Figures and data in Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls

Figures
Tables
Additional files

6 figures, 2 tables and 2 additional files

Figures

Figure 1

Download asset Open asset

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

Download asset Open asset

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
Download elife-48511-fig2-data1-v1.xlsx

Figure 2—figure supplement 1

Download asset Open asset

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

Download asset Open asset

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

Download asset Open asset

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

Download asset Open asset

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

Download asset Open asset

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

Download asset Open asset

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
Download elife-48511-fig4-data1-v1.xlsx

Figure 4—figure supplement 1

Download asset Open asset

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.

Figure 5

Download asset Open asset

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).

Figure 6

Download asset Open asset

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
Download 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.

Controls				All targets
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
(Low) Frequency < 1.6%		< 2	> 2	(Low) Frequency < 1.6%		< 2	> 2
Alleles Lost	22	5	17	Alleles Lost	60	24	36
Alleles Preserved	53	35	18	Alleles Preserved	94	71	23
Sum	75	40	35	Sum	154	95	59
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
Frequency > 1.6%		< 2	> 2	Frequency > 1.6%		< 2	> 2
Alleles Lost	0	0	0	Alleles Lost	2	0	2
Alleles Preserved	17	15	2	Alleles Preserved	20	13	7
Sum	17	15	2	Sum	22	13	9
Total alleles: 92				Total alleles: 176
fetuin-b				All targets except fetuin-b
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
(Low) Frequency < 1.6%		< 2	> 2	(Low) Frequency < 1.6%		< 2	> 2
Alleles Lost	20	9	11	Alleles Lost	40	15	25
Alleles Preserved	25	21	4	Alleles Preserved	69	50	19
Sum	45	30	15	Sum	109	65	44
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
Frequency > 1.6%		< 2	> 2	Frequency > 1.6%		< 2	> 2
Alleles Lost	2	0	2	Alleles Lost	0	0	0
Alleles Preserved	1	0	1	Alleles Preserved	19	13	6
Sum	3	0	3	Sum	19	13	6
Total alleles: 48				Total alleles: 128
catalase				All other targets except catalase
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
(Low) Frequency < 1.6%		< 2	> 2	(Low) Frequency < 1.6%		< 2	> 2
Alleles Lost	6	1	5	Alleles Lost	54	23	31
Alleles Preserved	1	1	0	Alleles Preserved	93	70	23
Sum	7	2	5	Sum	147	93	54
Allele Frequency		Log of fold change		Allele Frequency		Log of fold change
Frequency > 1.6%		< 2	> 2	Frequency > 1.6%		< 2	> 2
Alleles Lost	0	0	0	Alleles Lost	2	0	2
Alleles Preserved	1	0	1	Alleles Preserved	19	13	6
Sum	1	0	1	Sum	21	13	8
Total alleles: 8				Total alleles: 168

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Ambystoma mexicanum)	cagg:egfp	Ambystoma Genetic Stock Center (Sobkow et al., 2006)	AGSC Cat# 110A, RRID:AGSC_110A
Genetic reagent (Ambystoma mexicanum)	cagg:nls-mcherry	Ambystoma Genetic Stock Center (Kragl et al., 2009)	AGSC Cat# 112A, RRID:AGSC_112A
Chemical compound, drug	MS-222	Western Chemical	ANADA #200–226
Chemical compound, drug	Human chorionic gonadotropin (Chorulon,)	Merck Animal Health	NADA 140–927
Gene (Ambystoma mexicanum)	msx2	Axolotl transcriptome assembly 3.4	AMEXTC_0340000067092
Gene (Ambystoma mexicanum)	prmt1	Axolotl transcriptome assembly 3.4	AMEXTC_0340000062704
Gene (Ambystoma mexicanum)	myl6	Axolotl transcriptome assembly 3.4	AMEXTC_0340000067862
Gene (Ambystoma mexicanum)	fetub	Axolotl transcriptome assembly 3.4	AMEXTC_0340000227254
Gene (Ambystoma mexicanum)	hoxc8	Axolotl transcriptome assembly 3.4	AMEXTC_0340000065333
Gene (Ambystoma mexicanum)	akap8l	Axolotl transcriptome assembly 3.4	AMEXTC_0340000192860
Gene (Ambystoma mexicanum)	hrnrpa0	Axolotl transcriptome assembly 3.4	AMEXTC_0340000081837
Gene (Ambystoma mexicanum)	hsd17b10	Axolotl transcriptome assembly 3.4	AMEXTC_0340000257015
Gene (Ambystoma mexicanum)	hoxb9	Axolotl transcriptome assembly 3.4	AMEXTC_0340000035333
Gene (Ambystoma mexicanum)	tyrosinase	Axolotl transcriptome assembly 3.4	AMEXTC_0340000179254
Gene (Ambystoma mexicanum)	etv4	Axolotl transcriptome assembly 3.4	AMEXTC_0340000233035
Gene (Ambystoma mexicanum)	cacng1	Axolotl transcriptome assembly 3.4	AMEXTC_0340000081988
Gene (Ambystoma mexicanum)	catalase	Axolotl transcriptome assembly 3.4	AMEXTC_0340000186723
Gene (Ambystoma mexicanum)	hoxb13	Axolotl transcriptome assembly 3.4	AMEXTC_0340000007929
Gene (Ambystoma mexicanum)	zic5	Axolotl transcriptome assembly 3.4	AMEXTC_0340000057641
Gene (Ambystoma mexicanum)	ecm1	Axolotl transcriptome assembly 3.4	AMEXTC_0340000123229
Gene (Ambystoma mexicanum)	cornifelin	Axolotl transcriptome assembly 3.4	AMEXTC_0340000173184
Gene (Ambystoma mexicanum)	dsg-like	Axolotl transcriptome assembly 3.4	AMEXTC_0340000056512
Gene (Ambystoma mexicanum)	enpp2	Axolotl transcriptome assembly 3.4	AMEXTC_0340000217071
Gene (Ambystoma mexicanum)	fabp2	Axolotl transcriptome assembly 3.4	AMEXTC_0340000084459
Gene (Ambystoma mexicanum)	pmp2	Axolotl transcriptome assembly 3.4	AMEXTC_0340000238807
Gene (Ambystoma mexicanum)	kcne1	Axolotl transcriptome assembly 3.4	AMEXTC_0340000121776
Gene (Ambystoma mexicanum)	krt6a	Axolotl transcriptome assembly 3.4	AMEXTC_0340000060835
Gene (Ambystoma mexicanum)	rcc1	Axolotl transcriptome assembly 3.4	AMEXTC_0340000210022
Recombinant DNA reagent	MLM3613	(Hwang et al., 2013)	RRID: Addgene plasmid 42251	Cas9 expression vector
Peptide, recombinant protein	Cas9	PNABio	Cat. #: CP04-500
Commercial assay or kit	mMessage mMachine Kit	ThermoFisher	Cat. #: Am1345
Commercial assay or kit	MAXIscript SP6/T7 Transcription Kit	ThermoFisher	Cat. #: Am1322
Chemical compound, drug	MS-222	Sigma Aldrich	SML1656
Software, algorithm	Geneious Software	Biomatters	RRID:SCR_010519