1. Genetics and Genomics
  2. Stem Cells and Regenerative Medicine
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Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls

  1. Lucas D Sanor
  2. Grant Parker Flowers
  3. Craig M Crews  Is a corresponding author
  1. Yale University, United States
Research Advance
Cite this article as: eLife 2020;9:e48511 doi: 10.7554/eLife.48511
6 figures, 2 tables and 2 additional files

Figures

Haploid-diploid chimeric generation and lineage analysis.

(A) Schematic of haploid-diploid chimera generation. Gynogenetic haploids are generated by in vitro activation of unfertilized eggs with UV-enucleated sperm and subsequently mutagenized using CRISPR/Cas9. Chimeric embryos are generated by replacing the limb buds of GFP+ diploid embryos with corresponding tissue from mutagenized haploid donors. (B) DNA is extracted from original and fully regenerated haploid limbs of juvenile chimeric axolotls, target sequences are PCR amplified, and these products are subjected to NGS. (C) Schematic depicting the contribution of mutant cell lineages to the original and regenerated limb. Cell lineages mutant for non-essential candidate genes (light blue, dark blue, yellow, purple, red, green) may participate normally in regeneration and therefore contribute to the regenerated limb and original limb in a similar proportion. Cell lineages harboring deleterious mutant alleles deleterious (red, far right) are predicted to be reduced in regenerated limbs. (D) A hypothetical linear regression plot of the log2 of reads per ten thousand (RP10K+1) of mutant alleles before and after regeneration. Mutant alleles of a neutral gene, tyrosinase (blue), are faithfully preserved between original and regenerated limbs. Mutagenized genes essential for regeneration (red) will show a decrease in allele frequency or a complete loss of alleles in the regenerated limb.

Figure 2 with 2 supplements
Haploid-diploid chimeric axolotl.

(A) Composite fluorescent image of a chimeric axolotl produced from a limb bud graft from an RFP+ haploid embryo to a GFP+ diploid host. Scale bar = 1 cm. (B) Composite fluorescent image of haploid (left) and diploid (right) limbs produced by embryonic limb bud grafting from a white donor embryo to a GFP+ diploid host. Both the GFP- haploid limb and GFP- diploid limb grafted to a GFP+ diploid host exhibit a GFP expression pattern that appears to be restricted to spinal nerves innervating the limb (yellow arrow) and individual sensory neurons and blood-derived cells (white arrows) stemming from the host body. Blue box is at 4x magnification (bottom right). Scale bars = 1 mm. Composite images were generated by manually compiling individual photos. Images have been adjusted with cropping, contrast, color correction, and gamma correction.

Figure 2—source data 1

The number of diploid white to diploid GFP+ grafts that were performed to determine the optimal embryonic stage for limb bud grafting.

https://cdn.elifesciences.org/articles/48511/elife-48511-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Characterization of haploid larvae.

(A) Fluorescent image of a chromosome squash of a diploid cell (2n = 28). A’ and A’’ Fluorescent images of chromosome squashes from two haploid cells (1n = 14). Chromosomes were stained with Hoescht 33342. (B) Light image of stage 25 haploid (left) and diploid (right) embryos. B’ Green fluorescent image of GFP- haploid and GFP+ diploid embryos. (C) Lateral view (upper) and dorsal view (lower) of a haploid embryo 14 days post fertilization (dpf). C’ Lateral view (upper) and dorsal view(lower) of diploid embryo 14 dpf. (D) Bright field image of a stage 36 haploid-diploid chimera embryo with green and red channel fluorescent overlays in the two panels below. The two lowest panels are fluorescent images of the GFP+; RFP- diploid host body and GFP-; RFP+ haploid tissue graft. All scale bars = 1 mm. Images have been cropped and color corrected with brightness and contrast adjusted as necessary.

Figure 2—figure supplement 2
Time course of haploid and diploid limb regeneration.

