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(A) Schematic of the inverted shhact allele and primers sites. (B) The inverted allele was not detected in untreated tcf21:CreER; shhact/ct embryos using 40 cycles of PCR. (C) The inverted allele was not detected in untreated adult tcf21:CreER; shhact/ct fish using 30 cycles of PCR, but it was detected when the cycle number was increased to 35. Each PCR sample used genomic DNA isolated from five pooled hearts as a template.
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| 99.94 |
10.7554/eLife.24635.017Figure 5—figure supplement 2.A redundant role for epicardial shha in epicardial migration and proliferation during heart regeneration.(A) In situ hybridization analysis of raldh2 expression in sections obtained from 4-HT-treated 7 dpi shhact/ct (control) and tcf21:CreER; shhact/ct hearts. Brackets, injury site. (B) Immunofluorescence staining of Raldh2 and DsRed2 using sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:DsRed; tcf21:CreER; shhact/ct hearts (right). Single-channel images of the rectangle are shown at the bottom. Dotted line, approximate amputation plane. (C) Immunofluorescence staining of DsRed2 and EdU in sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts. Inset, non-injured area. Arrows indicate proliferating tcf21+ epicardial cells, which were defined as epicardial cells colabeled DsRed2 and EdU. (D) Quantification of epicardial cell proliferation in the sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts shown in C (n = 5 each). The data represent the mean ± SEM (wound, p=0.754; remote, p=0.602; Mann–Whitney U test). N.S., not significant. Single confocal slice images are shown in B and C. Scale bar, 50 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.017
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| 100.0 |
(A) In situ hybridization analysis of raldh2 expression in sections obtained from 4-HT-treated 7 dpi shhact/ct (control) and tcf21:CreER; shhact/ct hearts. Brackets, injury site. (B) Immunofluorescence staining of Raldh2 and DsRed2 using sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:DsRed; tcf21:CreER; shhact/ct hearts (right). Single-channel images of the rectangle are shown at the bottom. Dotted line, approximate amputation plane. (C) Immunofluorescence staining of DsRed2 and EdU in sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts. Inset, non-injured area. Arrows indicate proliferating tcf21+ epicardial cells, which were defined as epicardial cells colabeled DsRed2 and EdU. (D) Quantification of epicardial cell proliferation in the sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts shown in C (n = 5 each). The data represent the mean ± SEM (wound, p=0.754; remote, p=0.602; Mann–Whitney U test). N.S., not significant. Single confocal slice images are shown in B and C. Scale bar, 50 μm.
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| 100.0 |
In contrast to epicardial cell proliferation, myocardial cell proliferation, defined as cardiomyocytes colabeled with immunofluorescence using anti-Mef2 and anti-proliferating cell nuclear antigen (PCNA) antibodies, was significantly decreased in the subepicardial area of injured epi-KO hearts (Figure 5D and E). However, myocardial cell proliferation was not reduced in the trabecular myocardium, an area distant from the epicardium (Figure 5D and E). To investigate the interplay between the epicardium and subepicardial myocardium, we crossed tcf21:DsRed2 fish with Tg(gata4:EGFP) (gata4:EGFP) fish (Heicklen-Klein and Evans, 2004), as gata4:EGFP labels the subepicardial myocardium during cardiac growth (Gupta et al., 2013) and regeneration (Kikuchi et al., 2010). Immunofluorescence analysis of injured tcf21:DsRed2; gata4:EGFP hearts revealed that epicardial cells and subepicardial cardiomyocytes directly interact (Figure 5F), and semi-qRT-PCR analysis revealed that the expression of Hh target genes was strongly induced in gata4:EGFP+ subepicardial cardiomyocytes but not in control cardiomyocytes purified from uninjured Tg(cmlc2:EGFP) (cmlc2:EGFP) hearts (Figure 5G). These findings support the role of epicardial Shha in transmitting direct, short-range signals that promote the proliferation of adjacent cardiomyocytes during zebrafish heart regeneration.
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| 100.0 |
In this study, we developed a streamlined method to efficiently generate conditional alleles in zebrafish using the invertible gene trap cassette Zwitch. We demonstrated that Zwitch can be inserted into a defined locus of the zebrafish genome via precise in vivo genome editing for inducible Cre-mediated gene disruption. In theory, the approach established in this study can be used to generate conditional alleles of any zebrafish gene, and it will expand the utility of zebrafish as a model organism.
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| 100.0 |
Recently, TALEN-mediated HR was used to generate a floxed kcnh6a allele in zebrafish (Hoshijima et al., 2016). The loxP sites were provided by a donor plasmid that expressed the α-crystallin:Venus reporter gene, which was used to identify founders carrying the modified alleles with an overall germline transmission efficiency of 12% (Hoshijima et al., 2016). Using Zwitch in this study, we achieved a germline transmission efficiency of 59% and confirmed that 89% of alleles were in the correct non-mutagenic orientation (Figure 1E). The efficiency of the Zwitch method was likely enhanced by screening for the LG marker at two different time points (7 and 45 dpf), as this excluded false positives resulting from unintegrated targeting vectors at 7 dpf (Figure 1E). Moreover, the ability of Zwitch to be inserted at any location in the target intron allowed us to screen for the TALEN pair associated with the greatest HR efficiency (Figure 1B and C). This feature may facilitate the efficient generation of conditional alleles in other genes, as a high DSB rate increases the rate of HR at target sites, thereby increasing the likelihood of successfully generating conditional alleles via genome editing (Hoshijima et al., 2016).
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study
| 100.0 |
After pioneering studies demonstrated the editing of zebrafish genome with long dsDNA (Bedell et al., 2012; Zu et al., 2013), several studies reported the insertion of large DNA fragments at defined zebrafish gene loci via DSB-mediated HR (Hisano et al., 2015; Hoshijima et al., 2016; Shin et al., 2014; Zu et al., 2013) or homology-independent repair mechanisms (Auer et al., 2014; Kimura et al., 2014; Li et al., 2015). The efficiency of inserting the Zwitch construct may be increased via combination with these reported approaches. For example, a recent report suggested that asymmetric homology arm size, that is, 1 kb for one arm and 2 kb for the other arm, and the presence of a DSB in the shorter homology arm ensure efficient HR, thereby increasing the germline transmission rate of mutations (Shin et al., 2014). Designing homology arms following this approach may increase the targeting efficiency of the Zwitch construct. Recent studies using the CRISPR/Cas9 system reported that concurrent cleavage of the donor vector and target genome site induces efficient integration of a DNA fragment into a defined locus via homology-independent repair mechanisms (Auer et al., 2014; Kimura et al., 2014). Although we could not efficiently induce DSBs in the shha intronic sequence using the CRISPR/Cas9 system, this approach may be useful for targeting Zwitch into other genes.
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| 99.94 |
We attempted to visualize mutant cells by connecting the TagRFP reporter gene to the splice acceptor site via a bicistronic 2A peptide sequence in Zwitch (Figure 1A and Figure 1—figure supplement 1A and B). We detected TagRFP expression in cells in the ganglion cell layer (GCL) in the retina (arrowheads, Figure 2—figure supplement 3), in which robust shha expression was previously reported (Shkumatava et al., 2004). However, the expression level was variable between GCL cells, compared with the uniform expression of endogenous shha mRNA in this tissue (Figure 2—figure supplement 3). Moreover, TagRFP expression was inconsistent in other tissues known to produce Shha at high levels, such as the floor plate and notochord. Given the transcription of shha-2A-TagRFP mRNA (Figure 2C and Figure 2—figure supplement 2B), the variability in the expression of TagRFP might be attributed, at least in part, to inefficient cleavage of the 2A peptide in the target cells. It is of interest to examine whether TagRFP expression would be enhanced in the target tissues by modifying the 2A sequence of Zwitch to include a glycine-serine-glycine spacer (GSG), which was recently found to significantly improve the cleavage efficiency of the 2A peptide (Wang et al., 2015b). We did not detect TagRFP expression in conditional shha mutant epicardial cells during development and regeneration (data not shown). We suspect that the failure to visualize shha-deficient epicardial cells expressing TagRFP was due to the low expression levels of endogenous shha in the epicardium during heart development and regeneration. Further studies are needed to improve the visualization of mutant cells for conducting mosaic studies using Zwitch.
