A crystal-clear zebrafish for in vivo imaging

Classified as probably not a substantial claim. May combine two claims.

Common strategies to reduce zebrafish skin pigmentation can be grouped into two categories: chemical compounds interfering with melanin synthesis, most popularly PTU, and pigmentation mutants.

“Common strategies to reduce zebrafish skin pigmentation can be grouped in two categories: 1) use of chemical compounds interfering with the synthesis of melanin, the most popular one being 1-phenyl-2-thiourea (PTU)8; 2) use of pigmentation mutants in which genes controlling either pigment formation, chromatophore formation or interactions between chromatophores have been inactivated469.”

• cites [8] Karlsson, J., von Hofsten, J. & Olsson, P. E. Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar Biotechnol (NY) 3, 522–527 (2001). sourceNot checked — no full text of the source
• cites [4] White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008). sourceSupported
• cites [6] Kelsh, R. N. et al. Zebrafish pigmentation mutations and the processes of neural crest development. Development 123, 369–389 (1996). sourceNot checked — no full text of the source
• cites [9] Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. & Raible, D. W. Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999). sourceNot checked — no full text of the source

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

Several tissue clearing techniques have been developed to reduce light scattering in fixed biological tissues by selectively removing lipids in a non-destructive way.

“Recently, several tissue clearing techniques have been developed to reduce light scattering in fixed biological tissues by selectively removing lipids in a non-destructive way3.”

• cites [3] Richardson, D. S. & Lichtman, J. W. Clarifying Tissue Clearing. Cell 162, 246–257 (2015). sourcePartly supported

  The Modern Clearing Techniques and Aqueous based clearing sections say that clearing reduces light scattering and that several approaches remove lipids. But the aqueous-based section also describes an approach that does not remove lipids, while the Solvent based clearing and Hydrogel embedding sections report shrinkage and risks to tissue proteins. The paper therefore does not support describing tissue-clearing techniques generally as selectively removing lipids in a non-destructive way. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

In addition to neural-crest-derived chromatophores, another population of pigment cells forms the retinal pigment epithelium and originates from optic lobe neuroepithelial cells.

“In addition to these three populations, which in zebrafish larvae derive from neural crest cells, there is another population of pigment cells forming the retinal pigment epithelium (RPE), which originates from optic lobe neuroepithelial cells (Fig. 1a)7.”

• cites [7] Bharti, K., Nguyen, M. T., Skuntz, S., Bertuzzi, S. & Arnheiter, H. The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye. Pigment Cell Res 19, 380–394 (2006). sourceSupported

May combine two claims.

The crystal mutant constitutes a significant improvement in the optical accessibility of both larval and adult zebrafish, even superior to casper.

“Overall, the crystal mutant constitutes a significant improvement in the optical accessibility of both larval and adult zebrafish, even superior to the previously described combinatorial mutant casper4, which is characterised by black-pigmented eyes.”

• cites [4] White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008). sourceSupported

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

The casper mutant was obtained by crossing nacrew2/w2 and roya9/a9 mutants.

“The casper mutant was obtained by crossing nacrew2/w2 and roya9/a9 mutants, as previously described4.”

• cites [4] White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008). sourcePartly supported

  The Results identify casper as a double mutant for nacre and roy, and the Discussion says both mutations must be homozygous. Our copy does not specify the w2 and a9 alleles or explicitly describe the cross used to produce casper. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

A previous study reported that adult zebrafish mutants with hypopigmented eyes show deficits in performing visual escape assays under defined luminance conditions.

“A previous study has reported that adult zebrafish mutants with hypopigmented eyes show deficits in performing visual escape assays under defined luminance conditions34.”

• cites [34] Ren, J. Q., McCarthy, W. R., Zhang, H., Adolph, A. R. & Li, L. Behavioral visual responses of wild-type and hypopigmented zebrafish. Vision Res 42, 293–299 (2002). sourceSupported

Classified as probably not a substantial claim.

The combinatorial crystal mutant (albb4/b4;nacrew2/w2;roya9/a9) was generated by sequentially crossbreeding three single mutant strains: nacrew2/w2, roya9/a9 and albb4/b4.

“The combinatorial crystal mutant (albb4/b4;nacrew2/w2;roya9/a9) was generated by sequentially crossbreeding three different single mutant strains, namely nacrew2/w2 (ref. 9), roya9/a9 (ref. 4) and albb4/b4 (ref. 18) mutants.”