Four diploid limbs and four haploid limbs of eight stage-matched animals were amputated and imaged on days 0, 3, 7, 10, 16, 23, 30, 37. Haploids (top two rows) regenerate complete limbs and retain the neural GFP expression pattern (last two panels), but show a slight delay around 23 days after amputation versus controls (red box). At this time point, most haploid limbs were still in the palette stage of regeneration (3/4) while diploid limbs were at the point of digital outgrowth (4/4). Haploid limbs are smaller and shorter than diploid limbs (PreAmp, first column). Scale bars = 1 cm. Images have been cropped and color corrected with brightness and contrast adjusted as necessary.

Figure 3 with 2 supplements
Control alleles.

(A) Comparison of all alleles generated in the controls (methyltransferase plus tyrosinase) in the original and regenerated haploid limbs of 12 animals. The log scores of the reads per ten thousand (RP10K) of every allele in the original limb are significantly correlated with those of the secondary limb (R2 = 0.544, p-value<0.0001). (B) Linear regression comparing the log scores of RP10K for alleles depicted in 3A, but separated by gene (methyltransferase-like in red and tyrosinase in blue). The slopes of the regression lines are not significantly different for the two genes (methyltransferase-like m = 0.740, tyrosinase m = 0.935, p-value=0.238, ANCOVA).

Figure 3—figure supplement 1
Comparison of all alleles generated in the controls (methyltransferase and tyrosinase) in the original and regenerated haploid limbs of 12 animals shown individually and compared to the entire remaining set of control alleles.

In each graph, the open red circles depicts the log scores of the RP10K of every allele in the original limb compared to that of the same allele in the regenerated limb of an individual animal, while the black circles depict those of the alleles for all other control animals. The best-fit line for the linear regression of the alleles of each individual animal and that of those of all other control animals are depicted. There are no significant differences between the slopes of any best-fit lines with those of the remaining controls (from top-left to bottom-right, p=0.408, 0.699, 0.490, 0.273, 0.614, 0.534, 0.597, 0.767, 0.203, 0.211, 0.737, 0.652).

Figure 3—figure supplement 2
Histograms depicting the log of fold change after regeneration for alleles detected in the controls (methyltransferase-like and tyrosinase).

(A) All alleles detected in the controls. (B) All alleles that occur with a mutation frequency of 1.60% or less. The majority of these alleles undergo less than a two-fold change after regeneration. (C) All alleles that occur with a mutation frequency of 1.60% or less and are preserved between the first and second limb. (D) All alleles with a mutation frequency less than 1.60% that occur in either the first limb or the second limb only.

Figure 4 with 1 supplement
Fetuin-b alleles compared to all other target gene and control alleles.

(A) Linear regression plot of the log2(RP10K) score for all alleles of fetuin-b detected in the first and regenerated haploid limbs of 11 animals. The log scores of alleles in the primary limb poorly predict the log scores of alleles in the secondary limb. (R2 = 0.069, p-value=0.046). (B) Linear regression plot of the log2(RP10K) score for all alleles of all targets detected in the primary and regenerated limb (R2 = 0.264, p<0.0001). (C) Comparison of linear regression plots of fetuin-b (pink) with controls (gray). The slopes of the regression lines are significantly different (fetuin-b m = 0.254, controls m = 0.861, p-value<0.0001, ANCOVA). (D) Comparison of linear regression plots of fetuin-b (pink) with all other targets (green). The slopes of the regression lines are significantly different (fetuin-b m = 0.254, all other targets m = 0.619, p-value=0.009, ANCOVA).

Figure 4—source data 1

Raw number of reads, normalized reads, and log2(RP10K) score for all mutant alleles of every targeted gene in each mutant limb in this study.

https://cdn.elifesciences.org/articles/48511/elife-48511-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Linear regression plot of the log2(RP10K) score for all alleles in primary and secondary of each targeted gene for which no significant deviation was detected from that of control alleles (akap8l, p=0.069; cacng, p=0.166; hnrnpa0, p=0.371; hoxa9, p=0.637; hoxb13, p=0.053; myl6, p=0.850; pmp2, p=0.624; rcc, p=0.176; tyr, p=0.532,; zic5, p=0.480; ANCOVA).

Best-fit lines are added for genes for which more than one allele was detected. Numbers of animals in which mutations were detected are listed under each gene name.

Catalase alleles compared to all other target gene and control alleles.