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study
| 100.0 |
Previously inaccessible aspects of organ morphogenesis can be addressed by combining conditional genetic analysis with established embryological, pharmacological, and transgenic manipulations in the zebrafish system. Moreover, the cKO approach will enhance the utility of zebrafish in elucidating the mechanisms underlying biological phenomena in adults that cannot be adequately investigated using a global KO approach. As an example, we used the shha gene trap line with an epicardium-specific inducible Cre driver and provided the first direct evidence that shha expression in the epicardium is required for the synthesis of RA and cardiomyocyte mitogens in the epicardium during heart development (Figure 4G and H). We further provided evidence that Shha may transmit a short-range signal to induce subepicardial muscle cell proliferation in the regenerating heart (Figure 5D and E), a mechanism that was not identified in studies of cyclopamine-mediated global Hh signaling inhibition (Choi et al., 2013). In situ hybridization analysis of embryo and adult hearts detected the expression of shha in tcf21:DsRed-negative cells (Figure 4—figure supplement 1A–F), suggesting that non-epicardial cells also produce Shha. Moreover, semi-qRT-PCR analysis detected the expression of dhh and ihhb in epicardial cells purified from epi-KO hearts (Figure 5C). Investigations of shha function using broader Cre driver lines and conditional inactivation of other Hh ligand genes will be needed in the future to definitively clarify the function of Shha during heart development and regeneration. However, the results described in this study demonstrate that conditional genetic analysis is a feasible, effective approach to elucidate developmental and regenerative mechanisms in zebrafish, and it might also be useful for deciphering other complex phenomena such as immune responses, metabolism, and behavior.
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study
| 99.94 |
In conclusion, we established a simple and efficient genome editing approach to engineer conditional alleles in zebrafish via HR-mediated Zwitch insertion. The tools and methods described in this study can be used to generate conditional alleles of other zebrafish genes, and they may be generally applicable to any experimental system in which CRISPR/TALEN gene editing is available.
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study
| 99.94 |
The zebrafish used in this study were outcrossed from the Ekkwill (EK) background. All transgenic strains were analyzed as hemizygotes. The following published transgenic strains were used: Tg(cmlc2:DsRed2)pd15 (RRID:ZFIN_ZDB-ALT-110210-15) (Kikuchi et al., 2010), Tg(gata5:EGFP)pd25 (RRID:ZFIN_ZDB-ALT-110408-5) (Kikuchi et al., 2011), Tg(tcf21:CreER)pd42 (RRID:ZFIN_ZDB-ALT-110818-7) (Kikuchi et al., 2011), Tg(gata4:EGFP)ae1 (RRID:ZFIN_ZDB-ALT-051123-4) (Heicklen-Klein and Evans, 2004), and Tg(cmlc2:EGFP)f1 (RRID:ZFIN_ZDB-GENO-080403-2) (Burns et al., 2005). Tg(ubb:iCRE-GFP)vcc9 was generated by co-injecting pUbb-iCRE-GFP (Figure 2—figure supplement 1A) with I-SceI into single cell-stage–embryos. Zebrafish were used for regeneration experiments at 4–12 months of age. The heart injury experiments were conducted as previously described (Poss et al., 2002). The animals were maintained at a density of 3–5 fish per liter, and clutch-mates of the appropriate genotypes were used as controls. The zebrafish husbandry procedures and all experiments were conducted in accordance with institutional and national animal ethics guidelines.
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study
| 99.94 |
The TALENs used to facilitate the insertion of Zwitch were designed using TAL Effector Nucleotide Targeter 2.0 software (Doyle et al., 2012) and constructed using the Golden Gate assembly method (Cermak et al., 2011; Sakuma et al., 2013). The TALENs were cloned into pCS2TAL3DDD and pCS2TAL3RRR vectors (provided by Dr. David Grunwald, University of Utah) (Dahlem et al., 2012). TALEN mRNAs were synthesized from linearized vectors using the mMESSAGE mMACHINE SP6 Transcription Kit (Thermo Fisher Scientific, Waltham, MA), and they were co-injected with pZwitch-shha-int1 into one-cell-stage embryos. The modified alleles were characterized using genomic PCR. The sequences of the PCR primers used to characterize the alleles are listed in Supplementary file 1. The offspring of a single founder were propagated and used in all subsequent experiments. The established conditional gene trap line Tg(shha:Zwitch)vcc8Gt is referred to as shhact. A DIG DNA labeling and detection kit (Roche) was used for Southern blot analysis of genomic DNA isolated from adult shhact/+ fish and WT clutch-mates. The detection probe was generated using PCR with WT genomic DNA and the following primers:
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study
| 100.0 |
Embryos and adult shhact fish were genotyped using genomic PCR. The primers are listed in Supplementary file 1. Embryos were generated by intercrossing shhact/+ fish, crossing tcf21:CreER; shhact/+ with shhact/+ fish, or crossing tcf21:CreER; shhact/+ with tcf21:DsRed2; shhact/+ fish. Embryos were genotyped individually by PCR with template DNAs prepared from single embryos as described subsequently (sample preparation for embryo genotyping and analysis). The WT allele was detected by PCR using F1 and R1 primers (Figure 1B). Amplification by F1/R1 primers was intervened in the conditional trap allele with the inserted Zwitch (4448 bp; Figure 3—figure supplement 1A). The conditional trap allele was detected by PCR using F2 and R2 primers for assessing germline transmission (Figure 1D) or F1 and R2 primers for genotyping (Figure 3—figure supplement 1A). The inverted, hence mutagenic, allele was detected by PCR using F4 and F5 primers (Figure 2B). PCR using Cre-scr-F/R primers was performed to confirm tcf21:CreER and ubb:Cre-GFP transgenes (Figure 3—figure supplements 1C and 2B). Examples of the genotyping results are shown in Figure 3—figure supplements 1 and 2.
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study
| 100.0 |
Cre DNA and mRNA injection (Figure 3A–C and Figure 4—figure supplement 2B and C) or tamoxifen treatment (Figure 4B–H) was performed on embryos prior to genotyping PCR. Imaging of embryos (Figures 3B and 4C) and screening of the tcf21:DsRed2 transgene (Figure 4G) or GFP+ embryos after Cre DNA injection (Figure 3A–C) were also performed on anesthetized live embryos prior to the PCR genotyping using an epifluorescence microscope.
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study
| 99.94 |
For histologic analysis (Figure 4D and G), the upper body of embryos, including the heart, was separated from the lower body at the caudal end of the yolk sac extension using sharp forceps. The upper body was used for analysis, and the lower body was used for genotyping. The upper body of each embryo was transferred to an individual 1.5 ml tube containing 100 μl of 4% paraformaldehyde (PFA) and fixed at room temperature for 60 min, followed by rinsing with fix buffer (100 mM Na2HPO4 [pH 7.4], 4% sucrose, and 0.12 µM CaCl2). The samples were stored in fix buffer at 4°C until embryo genotyping was performed. The lower body was collected into a PCR tube containing 50 μl of DNA extraction buffer (10 mM Tris-Cl [pH 8.0], 2 mM EDTA, 0.2% NP-40, and 200 μg/ml proteinase K) and used for genomic DNA extraction. Genomic DNA was extracted from the collected tissues by incubating the sample tubes at 50°C for 60 min, followed by proteinase inactivation at 95°C for 5 min. One microliter of the DNA solution was used as a PCR template, and genotyping PCR was conducted using a PrimeSTAR 1 GXL kit (Clontech, Mountain View, CA).