• cites [9] Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. & Raible, D. W. Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999). sourceNot checked — no full text of the source
• cites [4] White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008). sourcePartly supported

  The Results (page unavailable) describe a transparent double mutant combining nacre and roy, and the Generation of Compound Mutant Lines section mentions an albino line. Neither passage describes a crystal triple mutant with the stated alleles or says it was generated by sequentially crossbreeding all three strains. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [18] Streisinger, G., Singer, F., Walker, C., Knauber, D. & Dower, N. Segregation analyses and gene-centromere distances in zebrafish. Genetics 112, 311–319 (1986). sourcePartly supported

  The Results describe alb-1 as a recessive zebrafish pigment-pattern mutant, supporting the citation’s identification of an alb mutant strain. Our copy does not establish the specific b4/b4 allele or describe the sequential crosses used to generate the combinatorial crystal mutant. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: The formation of each pigment type is controlled independently of the others.

May combine two claims.

The crystal mutant lacks all melanophores and iridophores and also has a non-pigmented retinal pigment epithelium.

“Here, we further develop this strategy to generate a fully optically clear combinatorial mutant (crystal) that not only lacks all melanophores and iridophores, but also has a non-pigmented RPE.”

May combine two claims.

Crystal larvae exhibited an optomotor response rate equivalent to wild type larvae, whereas PTU-treated larvae showed a dramatic behavioural impairment.

“Notably, crystal larvae exhibited a response rate equivalent to wild type larvae as opposed to PTU-treated larvae, which instead showed a dramatic behavioural impairment (Fig. 2b).”

May combine two claims.

Crystal allows fully unobstructed optical access to the larval zebrafish brain in its entirety, including the eyes, whereas nacre and casper mutants have a substantial portion of the nervous system (~18%) inaccessible through standard light-sheet imaging.

“Therefore, crystal allows a fully unobstructed optical access of the larval zebrafish brain in its entirety (i.e., including the eyes), as opposed to nacre and casper mutants where a substantial portion of the nervous system (~18%) is not accessible through standard light-sheet imaging”

May combine two claims.

The mean accessible volume in light-sheet imaging was crystal 3.04 ± 0.12 × 10^7 μm3 (n = 4), casper 2.51 ± 0.07 × 10^7 μm3 (n = 4), and nacre 2.47 ± 0.10 × 10^7 μm3 (n = 5), with p = 0.0040, F2,10 = 10.09, one-way ANOVA with post-hoc Tukey's HSD test.

“mean volume ± SEM, crystal 3.04 ± 0.12 × 107 μm3, n = 4; casper 2.51 ± 0.07 × 107 μm3, n = 4; nacre 2.47 ± 0.10 × 107 μm3, n = 5; p = 0.0040, F2,10 = 10.09, one-way ANOVA with post-hoc Tukey’s HSD test”

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

No qualitative differences between light and dark bars were noted, so data obtained using the two stimuli were combined.

“As no qualitative differences between light and dark bars were noted, data obtained using the two stimuli were combined.”

May combine two claims.

The spatial definition of brain regions between the eyes in crystal larvae appears lower than in other brain regions, likely due to scattering of excitation light by the lenses.

“Even though the optical accessibility of crystal larvae through light-sheet imaging is significantly higher than nacre and casper mutants, it is noteworthy that the spatial definition of brain regions between the eyes appears lower than other regions of the brain, likely due to scattering of excitation light by the lenses.”

May combine two claims.

Compared to untreated larvae, 4 dpf PTU-treated larvae showed a large decrease in visual responses, with direction- and orientation-selective neural responses being absent or severely impaired.

“Compared to untreated Tg(elavl3:GCaMP5G) larvae, 4 dpf PTU-treated larvae (treatment from 1 dpf to 3 dpf) showed a large decrease in visual responses with direction- and orientation-selective neural responses being absent or severely impaired, respectively (Fig. 2c).”

May combine two claims.

The optical clarity of crystal larvae is superior to that of larvae treated with 200 μM PTU, because PTU inhibits melanin production but does not interfere with iridophore function.

“Importantly, the optical clarity of crystal larvae is even superior to that of larvae treated with 200 μM PTU (Fig. 1c,d), since PTU inhibits melanin production but does not interfere with iridophore function8.”

• cites [8] Karlsson, J., von Hofsten, J. & Olsson, P. E. Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development. Mar Biotechnol (NY) 3, 522–527 (2001). sourceNot checked — no full text of the source

May combine two claims.

The eyes of crystal fish are considerably less pigmented than wild type, single mutant and casper fish, making them easily accessible to optical investigation.

“Consequently, the eyes of crystal fish are considerably less pigmented than wild type, single mutant and casper fish, therefore resulting easily accessible to optical investigation (see right insets in Fig. 1b–d).”

May combine two claims.

Direction-selective (DSI > 0.5) and orientation-selective (OSI > 0.5) responses were observed in the retina of crystal larvae, indicating that the retina is functional in crystal larvae.

“Interestingly, not only we could record stimulus-locked responses, but also we observed the presence of direction-selective (direction selectivity index (DSI) > 0.5, ROI# 4 and 5) and orientation-selective (orientation selectivity index (OSI) > 0.5, ROI# 1–3) responses (Fig. 4c,d), indicating that the retina is functional in crystal larvae.”