(A) Linear regression plot of the log2(RP10K) score for all alleles of catalase detected in the first and regenerated haploid limbs of three animals. The log scores of alleles in the primary limbs do not predict the log scores of alleles in the secondary limbs. (R2 = 0.002, p-value=0.898). (B) Comparison of linear regression plots of catalase (red) with all other targets excluding fetuin-b (teal). The slopes of the regression lines are significantly different (catalase m = 0.018, all other targets excluding fetuin-b m = 0.645, p-value=0.029, ANCOVA). (C) Comparison of linear regression plots of catalase (red) with controls (gray). The slopes of the regression lines are significantly different (catalase m = 0.018, controls m = 0.861, p-value=0.005). (D) Comparison of linear regression plots of catalase (red) with all other targets (green). The slopes of the regression lines are not significantly different (catalase m = 0.018, all other targets m = 0.550, p-value=0.073, ANCOVA).

Larval tail regeneration in tyrosinase, catalase, and fetuin-b mutants.

(A) Regenerative outgrowth of tail in high-level tyrosinase, catalase, and fetuin-b F0 mutants. While no significant difference is detected at early time points, both fetuin-b and catalase mutants display tail reduced tail regeneration compared to tyrosinase mutants at later time points (catalase vs tyrosinase, Day 4, p=0.205, Day 6, p=0.400, Day 10, p=0.111. Day 14, p=0.026, Day 18, p=0.011; fetuin-b vs tyrosinase, Day 4, p=0.450, Day 6, p=0.129, Day 10, p=0.047, Day 14, p=0.109, Day 18, 0 = 0.002, Welch’s t-test). Bars indicate standard deviation. (B) Plots of lengths of regenerate in individual tyrosinase, catalase, and fetuin-b F0 mutants at 18 days post-amputation; **=fetuin b, p=0.002, *=catalase, p=0.011. (C) Brightfield images of individual tyrosinase, catalase, and fetuin-b F0 mutants at 18 days post-amputation (dpa) showing median amount of tail regeneration at 18 dpa. Dotted line indicates the amputation plane.

Figure 6—source data 1

Regenerative outgrowth measurements and genotyping data for tyrosinase, catalase, and fetuin-b F0 mutants.