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study
| 100.0 |
For qRT-PCR analysis (Figure 3A), the embryos were dissected similarly, but RNAlater (Thermo Fisher Scientific, Waltham, MA) was used for fixation. The upper body of each embryo was transferred to an individual 1.5 ml tube containing 100 μl of RNAlater, fixed at room temperature for 10 min, and stored at −80°C until embryo genotyping was performed. The lower body was used for PCR genotyping as described above. After genotyping, 10 pooled upper bodies of the same genotype were transferred to an individual 1.5-ml tube containing 1 ml of TRIzol (Invitrogen, Carlsbad, CA) and used for qRT-PCR analysis as described subsequently (RT-PCR).
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study
| 99.94 |
For semi-qRT-PCR analysis (Figure 4B,F,H), the embryos were fixed with RNAlater at room temperature for 10 min, and the heart was dissected using sharp forceps. Each heart was transferred to an individual 1.5-ml tube containing 100 μl of RNAlater, and the samples were stored at −80°C until embryo genotyping was performed. The remaining body was collected into a PCR tube containing 50 μl of DNA extraction buffer and used for PCR genotyping as described above. After genotyping, 15 to 20 pooled hearts of the same genotype were transferred to an individual 1.5-ml tube containing 1 ml of TRIzol and used for semi-qRT-PCR analysis as described subsequently (RT-PCR).
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study
| 100.0 |
Zebrafish were treated with 4-HT as previously described (Kikuchi et al., 2011). Briefly, at 24 hpf, the embryos were placed in embryo medium supplemented with 5 μM 4-HT produced from a 1 mM 4-HT stock solution in 100% ethanol. After 24 hr, the embryos were transferred to fresh 4-HT-containing medium for an additional 24 hr.
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study
| 99.94 |
Adult zebrafish were placed in a small beaker of aquarium water supplemented with 5 μM 4-HT for 12 hr and subsequently transferred to fresh 4-HT-containing medium for an additional 12 hr. Then, the fish were rinsed with fresh aquarium water and returned to the recirculating water system. Vehicle- or 4-HT-treated fish were used for regeneration experiments or epicardial cell isolation 3 days after the treatment.
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study
| 99.94 |
We estimated the inversion efficiency using a PCR-based approach as follows. First, a DNA segment of the non-mutagenic or the mutagenic allele was amplified by PCR using F7 and R7 primers (Figure 4—figure supplement 2A). The PCR products were purified using a Wizard SV PCR and Gel Purification kit (Promega, Madison, WI) and digested with BglI. Via this digestion, the PCR products specific to the mutagenic allele (M1 and M2 bands; Figure 4—figure supplement 2B) were separated from those specific to the non-mutagenic allele upon gel electrophoresis (N1 and N2 bands; Figure 4—figure supplement 2B). The intensity of each band was quantified using ImageJ software, and the value was normalized by the size (bp) of the band, providing the relative mole quantity (q) of DNA molecules in each band. The inversion efficiency was obtained as the percentage of the ratio of the sum of the relative mole quantity of M1 and M2 (qM1 + qM2) to that of all DNA bands (qM1 + qM2 +qN1 + qN2). Examples of the estimated inversion rate and its correlation to shha expression levels are shown in Figure 4—figure supplement 2C.
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study
| 100.0 |
The LG cassette was PCR amplified from an α-crystallin:EGFP cassette (Kikuchi et al., 2011), and the PCR product was inserted between two FRT sites of pL451 (Liu et al., 2003) (provided by Dr. Stephan Creekmore, NCI-Frederick). The resulting vector was referred to as pL451-FRT-LG-FRT. Tandem LoxP-Lox5171 sites, the splice acceptor (SA) sequence of pFT1 (provided by Dr. Wenbiao Chen, Vanderbilt University) (Ni et al., 2012), and the P2A sequence (SA-P2A) were synthesized using Ultramer Oligo Synthesis by IDT Technologies. TagRFP cDNA was PCR-amplified from pTagRFP-C (Evrogen, Moscow, Russia), and 5× BGHpA repeats were derived from pFT1 via restriction enzyme digestion. The components were inserted into pL451-FRT-LG-FRT via restriction enzyme digestion, and the resulting construct was referred to as pZwitch. As this version was designed for +1 reading frame, it was designated pZwitch+1. To use different reading frames, we created pZwitch+2 and pZwitch+3 using SA-P2A oligonucleotides with the addition of one or five base pairs upstream of the P2A sequence (Figure 1—figure supplement 1B).
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study
| 100.0 |
Purified DNA from a BAC clone (CH211-202A12; BACPAC Resources Center, CHORI, CA) encoding ubb was used as the PCR template. Codon-improved Cre (iCRE) cDNA was PCR amplified from pDIRE (Addgene plasmid #26745; provided by Dr. Rolf Zeller, University of Basel) (Osterwalder et al., 2010), and EGFP cDNA was PCR-amplified using synthetic oligonucleotides with the P2A sequence as the 5′ primer. The components were assembled into pL451 using restriction enzyme digest cloning, and synthetic oligonucleotides with I-SceI (NEB, Ipswich, MA) sites were inserted at the 5′ and 3′ ends of the construct.
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study
| 99.94 |
Codon-optimized FLP (FLPo) was PCR amplified from pDIRE and inserted into pCS2+ using restriction enzyme digest cloning. To remove the LG tag, Flp mRNA was synthesized from linearized pCS2-FLPo using the mMESSAGE mMACHINE SP6 kit, and the resulting mRNA was injected into single-cell embryos.
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study
| 99.9 |
iCRE cDNA was PCR amplified from pDIRE and TagBFP cDNA was PCR amplified using synthetic oligonucleotides with the P2A sequence as the 5′ primer. The components were inserted into pCS2+ using restriction enzyme digest cloning. Cre mRNA was synthesized from linearized pCS2-iCRE-BFP using the mMESSAGE mMACHINE SP6 kit, and the resulting mRNA was injected into single-cell embryos.
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study
| 99.94 |
To increase the mosaicism of Cre expression, pUbb-iCRE-GFP DNA was co-injected with I-SceI into one-cell–stage embryos as previously described (Thermes et al., 2002). The injected embryos were examined at 3 dpf using an MVX10 microscope (Olympus, Tokyo, Japan). Embryos that expressed EGFP on approximately >80% of the total body surface area were selected for qRT-PCR and phenotypic analysis (Figure 3A–C). The embryos expressing EGFP at these levels exhibited the Zwitch inversion at more than 90% efficiency (Figure 4—figure supplement 2C).
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study
| 100.0 |
Embryonic epicardial cells and cardiomyocytes were isolated using a previously published protocol (Burns and MacRae, 2006). We extracted hearts from approximately 500 transgenic embryos harboring tcf21:DsRed2; gata5:EGFP (Kikuchi et al., 2011) or cmlc2:DsRed2 (Kikuchi et al., 2010). In tcf21:DsRed2; gata5:EGFP embryos, EGFP and DsRed2 colocalization was restricted to the epicardium (Kikuchi et al., 2011), and this effect enabled us to isolate highly purified epicardial cells (Figure 4A). The extracted hearts were placed in a Petri dish containing ice-cold 1× Hanks’ Balanced Salt Solution (HBSS) and examined using an MVX10 microscope. To prepare single-cell suspensions, hearts expressing the fluorescent reporter were collected with a pipette and transferred to a 1.5-ml tube containing 1× HBSS with 1 mg/ml collagenase type 2 (Worthington Biochemical, Lakewood, NJ). The samples were incubated for 40 min at room temperature and mixed by gentle pipetting every 10 min. The dissociated cells were washed and resuspended in ice-cold 1× HBSS containing 5% fetal bovine serum (FBS).
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study
| 100.0 |
To isolate adult epicardial cells and cardiomyocytes, ventricles were extracted from adult zebrafish and minced using sharp forceps in ice-cold 1× HBSS. To obtain single-cell suspensions, minced ventricle tissues were transferred to a 1.5-ml tube containing 1× HBSS with 1 mg/ml collagenase type 2. The samples were incubated for 40 min at room temperature and mixed by gentle pipetting every 10 min. Dissociated cells were washed and suspended in ice-cold 1× HBSS containing 5% FBS.