May combine two claims.

The combinatorial triple mutant nacrew2/w2;albb4/b4;roya9/a9, named crystal, lacks the vast majority of dark and reflective pigments normally present at the cutaneous level and appears optically clear.

“The resulting combinatorial triple mutant (nacrew2/w2;albb4/b4;roya9/a9; red boxes in Fig. 1b–d), which we name crystal, lacks the vast majority of dark and reflective pigments normally present at the cutaneous level and, as a consequence, appears optically clear.”

• AssumptionRests on an assumption: The formation of each pigment type is controlled independently of the others.

May combine two claims.

Both larvae and adult crystal zebrafish are viable and do not display visible morphological, functional or behavioural abnormalities other than the pigmentation phenotype.

“Importantly, both larvae and adult crystal zebrafish are viable and do not display visible morphological, functional or behavioural abnormalities other than the pigmentation phenotype.”

May combine two claims.

Unlike PTU-treated animals, adult crystal mutants are viable and produce normal numbers of offspring.

“Moreover, unlike PTU-treated animals, adult crystal mutants are viable and produce normal numbers of offspring.”

May combine two claims.

The nacre mutation does not affect the population of cells forming the retinal pigment epithelium, so this structure is still pigmented in the nacre single mutant.

“Since the nacre mutation does not affect the population of cells forming the RPE, this structure is still pigmented in this single mutant.”

• cites [9] Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. & Raible, D. W. Nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999). sourceNot checked — no full text of the source

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

A 10-second inter-epoch interval enables GCaMP5G signals to return to baseline.

“Each inter-epoch interval was 10 seconds to enable GCaMP5G signals to return to baseline.”

Probably not a substantial claim: may not pass S4 (based).

Waiting 60 seconds after activating laser scanning before visual stimulation ensures the retina adapted to the background light level caused by the multi-photon laser.

“Following activation of the laser scanning, we waited 60 seconds before starting the visual stimulation to ensure the retina adapted to the background light level caused by the multi-photon laser.”

• Also stated in: § Visual stimulation · plain

  “Following activation of the laser scanning, we waited 60 seconds before starting the visual stimulation to ensure the retina adapted to the background light level caused by the multi-photon laser.”

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

This work was approved by the local Animal Welfare and Ethical committee.

“This work was approved by the local Animal Welfare and Ethical”

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

Although crystal larvae perform the optomotor response as well as wild type larvae, it cannot be excluded that crystal mutants might show abnormal retinal responses under certain luminance conditions.

“Thus, even though our visual behaviour assays demonstrated that crystal larvae perform the optomotor response as well as wild type larvae (Fig. 2a,b), we cannot exclude that crystal mutants might show abnormal retinal responses under certain luminance conditions.”

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

A 10-second inter-epoch interval enables GCaMP6f signals to return to baseline.

“Each of the 12 directions of motion was presented once (6 seconds) with and inter-epoch interval of 10 seconds to enable GCaMP6f signals to return to baseline.”

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

The integral within each epoch window is a summary metric more resistant to saturation effects of the calcium probe than maximum signal change.

“The integral within each epoch window is a summary metric more resistant to saturation effects of the calcium probe than maximum signal change.”

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

Before performing statistical tests, descriptive statistics (e.g., normality tests, F-test) were used to choose the appropriate statistical test.

“Before performing statistical tests, descriptive statistics (e.g., normality tests to see whether values come from a Gaussian distribution or F-test to compare variances) were used to choose the appropriate statistical test (reported in Figure legends together with test results).”

Classified as probably not a substantial claim.

To minimize cross talk and over-fitting associated with DSI and OSI metrics, a stringent approach was undertaken.

“To minimize cross talk and over-fitting associated with DSI and OSI metrics, a stringent approach was undertaken.”

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

The agarose was sufficient to restrain the larvae so that anaesthesia was not required.

“The agarose was sufficient to restrain the larvae so that anaesthesia was not required.”

May combine two claims.

PTU can cause deleterious consequences on both zebrafish behaviour and neural function, and should be avoided whenever these two biological processes are under investigation.

“From these data, we conclude that PTU can cause deleterious consequences on both zebrafish behaviour and neural function, and should be therefore avoided whenever these two biological processes are under investigation.”

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

The study was approved by the local Animal Welfare and Ethical Review Body (King's College London) and carried out in accordance with the Animals (Scientific Procedures) Act 1986 under UK Home Office license PPL70/8057.

“This work was approved by the local Animal Welfare and Ethical Review Body (King’s College London), and was carried out in accordance with the Animals (Scientific Procedures) Act 1986, under license from the United Kingdom Home Office (PPL70/8057).”