We generated gynogenetic haploids through in vitro activation of eggs from white or transgenic RFP+ females using UV-enucleated sperm from a transgenic GFP+ male (Figure 1A,B). Haploidy was confirmed by karyotype (n = 14, 3/3 embryos, three squashes/embryo, Figure 2—figure supplement 1A), the universal appearance of the haploid syndrome embryonic phenotype (120/120 embryos, Figure 2—figure supplement 1B,C; Hronowski et al., 1979), and complete absence of paternally-derived GFP expression in donor embryos (156/156 GFP-, Figure 2—figure supplement 1B). Adult haploid axolotls are not viable, so we developed reliable whole limb bud grafting techniques to generate chimeric axolotls with haploid limbs (Figure 1A, Figure 2—figure supplement 1D). To find the optimal embryonic stage for limb bud grafting, we performed reciprocal grafts between stage-matched white and GFP+ diploid embryos across a range of developmental stages (Figure 2—source data 1). Diploid-diploid chimera (DDC) graft limbs were scored for the presence or absence of GFP+ host-derived cells using a fluorescent microscope. Embryos grafted at stage 23–25 produced normally developed limbs with a consistent host-derived neural GFP+ expression pattern (Figure 2B; Figure 2—source data 1). We adapted the DDC grafting protocol for haploids by substituting diploid tissue with that of haploid donors. We found that cleanly grafted haploid limbs develop fully, but are smaller and shorter than the opposing diploid limbs of the same animals (Figure 2A, Figure 2—figure supplement 2). Furthermore, haploid-diploid chimeras (HDCs) exhibited a neural-GFP expression pattern similar to DDCs (Figure 2B).

Figure 2 with 2 supplements with 2 supplements see all 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.

Next, we tested the regenerative capacity of HDC and DDC graft limbs. We amputated HDC and DDC limbs and found that both fully regenerate and retain their neural GFP expression pattern (2/2 HDC limbs, 2/2 DDC limbs). While the gross morphology of regenerated haploid limbs is identical to that of the original limbs, haploid limb regeneration is slightly delayed relative to diploid limb regeneration (Figure 2—figure supplement 2). To quantify the fidelity of haploid limb regeneration, we generated HDCs using haploid donors mutagenized at one of two genomic loci non-essential for regeneration, tyrosinase and methyltransferase-like, for which we had previously observed faithful recapitulation of mutant allele frequencies between original and regenerated diploid limbs. NGS of targeted loci in 12 HDCs mutagenized with one of two highly active guide RNAs (gRNAs) revealed 92 total alleles with a mean mutation frequency of 3.46% per allele in the primary limbs (SE = + /- 1.19%). NGS of these targeted sites in DNA from regenerated limbs revealed that the log score of the normalized read numbers for each allele in the primary limbs predicts the log score of the normalized read numbers in the secondary limbs (R2 = 0.544, p<0.0001, Figure 3A,B, Figure 3—figure supplement 1), which is similar to observations made with these same targets in diploid mosaic limbs (Flowers et al., 2017). Thus, with respect to morphology and cell lineage contributions, haploid limb regeneration is similar to that of diploid limb regeneration.

Figure 3 with 2 supplements with 2 supplements see all 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).

The majority of tyrosinase and methyltransferase-like alleles (76.1%, 70/92) are detected in both the first and second haploid limbs. Most mutant alleles occur at a low frequency, comprising fewer than 1.6% of the total reads for a given haploid limb (81.5%, 75/92, Table 1). The majority of low-frequency alleles are detected in both primary and secondary limbs (70.7%, 53/75) and undergo less than a two-fold change in frequency after regeneration (69.3%, 52/75, Table 1, Figure 3—figure supplement 2A–D). Collectively, these results support the notion that, as in diploids, haploid limb regeneration is a high-fidelity process in which the majority of small cell lineages contribute to the regenerated limb in a manner similar to their contributions to the original developed limb.

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

Figure 4 with 1 supplement with 1 supplement see all Download asset Open asset Fetuin-b alleles compared to all other target gene and control alleles. (A) Linear regression plot of the log 2 (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 log 2 (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).