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study
| 99.94 |
The cells were sorted using a FACSAria IIU 16 platform with the purity mode (BD Biosciences, Franklin Lakes, NJ). The cells were directly collected into a 1.5-ml tube containing 1 ml of TRIzol reagent and subsequently used for RT-PCR analysis. Dead cells, defined as those stained with DAPI, were excluded from the cell sorting experiments.
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study
| 99.94 |
The qRT-PCR analysis used 10 upper bodies of the same genotype (Figure 3A), and the semi-qRT-PCR analysis used 15–20 hearts of the same genotype (Figure 4B,F,H). Total RNA was extracted using TRIzol, and cDNA was subsequently synthesized using a Transcriptor First-Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). qRT-PCR was conducted using a LightCycler 480 system (Roche, Basel, Switzerland). For semi-qRT-PCR, genes of interest were amplified using a PrimeSTAR GXL kit. cDNA levels were normalized to actb2/β-actin2 levels in both the qRT-PCR and semi-qRT-PCR experiments. The primers used for semi-qRT-PCR and qRT-PCR analysis are listed in Supplementary file 1.
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study
| 100.0 |
In situ hybridization for raldh2 and immunofluorescence staining assays were conducted as previously described (Kikuchi et al., 2011), and the results were imaged using a LeicaDM400 B LED microscope with an MC170 HD camera (Leica Microsystems, Wetzlar, Germany) or a Zeiss AXIO imager M1 microscope (Carl Zeiss AG, Oberkochen, Germany). Confocal images were captured using a Zeiss LSM 710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). In situ hybridization for shha was performed using RNAscope Reagent kits (Advanced Cell Diagnostics, Newark, CA), and the results were imaged using a LeicaDM400 B LED microscope and a Zeiss LSM 710 confocal microscope. EdU (8 mM) was intraperitoneally injected at 4, 5, and 6 dpi, and EdU incorporation was detected using a Click-iT EdU Alexa 647 Imaging Kit (Invitrogen, Carlsbad, CA).
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study
| 99.94 |
The following primary antibodies were used: rabbit anti-DsRed (Invitrogen, Carlsbad, CA), rabbit anti-tRFP (Evrogen, Moscow, Russia), chicken anti-GFP (Abcam, Cambridge, United Kingdom), rabbit anti-Mef2 (Santa Cruz Biotechnology, Dallas, TX), mouse anti-myosin heavy chain (Clone F59; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-PCNA (Sigma-Aldrich, St. Louis, MO), and rabbit anti-zf Raldh2 (Abmart, Berkeley Heights, NJ). The following secondary antibodies were used: Alexa Fluor 488 donkey anti-mouse IgG(H + L), Alexa Fluor 488 donkey anti-rabbit IgG(H + L), Alexa Fluor 555 donkey anti-mouse IgG(H + L), and Alexa 24 Fluor 555 donkey anti-rabbit IgG(H + L) (Invitrogen, Carlsbad, CA).
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other
| 99.9 |
The number of cardiomyocytes in embryonic hearts was determined using single confocal sections (1 μm thick) of ventricles that were captured using a Zeiss LSM 710 confocal microscope with a 25× objective. The number of Mef2+DAPI+ nuclei in ventricles stained with anti-MHC was manually quantified using ImageJ software. The number of cardiomyocytes was determined by calculating the average number of Mef2+DAPI+ nuclei in three sections from each heart.
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study
| 99.94 |
To quantify subepicardial myocardial cell proliferation, images of the injury border were captured using a Zeiss AXIO imager M1 microscope with a 10× objective (967 × 267 pixels). The subepicardial region was defined as the area within approximately 50 μm of the epicardium, and the trabecular area was defined as the area encompassing the injury, excluding the area used to quantify subepicardial myocardial proliferation. The numbers of Mef2+ and Mef2+PCNA+ cells in the subepicardial and trabecular regions were manually quantified using ImageJ software. The cardiomyocyte proliferation index was defined as the percentage of Mef2+PCNA+ cells in three selected sections from each heart.
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study
| 100.0 |
To quantify epicardial cell proliferation, images of the wound area and the uninjured right ventricular wall were captured at 7 dpi using a Zeiss AXIO imager M1 microscope with a 10× objective (967 × 267 pixels). The numbers of tcf21:DsRed2+DAPI+ nuclei and tcf21:DsRed2+EdU+ nuclei were manually determined using ImageJ software. The epicardial cell proliferation index was defined as the percentage of tcf21:DsRed2+EdU+ nuclei in three selected sections from each heart.
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study
| 99.94 |
Sample sizes were determined in previous publications, and experiment types and are indicated in the legends. Representative results of at least two repeated experiments are shown. All experiments were performed with at least five biological replicates. Individual fish or pooled individual embryos were used as biological replicates. No data were excluded unless the animal died during the procedure. The Mann–Whitney U test and Fisher’s exact test were used. The statistical methods used and p values are indicated in the legends. Raw quantification data are available in Supplementary file 2.
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study
| 99.9 |
Open the Ensembl genome browser (http://www.ensembl.org/Danio_rerio/Info/Index) and search for the target gene using the zebrafish genome. On the target gene page, choose ‘Transcript ID’ for each transcript variant. In the ‘Transcript-based displays panel’ on this page, select ‘Exons’ under ‘Sequence’ to obtain sequence information and identify the translated exon containing the ATG start codon. Repeat this step for all variants and identify the ATG exon included in all potential transcript variants. Any introns located downstream of this ATG exon can be used for Zwitch insertion. The exon usage may need to be experimentally confirmed using target cells and/or tissue samples.
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other
| 99.8 |
In the Ensembl page of the target gene, click ‘Configure this page’ and change the number at ‘Intron base pairs to show at splice sites’ to display the entire sequence of the intron. Open TAL Effector Nucleotide Targeter 2.0 software (https://tale-nt.cac.cornell.edu/node/add/talen) and paste the target intron sequence in the FASTA format in the ‘Sequence’ box. Select ‘Provide Custom Spacer/RVD length’ and search TALENs with the following parameters: Minimum Spacer Length, 13; Maximum Spacer Length, 20; Minimum Repeat Array Length, 15; Maximum Repeat Array Length 15; G Substitute, NH; Filter Options, Show TALEN pairs (hide redundant TALENs); Streubel et al. guidelines, Off; Count Predicted Targets in a Genome/Proteome, Danio rerio (genome), Scoring Matrix, Doyle et al; and Upstream Base, T only. Select several TALENs that have no or the smallest number of off-targets, contain a restriction enzyme site in its spacer sequence, and bind to the intronic sequence at least 100 bp from the exon to avoid interfering splicing machinery.
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other
| 99.7 |
Assemble TALENs using a Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene kit # 1000000024) (Cermak et al., 2011). Clone the assembled TALENs into pCS2TAL3DDD and pCS2TAL3RRR vectors (provided by Dr. David Grunwald, University of Utah) (Dahlem et al., 2012), and synthesize mRNAs using the mMESSAGE mMACHINE SP6 Transcription Kit.
