Salamanders are the only vertebrates known to regenerate complete limbs as adults. The axolotl, a species of salamander, can regenerate limbs, tails, and gills without scarring. Regeneration of these complex structures occurs through the formation of a blastema, a mass of proliferating dedifferentiated cells and pre-existing progenitor and stem cells (Currie et al., 2016; Kragl et al., 2009; Sandoval-Guzmán et al., 2014). Transcriptional profiling of the limb blastema has produced long lists of candidate genes that, to date, remain largely uncharacterized (Bryant et al., 2017; Campbell et al., 2011; Gerber et al., 2018; Knapp et al., 2013; Leigh et al., 2018; Monaghan et al., 2009; Voss et al., 2015).

The advent of CRISPR/Cas9 has made the axolotl a genetically tractable organism and the functional interrogation of these genes possible (Flowers et al., 2014; Fei et al., 2014). Although near-complete knockout F₀ animals can be generated, they appear to be universally mosaic, harboring a variety of mutant alleles (Flowers et al., 2014). Such embryonically generated mutations both perturb the function of the targeted gene and uniquely label affected cell lineages with a traceable genetic barcode. Previously, we measured the fidelity of limb regeneration by using next-generation sequencing (NGS) to quantify the mutant allele frequencies of multiple genomic loci before and after limb regeneration in mosaic mutant axolotls (Flowers et al., 2017). We found that the majority of very low-frequency alleles reoccur in a regenerated limb at a frequency strikingly similar to that of the original limb. These data indicate that limb regeneration is a high-fidelity process in which the contributions of small cell populations to the original limb are replicated in the regenerated limb (Figure 1C,D).

Figure 1

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Recent single-cell sequencing of the axolotl limb blastema demonstrated that cell identities converge at a transcriptional level during regeneration (Gerber et al., 2018). This suggests a shared genetic program across most blastemal cells. We anticipated that genetic perturbation of critical blastema-enriched genes would impair mutagenized cells’ ability to participate in the regenerative process. Negative selection screens are widely used to identify genes essential for cellular processes with CRISPR/Cas (Shalem et al., 2014; Wang et al., 2014; Yin and Chen, 2017). Screening can be improved by using haploid cells, which harbor a single copy of each gene, and thus require monoallelic inactivation to unveil loss-of-function phenotypes. We sought to determine whether we could detect negative selection of mutant alleles in regenerated haploid limbs of axolotls. (Figure 1C,D).

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).

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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.

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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.

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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.

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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.

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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).

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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.

Figure 6

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Figure 6—source data 1

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

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Collectively, our data suggests that cells lacking the limb blastema-enriched genes, fetub and catalase, have a reduced capacity to contribute to the regenerating limb. Catalase is an enzyme that plays a conserved role in protecting cells from oxidative damage by catalyzing the decomposition of hydrogen peroxide, a reactive oxygen species (ROS). Despite their potentially harmful effects, ROS are critical for normal tail, fin, and heart regeneration to proceed in xenopus and zebrafish (Love et al., 2013; Gauron et al., 2013; Han et al., 2014). However, prolonged ROS-exposure and ROS-induced cellular senescence impair tissue regeneration (Saxena et al., 2019). Overexpression of catalase impedes heart regeneration after infarction in zebrafish, and chemical inhibition of Catalase may transiently delay tail regeneration in xenopus larva, suggesting that ROS levels must be carefully regulated during regeneration (Han et al., 2014; von HAHN, 1959).

Fetuin-B and its paralogue Fetuin-A, are highly expressed in the liver, where they are secreted into the blood plasma, and are also expressed in the chondrocytes and muscle cells of developing limb buds in mouse, rat, and sheep (Terkelsen et al., 1998; Saunders et al., 1994; Dziegielewska et al., 1996; Denecke et al., 2003). Mammalian Fetuins belong to the cystatin superfamily of proteins, which include many protease inhibitors, yet these two proteins appear to have differing biochemical activities (Denecke et al., 2003; Karmilin et al., 2019). Fetuin-A is expressed in the growth plate chondrocytes of young mice and is required for proper long bone development, and Fetuin-A knockout mice exhibit severely foreshortened femora due to growth plate deformations and displaced distal epiphyses (Seto et al., 2012). Fetub, knockout mice, however, do not display these defects, and instead show female infertility (Dietzel et al., 2013); and mammalian Fetuin-B, unlike Fetuin-A, appears to function as a specific inhibitor of meprin and ovastacin metalloproteinases (Karmilin et al., 2019). Extracellular matrix remodeling by metalloproteinases is crucial for a variety of processes, including regeneration. Together, our results suggest that locally expressed Fetub is an important regulator of regeneration in the axolotl.

