic reconstitution after transplantation of luciferase-positive HSC populations.

“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”

• cites Welsh and Kay, 2005 sourceNot checked — no full text of the source
• cites Cao et al., 2006 sourcePartly 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)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The dose of radiation in both CT and PET that would need to be delivered to the fish to achieve adequate resolution may have independent biological effects.

“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”

• cites Paulus et al., 2000 sourcePartly 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)

  Experimental — accuracy not yet measured
• cites Pickhardt et al., 2005 sourcePartly 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)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The iridophores of the skin are a dermal-hypodermal structure normally intimately connected to surrounding xanthophores and lie just above the trunk skeletal muscle.

“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”

• cites Le Guellec et al., 2004 sourcePartly 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)

  Experimental — accuracy not yet measured

May combine two claims.

There is a complete absence of the scleral iridophore layer in casper, demonstrated by loss of refractive crystalline plates referred to as schemochromes.

“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”

• cites Clothier and Lythgoe, 1987 sourceNot checked — no full text of the source

May combine two claims.

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

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

May combine two claims.

In wild-type animals, the pigmented retinal epithelium is normally obscured by reflective iridophores; their absence in roy mutants exposes the black cells across the entire surface of the eye.

“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.”

May combine two claims.

The authors created a transparent adult zebrafish in which they transplanted either hematopoietic stem/progenitor cells or tumor cells.

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

May combine two claims.

The roy orbison (roy) zebrafish is a spontaneous mutant with a complete lack of iridophores, uniformly pigmented eyes, sparse melanocytes, and translucency of the skin.

“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.”

May combine two claims.

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

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

May combine two claims.

At 4 weeks posttransplant, the wild-type kidney shows a normal distribution of hematopoietic cells, and many of these cells are GFP positive, although no GFP can be visualized with a fluorescent stereoscope.

“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).”

May combine two claims.

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

“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.”

May combine two claims.

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

May combine two claims.

A single recipient fish was fully viable over 4 weeks of repeated imaging, and tumor volume could be assessed at each time point without sacrificing the animal.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At 4 hr posttransplant, a pool of blood surrounds the cardiac chambers, likely representing extravasated blood in the pericardial sac.

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

May combine two claims.

All hematopoietic cells are strongly GFP-positive by IHC, whereas none of the kidney tubule cells stain for GFP.

“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.”

May combine two claims.

Tumor cells at 10 days posttransplant continue to be strongly GFP positive and could be localized even in the absence of black pigmentation.

“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.”

May combine two claims.

Mediolateral and ventral views allow for three-dimensional calculation of tumor volume, in this case 1496 mm3.

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

May combine two claims.

In a representative fish, a small cluster of individual cells disseminated far away from the implantation site and became embedded in the dorsal skin.

“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.”

May combine two claims.

By 4 weeks posttransplant, a significant increase in the size and distribution of the GFP-labeled population can be easily visualized and is largely confined to the anatomical area of the kidney.

“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).”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the casper mutant, distinct phases of systole and diastole can be examined by light microscopy.

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

May combine two claims.

While wild-type fish show a substantial layer of reflective cells, the casper mutant has a complete absence of this layer, while remaining skin structures are relatively intact, including epidermal scales and dermal collagen fibers.

“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).”

May combine two claims.

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.

“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.”

Classified as probably not a substantial claim.

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.

“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.”

May combine two claims.

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.

“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.”

May combine two claims.

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.

“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.”

May combine two claims.

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.

“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.”

May combine two claims.

The transplantable hematopoietic stem/progenitor cell marrow population resides within the adult kidney and can be isolated using flow cytometric analysis of forward and side scatter.

“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”

• cites Traver et al., 2003 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S2 (checkable).

The casper fish will serve as a useful platform for researchers interested in processes such as stem cell engraftment and homing.

“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.”

May combine two claims.

Based on the scatter profile, multilineage engraftment has occurred, indicating that the engrafted cells represent a primitive hematopoietic population.

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

Probably not a substantial claim: may not pass S3 (load-bearing).

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

“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.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based). May combine two claims.

The zebrafish has become a more commonly utilized tool for studying stem cell and tumor biology and should be broadly applicable to stem cell researchers.

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

• cites Wakamatsu et al., 2001 sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Imaging of very deep tissues may require multiphoton microscopy, which is currently experimental in the zebrafish system.

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

Probably not a substantial claim: may not pass S2 (checkable).

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.

“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.”

• AssumptionRests on an assumption: The adult zebrafish is well suited to genetic and small molecule based screens.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Because the mutation in roy is currently unknown, 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 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.”

May combine two claims.

PET imaging can detect relatively small numbers of tumor cells, requires radioactive labeling of cell populations, is only moderately sensitive, and necessitates small animal PET scanners that are not broadly available.

“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”

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

May combine two claims.

Bioluminescence imaging has limited spatial resolution (on the order of 1-10 mm), requires several minutes of imaging per animal, has limited detection of light from deep tissues, and requires intravenous administration of substrate.

“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.”