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other
| 99.9 |
Inject approximately 50 pg of TALEN mRNAs into one-cell–stage embryos and purify genomic DNA after 48 hr from 10 to 20 pooled embryos using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Also prepare genomic DNA from embryos that were not injected with TALENs as a negative control for the next measurement step. Use a Restriction Fragment Length Polymorphism (RFLP) assay and measure TALEN activities as described previously (Ma et al., 2013). Other assays such as T7 Endonuclease I cleavage assay or high-resolution melt curve analysis can also be used, but we prefer using the RFLP assay because it is inexpensive and reasonably accurate in determining TALEN activities. Select the TALEN pairs displayed the highest efficiency in inducing DSBs for the subsequent steps.
|
study
| 99.94 |
Amplify 500–1000 bp DNA segment of 5′ upstream and 3′ downstream regions to the spacer sequence of the identified TALEN binding site using a PrimeSTAR GXL PCR Kit. Use primers including NheI or MluI sites at their 5′ ends to amplify the upstream DNA segment. Similarly, use primers including recognition sites for ApaI, XhoI, ClaI, EcoRV, or EcoRI at their 5′ end to amplify the downstream DNA segment. Insert the upstream DNA segment into the NheI and MluI sites of pZwitch as the left homology arm, and the downstream DNA segment into ApaI, XhoI, ClaI, EcoRV, and EcoRI sites as the right homology arm via restriction enzyme cloning. Purify the DNA of the resulting pZwitch construct and perform DNA sequencing of the cloned homology arms using the following primers:
|
study
| 98.1 |
Obtain genomic DNA by tail fin clipping from the clutch-mate fish of the fish used for homology arm cloning. Perform DNA sequencing and identify several male and female fish whose DNA sequence matches the cloned homology arms. Use these fish to produce embryos for the injection of the pZwtich construct. Inject approximately 50 pg each of pZwtich construct DNA and TALEN mRNAs into one-cell–stage embryos.
|
other
| 97.8 |
Screen LG expression in injected embryos between 4 and 7 dpf and transfer LG+ embryos to aquarium tanks. Re-screen LG expression between 30 and 45 dpf and select fish maintaining LG expression uniformly in the eye. Do not select fish exhibiting punctate LG expression, as these fish often do not carry the integrated gene trap cassette (data not shown).
|
other
| 99.5 |
Outcross adult LG+ fish with WT fish and assess LG expression in the offspring. It is critical to perform genomic PCR diagnosis for all LG+ F1 fish to confirm the correct insertion of Zwitch. To validate insertion at the 5′ end of the target site, perform PCR using a forward primer binding to DNA sequences outside of the left homology arm and the following primer as a reverse primer:
|
other
| 99.75 |
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
|
other
| 99.94 |
Thank you for submitting your article "Dissection of zebrafish shha function using site-specific targeting with a Cre-dependent genetic switch" for consideration by eLife. Your article has been favorably evaluated by K VijayRaghavan (Senior Editor) and four reviewers, one of whom, Alejandro Sánchez Alvarado (Reviewer #1), is a member of our Board of Reviewing Editors.
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other
| 99.94 |
In this manuscript, Kikuchi and colleagues generated a novel, invertible gene trap cassette, termed Zwitch, which they use as a part of a methodology for conditional gene abrogation in zebrafish. In the Zwitchconstruct they included a Lens GFP marker for determining transgene incorporation. Overall, the reviewers agree that there is potentially great significance to the application of the inversible gene trap cassettes that can be integrated site-specifically as described by the authors. However, all reviewers also expressed concerns regarding the biological findings reported. As such, we would like to recommend that the authors refocus their efforts in the requested revision to expand on the details of the methods so as to make Zwitch more accessible to the interested community.
|
review
| 99.9 |
2) Results. "Epicardium-specific shha deletion during zebrafish heart development" and "To demonstrate the utility of Zwitch, we investigated the functional consequences of shha deletion in the epicardium, the mesothelial layer covering the heart." The word "deletion" here is not accurate as Zwitch only inactivates gene expression and is not able to generate a real deletion for the shha locus. This may also explain why not all embryos develop severe phenotypes.
|
study
| 99.94 |
3) Subsection “Epicardium-specific shha deletion during zebrafish heart regeneration”, end of first paragraph. Authors should show the expression pattern of shha during heart development and regeneration to rule out or confirm the possibility that non-epicardium-derived shha is present. Importantly, other broad drivers should be used to generate conditional knockout to see if stronger phenotypes can be detected during heart regeneration. If redundant Hh proteins are present, what are they? Where are they being expressed? How do you confirm weak phenotype are caused by redundancy but not because authors did not find the right one?
|
other
| 98.2 |
4) The authors attribute the normal epicardial gene expression during heart regeneration in epicardial shha deficient adults to redundant expression of other Hh genes. It would be interesting to determine whether there is any compensatory increase of the expression of these genes in the mutant heart.
|
study
| 99.94 |
5) The inability of detecting tagRFP is somewhat disappointing. I agree that low level of expression of the endogenous gene in epicardium may very well be a contributing factor. However, shha is highly expressed in the floor plate and notochord during development. It is unclear if the authors detected tagRFP signal in developing embryos after global inversion by Cre mRNA injection.
|
review
| 51.56 |
7) Figure 3: it's unclear how the authors identified ct/ct embryos. Methods indicate that they did not raise homozygous adults, which then were incrossed. Rather it seems the experiments were done on individual embryos genotyped from an incross of ct/+ fish? Please clarify and make this also clear in the manuscript text. If the latter: an example of a genotyping PCR should be shown that can distinguish ct/+ from ct/ct embryos with and without Cre.
|
other
| 99.44 |
8) To prove that the inverted ct allele is a loss-of-function allele of shh, the authors should establish a non-mosaic line of heterozygous fish containing the mutagenic allele and analyze the phenotype of embryos derived from incrosses of these and quantify shha RNA expression (protein quantification is probably not possible due to non-availability of anti-Shha antibodies?)
|
other
| 98.6 |
11) This article aims to provide a novel construct for using Cre-mediated recombination to conditionally ablate genes in zebrafish. Overall, the ability to efficiently introduce targeted mutations would be an important advance for functional work in zebrafish. The utility of this strategy seems contingent on the efficiency of homologous recombination. The manuscript would benefit by including an additional description of how the efficiency of their strategy compares with other published approaches. Is this really any better than what is out there?
|
review
| 99.75 |
12) For a methods paper, little information is given on the approach used for designing the left arm and right arm homology sequences and the TALENs. For a general audience, this needs to be expanded. Additionally, the Materials and methods should be expanded to include all details (including relevant product numbers) for vector construction and the specific steps that would be required for using the Zwitch construct to target other genes, such that anyone with reasonable molecular biology skills could follow step-by-step.
|
other
| 99.7 |
We measured the inversion rate in the Cre-DNA–injected embryos used for the experiments in Figure 3, as mentioned in the subsection “Cre DNA injection” of the Materials and methods in the original manuscript. We used a PCR-based approach for this measurement, as it was difficult to prepare an adequate quantity of genomic DNA from embryos for Southern blot analysis. We have included a new subsection titled “Inversion rate measurement” in the Materials and methods in the revised manuscript and described how the inversion rate of the gene trap cassette was measured in embryos and epicardial cells. We presented the data in Figure 4—figure supplement 2.
|
study
| 96.44 |
2) Results. "Epicardium-specific shha deletion during zebrafish heart development" and "To demonstrate the utility of Zwitch, we investigated the functional consequences of shha deletion in the epicardium, the mesothelial layer covering the heart." The word "deletion" here is not accurate as Zwitch only inactivates gene expression and is not able to generate a real deletion for the shha locus. This may also explain why not all embryos develop severe phenotypes.
|
study
| 99.94 |
3) Subsection “Epicardium-specific shha deletion during zebrafish heart regeneration”, end of first paragraph. Authors should show the expression pattern of shha during heart development and regeneration to rule out or confirm the possibility that non-epicardium-derived shha is present. Importantly, other broad drivers should be used to generate conditional knockout to see if stronger phenotypes can be detected during heart regeneration. If redundant Hh proteins are present, what are they? Where are they being expressed? How do you confirm weak phenotype are caused by redundancy but not because authors did not find the right one?
|
other
| 98.2 |
We used tcf21:DsRed2 transgenic reporter zebrafish to confirm whether shha mRNA expression overlap with tcf21+ epicardial cells. In the developing heart, we detected shha mRNA expression (arrowheads) in the ventricle (ve), and confirmed that some expression in the ventricle was colocalized with tcf21-driven DsRed2 (arrows, Figure 4—figure supplement 1C). We also detected shha mRNA expression in the bulbus arteriosus (ba) but did not observe colocalization of tcf21-driven DsRed2 with these shha mRNA signals (arrows, Figure 4—figure supplement 1B). These signals may arise from non-epicardial cells. The expression of shha mRNA was also detected in non-cardiac tissues such as the skin and the epithelium of the esophagus the epithelium of the esophagus (es; Figure 4—figure supplement 1A).