Both genes for which mutant cells exhibited negative selection in this assay are not developmentally essential in mice; however, transcriptional profiling of axolotl limb blastemas across the time course of regeneration indicates that a considerable portion of blastema-enriched genes are known to participate in limb development or cell survival in other organisms (Monaghan et al., 2012; Knapp et al., 2013; Stewart et al., 2013). We anticipate that mosaic loss-of-function of many genes enriched in both the limb bud and limb blastema may result in a depletion of mutant cell lineages prior to limb formation. Thus, mutant alleles of genes required for both limb development and regeneration may not exhibit negative selection in this assay. Exclusion of mutant alleles prior to limb formation may potentially be investigated by comparing allele frequencies from targeted loci in non-grafted tissues in donor haploid embryos to those found within developed limbs arising from tissue grafts from the same embryos. The identification and validation of catalase and fetub in this assay suggests that this method is particularly useful for identifying genes that are not essential for limb development but critical for proper regeneration.

Recent single-cell analyses of blastema cells across the time course of regeneration indicate that connective-tissue-derived blastema cells transcriptionally converge to express a shared set of genes distinct from that expressed in developing limb buds; however, at later stages of regeneration these blastema cells recapitulate transcriptional programs found in developing limbs (Gerber et al., 2018). These findings indicate that the genes controlling early blastema formation do not substantially overlap with those required for proper limb development. There are several known examples demonstrating dissociation between the genetic control of limb of development and regeneration in the axolotl. The long-characterized recessive short-toes allele in axolotls produces animals that develop limbs but display a progressive decline in regenerative capacity (Del Rio-Tsonis et al., 1992; Gassner and Tassava, 1997). Similarly, loss of sox2 in axolotls produces an early spinal cord regeneration defect without perturbing spinal cord development (Fei et al., 2014). Nonetheless, because of the potential confounding effects that may arise in this assay when targeting genes essential for limb development, we largely excluded blastema-enriched genes expected to be required for limb development from the set of targeted genes in this study.

Although this negative selection assay in mosaic haploid mutant limbs led to the identification and subsequent confirmation of catalase and fetub as critical regeneration genes, we failed to detect significant evidence of negative selection of mutant alleles for other assessed candidate genes. For several of these targeted genes, there were insufficient mutant alleles to provide evidence of negative selection (Figure 4—figure supplement 1). The absence of significant negative selection for these targets should not be regarded as evidence that these genes are not critical for limb regeneration. As we conducted multiplex mutagenesis to permit analysis of multiple targets in limbs of an individual animal, we reduced the mass of injected gRNAs from that used in earlier CRISPR mutagenesis studies (Flowers et al., 2017) to prevent confounding effects caused by mutating multiple targets in single-cell lineages. While 16/25 gRNAs used in this study produced mutant alleles in the limb of at least one animal analyzed, we expect that in future studies injecting greater quantities of gRNA will produce more mutant alleles without confounding results.

Here, we provide a novel screening platform that couples targeted mutagenesis and lineage tracing to identify novel regulators of regeneration. This method relies upon the ability to generate chimeric animals that possess mutagenized haploid limbs. We find that the development and regeneration of these haploid limbs is comparable to that of diploid limbs. Using this approach, we find that catalase and fetuin-b are required for cells to participate in limb regeneration and for proper tail regeneration. As the axolotl possesses an impressive capacity to regenerate many parts of its body, future work should explore whether haploid chimeric approaches may be applied to the study the regeneration of these structures. To our knowledge, this is the first example of a true in vivo haploid selection screen conducted in a complex structure of a vertebrate.

All data generated or analyzed during this study are included in the manuscript or supporting files.

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Author details

1. Lucas D Sanor

   Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Grant Parker Flowers

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2756-0457

2. Grant Parker Flowers

   Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Lucas D Sanor

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7436-3531

3. Craig M Crews

   1. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, United States
   2. Department of Chemistry, Yale University, New Haven, United States
   3. Department of Pharmacology, Yale University, New Haven, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   craig.crews@yale.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8456-2005

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