https://cdn.elifesciences.org/articles/48511/elife-48511-fig6-data1-v1.xlsx

Tables

Table 1
The numbers of all alleles in the first limbs of controls, all targets, fetuin-b, all targets excluding fetuin-b, catalase, and all targets excluding catalase that are sorted by mutation frequency and log of fold change.
ControlsAll targets
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
(Low) Frequency < 1.6%< 2> 2(Low) Frequency < 1.6%< 2> 2
Alleles Lost22517Alleles Lost602436
Alleles Preserved533518Alleles Preserved947123
Sum754035Sum1549559
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
Frequency > 1.6%< 2> 2Frequency > 1.6%< 2> 2
Alleles Lost000Alleles Lost202
Alleles Preserved17152Alleles Preserved20137
Sum17152Sum22139
Total alleles: 92Total alleles: 176
fetuin-bAll targets except fetuin-b
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
(Low) Frequency < 1.6%< 2> 2(Low) Frequency < 1.6%< 2> 2
Alleles Lost20911Alleles Lost401525
Alleles Preserved25214Alleles Preserved695019
Sum453015Sum1096544
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
Frequency > 1.6%< 2> 2Frequency > 1.6%< 2> 2
Alleles Lost202Alleles Lost000
Alleles Preserved101Alleles Preserved19136
Sum303Sum19136
Total alleles: 48Total alleles: 128
catalaseAll other targets except catalase
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
(Low) Frequency < 1.6%< 2> 2(Low) Frequency < 1.6%< 2> 2
Alleles Lost615Alleles Lost542331
Alleles Preserved110Alleles Preserved937023
Sum725Sum1479354
Allele FrequencyLog of fold changeAllele FrequencyLog of fold change
Frequency > 1.6%< 2> 2Frequency > 1.6%< 2> 2
Alleles Lost000Alleles Lost202
Alleles Preserved101Alleles Preserved19136
Sum101Sum21138
Total alleles: 8Total alleles: 168
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Ambystoma mexicanum)cagg:egfpAmbystoma Genetic
Stock Center
(Sobkow et al., 2006)
AGSC Cat# 110A, RRID:AGSC_110A
Genetic reagent (Ambystoma mexicanum)cagg:nls-mcherryAmbystoma Genetic Stock Center
(Kragl et al., 2009)
AGSC Cat# 112A, RRID:AGSC_112A
Chemical compound, drugMS-222Western ChemicalANADA #200–226
Chemical compound, drugHuman chorionic gonadotropin (Chorulon,)Merck Animal HealthNADA 140–927
Gene (Ambystoma mexicanum)msx2Axolotl transcriptome assembly 3.4AMEXTC_0340000067092
Gene (Ambystoma mexicanum)prmt1Axolotl transcriptome assembly 3.4AMEXTC_0340000062704
Gene (Ambystoma mexicanum)myl6Axolotl transcriptome assembly 3.4AMEXTC_0340000067862
Gene (Ambystoma mexicanum)fetubAxolotl transcriptome assembly 3.4AMEXTC_0340000227254
Gene (Ambystoma mexicanum)hoxc8Axolotl transcriptome assembly 3.4AMEXTC_0340000065333
Gene (Ambystoma mexicanum)akap8lAxolotl transcriptome assembly 3.4AMEXTC_0340000192860
Gene (Ambystoma mexicanum)hrnrpa0Axolotl transcriptome assembly 3.4AMEXTC_0340000081837
Gene (Ambystoma mexicanum)hsd17b10Axolotl transcriptome assembly 3.4AMEXTC_0340000257015
Gene
(Ambystoma mexicanum)
hoxb9Axolotl transcriptome assembly 3.4AMEXTC_0340000035333
Gene (Ambystoma mexicanum)tyrosinaseAxolotl transcriptome assembly 3.4AMEXTC_0340000179254
Gene (Ambystoma mexicanum)etv4Axolotl transcriptome assembly 3.4AMEXTC_0340000233035
Gene (Ambystoma mexicanum)cacng1Axolotl transcriptome assembly 3.4AMEXTC_0340000081988
Gene (Ambystoma mexicanum)catalaseAxolotl transcriptome assembly 3.4AMEXTC_0340000186723
Gene (Ambystoma mexicanum)hoxb13Axolotl transcriptome assembly 3.4AMEXTC_0340000007929
Gene (Ambystoma mexicanum)zic5Axolotl transcriptome assembly 3.4AMEXTC_0340000057641
Gene (Ambystoma mexicanum)ecm1Axolotl transcriptome assembly 3.4AMEXTC_0340000123229
Gene (Ambystoma mexicanum)cornifelinAxolotl transcriptome assembly 3.4AMEXTC_0340000173184
Gene (Ambystoma mexicanum)dsg-likeAxolotl transcriptome assembly 3.4AMEXTC_0340000056512
Gene (Ambystoma mexicanum)enpp2Axolotl transcriptome assembly 3.4AMEXTC_0340000217071
Gene (Ambystoma mexicanum)fabp2Axolotl transcriptome assembly 3.4AMEXTC_0340000084459
Gene (Ambystoma mexicanum)pmp2Axolotl transcriptome assembly 3.4AMEXTC_0340000238807
Gene (Ambystoma mexicanum)kcne1Axolotl transcriptome assembly 3.4AMEXTC_0340000121776
Gene (Ambystoma mexicanum)krt6aAxolotl transcriptome assembly 3.4AMEXTC_0340000060835
Gene (Ambystoma mexicanum)rcc1Axolotl transcriptome assembly 3.4AMEXTC_0340000210022
Recombinant DNA reagentMLM3613(Hwang et al., 2013)RRID: Addgene plasmid 42251Cas9 expression vector
Peptide, recombinant proteinCas9PNABioCat. #: CP04-500
Commercial assay or kitmMessage mMachine KitThermoFisherCat. #: Am1345
Commercial assay or kitMAXIscript SP6/T7 Transcription KitThermoFisherCat. #: Am1322
Chemical compound, drugMS-222Sigma AldrichSML1656
Software, algorithmGeneious SoftwareBiomattersRRID:SCR_010519

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