|
study
| 100.0 |
In the regenerating heart, we detected the majority of shha mRNA signals in the subepicardial area of the ventricle (bracket, Figure 4—figure supplement 1D) and confirmed these signals largely overlapped with tcf21-driven DsRed2 (arrows, Figure 4—figure supplement 1E). We also identified a few cells expressing shha mRNA that were not labeled with DsRed2 (arrow, Figure 4—figure supplement 1F), suggesting that Shha may be produced at low level by non-epicardial cells during regeneration.
|
study
| 100.0 |
We agree that additional Cre driver lines should be used to delineate shha functions in other tissues, however importing such Cre driver lines and analyzing adult regeneration in the homozygous mutant background could not be completed within the given period for this revision. While our data does not rule out a role for extra-epicardial shha or the redundant action of other Hh ligands, the major focus of our study is to establish a genetic method facilitating an inducible, tissue-specific gene knockout analysis in zebrafish and demonstrate its utility in embryo and adult zebrafish. Further investigation of Shha function during regeneration using different Cre driver lines is important, but it will be a focus of a future study.
|
study
| 98.0 |
A previous study reported that the expression of Hh ligand genes, dhh and ihhb, is upregulated in injured zebrafish hearts (Wang J, et al. Nature 2015). Consistent with this result, our new semi-qRT-PCR result revealed the upregulation of dhh and ihhb expression in purified epicardial cells after injury (Figure 5B). The expression of these genes was unchanged after the inactivation of shha expression (Figure 5C). Figure 5B and 5C have been modified to include these results in the revised manuscript. The other Hh ligand genes shhb and ihha were undetectable, as previously reported (Wang J, et al. Nature 2015). Thus, the redundant Hh proteins present in injured zebrafish hearts are likely Dhh and Ihhb, and these ligands are expressed at least in the epicardium.
|
study
| 100.0 |
Among the Hh ligand genes expressed in the zebrafish heart, Shha is the ligand most strongly upregulated during regeneration (Wang J, et al. Nature 2015) (Figure 5B). Therefore, we focused on Shha as the major Hh ligand in heart regeneration. Future studies employing conditional and combinatorial inhibition of Hh ligands will be necessary to definitively determine the relative contribution of each ligand.
|
study
| 100.0 |
“In situ hybridization analysis of embryo and adult hearts detected the expression of shha in tcf21:DsRed-negative cells (Figure 4—figure supplement 1A–F), suggesting that non-epicardial cells also produce Shha. […] Investigations of shha function using broader Cre driver lines and conditional inactivation of other Hh ligand genes will be needed in the future to definitively clarify the function of Shha during heart development and regeneration.”
|
study
| 100.0 |
4) The authors attribute the normal epicardial gene expression during heart regeneration in epicardial shha deficient adults to redundant expression of other Hh genes. It would be interesting to determine whether there is any compensatory increase of the expression of these genes in the mutant heart.
|
study
| 99.94 |
We analyzed the expression of the Hh ligand genes shhb, dhh, ihha, and ihhb using 4-HT–treated injured shhact/ctand tcf21:CreER; shhact/ctheart samples via qRT-PCR. shhb and ihha expression was not detectable, consistent with the result described in a previous study (Wang J, et al. Nature 2015). The expression of dhh and ihhb was detected in the injured heart, but their expression was not significantly changed by the inactivation of shha expression in the epicardium. As detailed in the response to comment #3, we also examined the expression of dhh and ihhb in purified epicardial cells via semi-qRT-PCR. We essentially observed the same result as that of the qRT-PCR analysis; namely, we did not observe a clear increase in the expression of these Hh ligand genes in shha-deficient epicardial cells.
|
study
| 100.0 |
5) The inability of detecting tagRFP is somewhat disappointing. I agree that low level of expression of the endogenous gene in epicardium may very well be a contributing factor. However, shha is highly expressed in the floor plate and notochord during development. It is unclear if the authors detected tagRFP signal in developing embryos after global inversion by Cre mRNA injection.
|
review
| 51.06 |
We observed only weak TagRFP expression in floor plate cells in some embryos after the global inversion of the gene trap cassette. We considered this expression inconclusive, and we also examined other tissues to confirm the expression of TagRFP from the inverted cassette. We detected TagRFP expression most convincingly in the ganglion cell layer (GCL) of the retina, in which robust shha mRNA expression was reported previously (Shkumatava A, et al. Development 2004), and presented the result in Figure 2—figure supplement 3.
|
study
| 100.0 |
It is unclear why TagRFP expression does not fully recapitulate endogenous shha mRNA expression. We currently speculate that this is attributable to inefficient cleavage of the 2A peptide sequence. A glycine-serine-glycine spacer was recently revealed to significantly improve the cleavage efficiency of the 2A peptide (Wang Y, et al. Scientific Report2015), but we were unable to use this spacer because it was reported after the construction of Zwitch. We will address this issue in a future study to improve the visualization of mutant cells using Zwitch.
|
study
| 100.0 |
“We detected TagRFP expression in cells in the ganglion cell layer (GCL) in the retina (arrowheads, Figure 2—figure supplement 3), in which robust shha expression was previously reported (Shkumatava et al., 2004). […] It is of interest to examine whether TagRFP expression would be enhanced in the target tissues by modifying the 2A sequence of Zwitch to include a glycine-serine-glycine spacer (GSG), which was recently found to significantly improve the cleavage efficiency of the 2A peptide (Wang et al., 2015b).”
|
study
| 100.0 |
We appreciate the reviewer’s comment. We repeated the experiment described in Figure 1F with an appropriate control. Regarding the experiments described in Figure 2B, C, and E, we had performed the experiments with the controls, but we did not include the data in the original manuscript. We have included the control data in each figure in the revised manuscript.
|
other
| 99.9 |
7) Figure 3: it's unclear how the authors identified ct/ct embryos. Methods indicate that they did not raise homozygous adults, which then were incrossed. Rather it seems the experiments were done on individual embryos genotyped from an incross of ct/+ fish? Please clarify and make this also clear in the manuscript text. If the latter: an example of a genotyping PCR should be shown that can distinguish ct/+ from ct/ct embryos with and without Cre.
|
other
| 99.44 |
In this experiment, we used shhact/ctembryos prepared from incrosses of shhact/+fish and genotyped the embryos individually via PCR. We presented examples of the PCR genotyping results of embryos from incrosses of shhact/+fish and crosses of shhact/+fish with tcf21:CreER; shhact/+fish in Figure 3—figure supplement 1. Furthermore, we clarified that the screening was performed using individual embryos in the manuscript text as follows:
|
study
| 99.6 |
“Next, we crossed shhact/+fish with shhact/+fish carrying the epicardium-specific inducible Cre transgene Tg(tcf21:CreER) (tcf21:CreER) (Kikuchi et al., 2011) and obtained tcf21:CreER; shhact/ctembryos after PCR genotyping of individual embryos (Figure 3—figure supplement 1C).”
|
study
| 99.94 |
“To analyze epicardial cell development and raldh2 expression in epi-KO hearts, we crossed tcf21:CreER; shhact/+with shhact/+fish carrying the epicardium-specific DsRed2 reporter transgene Tg(tcf21;DsRed2) (tcf21:DsRed2) (Kikuchi et al., 2011) and obtained tcf21:DsRed2; tcf21:CreER; shhact/ctembryos after PCR genotyping of individual embryos.”
|
study
| 99.94 |
Moreover, we have significantly revised the subsection “Genotyping” and included a new subsection “Sample preparation for embryo genotyping and analysis” in the Materials and methods. We have provided more detailed descriptions of the methods by which embryos were processed for genotyping PCR.