We found two genes, fetuin-b and catalase, that exhibited signs of negative selection, showing both a loss of mutant alleles and a decline in the contribution of mutant alleles from primary to secondary limbs (Table 1). We compared the linear regression line slopes of all mutant alleles between primary and secondary limbs for each target gene with those of the inessential controls (methyltransferase-like and tyrosinase) and found that fetuin-b (fetub) was significantly different (n = 48 mutant alleles, fetub m = 0.254, controls m = 0.861, p<0.0001, Figure 4A,C). Further comparison of fetub with all other target genes combined reveals that the slope of the linear regression of fetub is lower than that of all other target genes combined (fetub m = 0.254, All other target genes m = 0.619, p=0.009, ANCOVA, Figure 4D). Linear regression analysis of fetub reveals that the log scores of the normalized read numbers for each allele in the second limb poorly predict the log scores of the normalized read numbers in the primary limb (R2 = 0.069, p=0.046, Figure 4A). Alleles of fetub detected in the primary limb are more likely to be absent in the secondary limb (45.8%, 22/48) than alleles detected in controls (23.9%, 22/92) and this difference is significant (χ2 = 7.03, p=0.008). Fetub alleles (45.8%, 22/48) are more likely to be absent from the second limb than alleles of all other targets combined (31.3%, 40/128), but this effect is not significant, except when the other outlier, catalase, is excluded (χ2 = 3.25, p=0.071 and χ2 = 5.23, p=0.022, respectively).

Similarly, the slope of the linear regression of catalase alleles differed from control genes (n = 8 mutant alleles, catalase m = 0.018, controls m = 0.861, p=0.005, ANCOVA, Figure 5A,C). The slope of the linear regression of catalase did not differ from that of all other target genes combined, except when fetub was excluded (catalase m = 0.018, all other target genes m = 0.550, p=0.073, all other target genes excluding fetub m = 0.645, p=0.029, ANCOVA, Figure 5B,D). A significantly greater proportion of catalase alleles are lost (75.5%, 6/8) than both those of controls and all other targets combined (χ2 = 9.53, p=0.002 and χ2 = 5.81, p=0.016, respectively).

Figure 5 Download asset Open asset Catalase alleles compared to all other target gene and control alleles. (A) Linear regression plot of the log 2 (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).

To increase the total number of catalase and fetub mutants analyzed, we next addressed whether loss of these genes perturbs regeneration at a whole organismal level. We produced early embryonic mutants for catalase, fetub, and tyrosinase by injecting gRNAs against each with Cas9 protein into zygotes. At stage 44, we amputated the posterior 2 mm of the tails of each larva and monitored its regeneration. We extracted DNA from the amputated tails and confirmed the high-level mutagenesis of fetub and catalase by fluorescent PCR fragment analysis (fetub, n = 12, mean = 7.3% wildtype alleles, SD = + /- 5.2%;. catalase, n = 16, mean = 3.0% wildtype alleles, SD = + /- 5.8%, Figure 6—source data 1). fetub and catalase mutants did not display regeneration growth defects compared to tyrosinase mutants at early time points, but the total regenerative outgrowth of both fetub and catalase mutant tails were reduced compared to tyrosinase mutants at 18 days post-amputation (n = 16 tyrosinase mutants, p=0.002, fetub; p=0.012, catalase; Welch’s t-test, one-tailed; Figure 6A–C), with the reduction in regeneration also evident at 14 days post-amputation in catalase mutant tails (p=0. 025, Welch’s t-test, one-tailed, Figure 6A). These data indicate that, while catalase and fetub are not essential for the onset of regeneration, the process of regeneration is slower in the tails of catalase and fetub mutants. These findings are consistent with the apparent loss of catalase and fetub mutant cells within the context of regenerating mosaic mutant haploid limbs and suggest a broader role for these genes in the regeneration of multiple tissues and structures. As cell competition in developing tissues can result in the elimination of cells lacking genes controlling the rate of growth at a whole organismal level (Johnston et al., 1999; Morata and Ripoll, 1975), these findings support the validity of this assay as a means to identify genes critical for proper regeneration.