|
other
| 99.9 |
8) To prove that the inverted ct allele is a loss-of-function allele of shh, the authors should establish a non-mosaic line of heterozygous fish containing the mutagenic allele and analyze the phenotype of embryos derived from incrosses of these and quantify shh RNA expression (protein quantification is probably not possible due to non-availability of anti-shha antibodies?)
|
other
| 98.7 |
We thank the reviewer for this important suggestion. However, establishing a new non-mosaic line containing the heterozygous mutagenic allele and analyzing its offspring could not be performed within the given period for this revision. Instead, we performed the following experiment, which we believe addresses this issue. We presented the result as Figure 3—figure supplement 2 in the revised manuscript.
|
other
| 99.94 |
We established shhact/+fish carrying the transgene Tg(ubb:iCRE-GFP), in which the expression of codon-improved Cre (iCRE) DNA is expressed by the ubiquitin B (ubb) promoter (Figure 2—figure supplement 1A). Although the ubb:Cre-GFP; shhact/+line is not the non-mosaic line that the reviewer suggested we establish, we suspect that the gene trap allele is inverted in all cells, including germ cells, of this line due to the strong and ubiquitous activity of the ubbpromoter. Consistent with global inversion in this strain, PCR genotyping of 48 single embryos prepared from incrosses of this line showed that all embryos contained the wild-type and/or the inverted mutagenic allele, and no embryos maintained the non-mutagenic allele, irrespective of the presence of the Cre transgene (Figure 3—figure supplement 2A and B). Thus, mosaicism with the mutagenic allele was virtually undetectable in the offspring of the ubb:Cre-GFP; shhact/+line.
|
study
| 100.0 |
We analyzed the phenotype of Cre transgene-negative embryos carrying wild-type shha alleles, heterozygous mutagenic alleles, or homozygous mutagenic alleles. Pectoral fin development was normal in all wild-type embryos (all five analyzed fish were normal) and most heterozygous mutants (six of seven fish were normal), but severely hampered in all homozygous mutants (all three analyzed fish were abnormal) (Figure 3—figure supplement 2C). We also performed semi-qRT-PCR analysis of shha expression in these embryos (Figure 3—figure supplement 2D). Densitometric quantification of the PCR result demonstrated that shha expression was reduced in a concentration-dependent manner with the mutagenic allele (Figure 3—figure supplement 2E). Together with the observation from the Cre DNA injection experiment (Figure 3A–C), we believe these results strongly support our conclusion that the inverted shhactallele is a loss-of-function allele of shha. We have described this result in the revised manuscript as follows:
|
study
| 100.0 |
“To confirm this result, we established shhact/+fish carrying the transgene Tg(ubb:iCRE- GFP) (ubb:Cre-GFP), in which codon-improved Cre (iCRE) DNA was expressed by a strong, ubiquitously expressed ubiquitin B (ubb) promoter (Mosimann et al., 2011) (Figure 2—figure supplement 1A). […] We also performed semi-qRT-PCR analysis of shha expression and confirmed that its expression was reduced to nearly 50% of WT levels in the heterozygous mutants and to an undetectable level in the homozygous mutants (Figure 3—figure supplement 2D and E).”
|
study
| 100.0 |
We analyzed vehicle- or 4-HT–treated tcf21:CreER; shhact/ctembryos as shown in Figure 4C. We did not observe cardiac edema in the vehicle-treated embryos examined (0 abnormal in 8 analyzed) but found severe cardiac edema in six 4-HT–treated embryos (6 abnormal in 8 analyzed; p< 1.0 × 10-8, Fisher’s exact test). We have included this information in Figure 4C and the Figure 4 legend in the revised manuscript.
|
study
| 99.94 |
We observed severe cardiac edema and reductions in cardiomyocyte cell numbers in the conditional mutant heart at 96 and 120 hpf (Figure 4C–E). The cardiac edema phenotype was not evident or extremely weak at 72 hpf in the conditional mutant heart. We analyzed the global shha mutant embryos at 72 hpf (Figure 3A–C) but not at later time pointes, as proper characterization was nor possible due to developmental abnormalities associated with global inactivation of shha expression. We suspect that inactivation of shha expression in the epicardium leads to heart defects mainly at later developmental stages, and such phenotypes may be obscured in non-conditional mutant embryos by pleiotropic effects of global shha inactivation. We included this interpretation in the revised manuscript as follows:
|
study
| 100.0 |
“Cardiac edema was unclear or extremely weak in epi-KO hearts at 72 hpf, suggesting that epicardial inactivation of shha expression leads to heart defects at later developmental stages. We could not determine whether a similar cardiac phenotype was also observed in the global shha mutant embryos, as proper characterization was not possible due to the pleiotropic effects of global inactivation of shha expression at later time points.”
|
study
| 100.0 |
11) This article aims to provide a novel construct for using Cre-mediated recombination to conditionally ablate genes in zebrafish. Overall, the ability to efficiently introduce targeted mutations would be an important advance for functional work in zebrafish. The utility of this strategy seems contingent on the efficiency of homologous recombination. The manuscript would benefit by including an additional description of how the efficiency of their strategy compares with other published approaches. Is this really any better than what is out there?
|
review
| 99.75 |
The targeting efficiency and germline transmission rate of genome editing was likely affected by a number of experimental parameters such as the targeted gene loci, inserted DNA size, and donor vector design. Therefore, it is difficult to directly compare the efficiency achieved in our study to those in other studies. Rather, we included one paragraph to discuss how other published approaches can be used with Zwitch to facilitate functional work in zebrafish as follows:
|
study
| 91.4 |
“After pioneering studies demonstrated the editing of zebrafish genome with long dsDNA (Bedell et al., 2012; Zu et al., 2013), several studies reported the insertion of large DNA fragments at defined zebrafish gene loci via DSB-mediated HR (Hisano et al., 2015; Hoshijima et al., 2016; Shin et al., 2014; Zu et al., 2013) or homology-independent repair mechanisms (Auer et al., 2014; Kimura et al., 2014; Li et al., 2015). […] Although we could not efficiently induce DSBs in the shha intronic sequence using the CRISPR/Cas9 system, this approach may be useful for targeting Zwitch into other genes.”
|
study
| 99.94 |
12) For a methods paper, little information is given on the approach used for designing the left arm and right arm homology sequences and the TALENs. For a general audience, this needs to be expanded. Additionally, the Materials and methods should be expanded to include all details (including relevant product numbers) for vector construction and the specific steps that would be required for using the Zwitch construct to target other genes, such that anyone with reasonable molecular biology skills could follow step-by-step.
|
other
| 99.7 |
We have included a subsection titled “Targeting Zwitch into other genes” in the Materials and methods, in which we described the design of the TALENs and homology arms and explained key steps for Zwitch construction for other genes, including details of the reagents used.
|
other
| 99.9 |
Diffusion‐weighted (DW) magnetic resonance imaging (MRI) can provide unique insights into the microstructure of living tissue and is increasingly used to study the microanatomy and development of normal functioning tissue as well as its pathology. Mathematical models for analysis and interpretation have been crucial for the development and translation of DW‐MRI. Even though diffusion tensor imaging (DTI),1 which is based on a simple Gaussian model of the DW‐MRI signal, has shown promise in clinical applications,2 e.g. Alzheimer's disease,3 multiple sclerosis4 or brain tumors,5 a much wider variety of DW‐MRI models has been proposed to extract more information from the DW signal.
|
review
| 99.9 |
Models generally fall between two extremes: ‘models of the tissue’ and ‘models of the signal’. Models of the tissue6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 describe the underlying tissue microstructure in each voxel explicitly with a multi‐compartment approach.18, 19, 20 Models of the signal focus on describing the DW signal attenuation without describing the underlying tissue composition that gives rise to the signal explicitly.21, 22, 23, 24, 25, 26, 27, 28, 29 Other approaches fall between these two classes and include some features of the tissue, such as the distribution of fibre orientations, but often describe the signal from individual fibres without modelling the fibre composition explicitly.30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40
|
review
| 99.7 |
Despite this explosion of DW‐MRI models, a broad comparison on a common dataset and within a common evaluation framework is lacking, so little is understood about which models are more plausible representations or explanations of the signal. Panagiotaki et al.18 established a taxonomy of diffusion compartment models and compared 47 of them using data from the fixed corpus callosum of a rat acquired on a pre‐clinical system. Later, Ferizi et al.39 performed a similar experiment using data from a live human subject, while Ferizi et al.41, 42 explored a different class of models, which aim to capture fibre dispersion. Rokem et al.43 compared two classes of models using cross‐validation and test–retest accuracy. All these studies18, 43, 44 aim to evaluate variations with specific classes of models with all other variables of the parameter estimation pipeline (i.e. noise model, fitting routine, etc.) fixed. While this provides fundamental insight into which compartments are important in compartment models, questions remain about the broader landscape of models; in particular, which classes of models explain the signal best and how strongly performance depends on the choice of parameter‐estimation procedure.
|
review
| 99.8 |
Publicly organized challenges provide a unique opportunity to bring a research community together to gain a quantitative and unbiased comparison of a diverse set of methods applicable to a particular data‐processing task. Such publicly organized challenges have helped to establish a common ground for the evaluation of competing methods in a variety of imaging‐related tasks, e.g. in brain MR image registration45 and segmentation.46 In DW‐MRI, public challenges have focused on recovering synthetic intra‐voxel fibre configurations47 or evaluating tractography techniques48, 50 and have been very successful at driving research and translation forward. Another interesting comparison of reconstruction methods using DW‐MRI data was based on the signal acquired from a physical phantom.49 Here we report on such a community‐wide challenge to model the variation of DW‐MRI signals at the voxel level in the in vivo human brain.
|
study
| 99.94 |
Modelling the diffusion signal is a key step in realizing practical and reliable quantitative imaging techniques based on diffusion MRI. The challenge in the area is to extract the salient features from the diffusion signal and relate them to the principal features of the underlying tissue (e.g. in the case of brain white matter (WM) the fibre orientation, axonal packing and axonal size). Three distinct questions arise. Given the richest possible dataset that samples the space of achievable measurements as widely as possible, which mathematical model can capture best the intrinsic variation of the acquired signal?Which tissue features can be derived from the model?What subset of those features can we estimate from limited acquisition time on a standard clinical scanner and what dataset best supports such estimates?
|
study
| 50.38 |
The intuition gained from (i) is generalizable over a wide range of applications, while (ii) and (iii) are highly dependent on the MRI study design and the available hardware. Therefore, our challenge focuses on question (i), as an understanding of (i) is necessary to inform (ii) and (iii). To that end, we acquire the richest possible dataset using the most powerful hardware available and the most motivated subject available (UF). Specifically, we use the Connectome scanner,51 which is unique among human scanners in having 300 mT/m gradients, rather than 40 mT/m as is typical of state‐of‐the‐art human scanners. Preclinical work by Dyrby et al.13 highlights the benefits of such strong gradients and the first results from the Connectome scanner42, 52, 53, 54 are now starting to verify those findings.
|
study
| 99.94 |
This kind of model comparison, based on prediction error, is a common and crucial part of the development of any statistical model‐based estimation applications. Burnham and Anderson55 explain how and why such comparisons should be performed to reject models that are theoretically plausible but not supported by the data. To that end, we used a uniquely rich dataset acquired on the Connectome system42 composed of around 5000 points in q space with, for each shell, a unique combination of gradient strength, diffusion time, pulse width and echo time. This offers the opportunity for the comparison of the many different types of models within a common framework, over a very wide range of measurement space. Using this rich dataset, we organized the White Matter Modeling challenge, held during the 2015 International Symposium on Biomedical Imaging (ISBI) in New York. The goal of the challenge was to evaluate and compare the models in two different tissue configurations that are common in the brain: (1) a WM region of interest where fibres are relatively straight and parallel, specifically the genu of the corpus callosum; and (2) a region in which the fibre configuration is more complex, specifically the fornix. Challenge participants had access to three‐quarters of each whole dataset; the participating models were evaluated on how well they predicted the remaining ‘unseen’ part of the data. As announced before the challenge, the final ranking was based exclusively on the performance on the genu data. In this article, however, we include results from both the genu and the fornix.
|
study
| 100.0 |
The article is organized as follows. We first describe in section 2 the experimental protocol, data post‐processing and preparation of the training and testing data for the challenge. We then present the methods for ranking the models and tabulate the various models involved in the competition succinctly. We report the challenge results in section 3 and discuss these results in section 4; a more detailed description of the models follows in the Appendix.
|
other
| 99.7 |
One healthy volunteer was scanned over two non‐stop 4 h sessions. The imaged volume comprised twenty 4 mm thick whole‐brain sagittal slices covering the corpus callosum left–right. The image size was 110×110 and the in‐plane resolution 2×2 mm2. 45 unique and evenly distributed diffusion directions (taken from http://www.camino.org.uk) were acquired for each shell, with both positive and negative polarities; these directions were the same in each shell. We also included 10 interleaved b=0 measurements, leading to a total of 100 measurements per shell. Each shell had a unique combination of Δ={22,40,60,80,100,120} ms, δ={3,8} ms and |G|={60,100,200,300} mT/m (see Table 1). The measurements were randomized within each shell, whereas the gradient strengths were interleaved. We inspected the images visually and did not observe any obvious shifts from gradient heating. The minimum possible echo time (TE) for each gradient duration and diffusion time combination was chosen to enhance signal‐to‐noise ratio (SNR) and potential estimation of compartment‐specific relaxation constants. The SNR of b=0 images was 35 at TE = 49 ms and 6 at TE = 152 ms. The SNR was computed by assessing the signal mean and noise variance across the selected WM voxels on multiple b=0 images. In both cases these estimates matched reasonably well. More details about the acquisition protocol can be found in Ferizi et al.42
|
study
| 100.0 |
All post‐processing was performed using Software Library (FSL).56 The DW images were corrected for eddy current distortions separately for each combination of δ and Δ using FSL's E d d y module (www.fmrib.ox.ac.uk/fsl/eddy) with its default settings. The images were then co‐registered using FSL's F n i r t package. As the 48 shells were acquired across a wide range of TEs, over two days, we chose to proceed in two steps. First, within each quarter of the dataset (different day, different δ) we registered all the b=0 images together. We then applied these transformations to their intermediary DW images, using a trilinear resampling interpolation. The second stage involved co‐registering the four different quarters. To help the co‐registration, especially between the two days images that required some through‐plane adjustment as well, we omitted areas of considerable eddy‐current distortions by reducing the number of slices from 20 to 5 (i.e. leaving two images either side of the mid‐sagittal plane) and reducing the in‐plane image size to 75×80.
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The data for this work originated from two regions of interest (ROIs), each containing 6 voxels (see Figure 1). The first ROI was selected in the middle of the genu in the corpus callosum, where the fibres are mostly straight and coherent. The second ROI's fibre configuration is more complex: it lies in the body of fornix, where two bundles of fibres bend and bifurcate.
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The dataset was split into two parts: the training dataset and the testing dataset. The training dataset was fully available for the challenge participants. The testing dataset was retained by one of the organizers (UF). The DW signal of the training dataset (36 shells, with acquisition parameters shown in black in Table 1) was provided together with the gradient scheme on the challenge website (http://cmic.cs.ucl.ac.uk/wmmchallenge/). This data was used by the participants to estimate their DW‐MRI model parameters. The signal attenuation in the testing dataset (12 shells, with acquisition parameters shown in red in Table 1) was kept unseen. It contained one shell, chosen at random, from each TE‐specific set of four shells (i.e of the same combination of δ and Δ). The challenge participants were then asked to predict the signal for the corresponding gradient scheme. They were free to use as much or as little of the training data provided as they wished to predict the signal of the test dataset for the six voxels in each ROI.
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study
| 100.0 |
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