Smith SJ , Towers N , Demetriou K , Mohun TJ .

Cancer Research UK, U117562103 Medical Research Council , Wellcome Trust

             cardiac ventricle morphogenesis
             heart development

Xla.Tg(myl7:Gal4;UAS:Hsa.adprhl1-Xla.adprhl1){Mohun} (Fig. 2. E,F)
Xla.Tg(myl7:Gal4;UAS:Hsa.adprhl1-Xla.adprhl1){Mohun} (Fig.S2.3B-3E)
Xla Wt + adprhl1 CRISPR (Fig. 3. B)
Xla Wt + adprhl1 CRISPR (Fig. 6. CD)
Xla Wt + adprhl1 CRISPR (Fig.7.A-D,I-L)
Xla Wt + adprhl1 CRISPR (Fig.S15.A-H)
Xla Wt + adprhl1 CRISPR (Fig.S15.I-P)
Xla Wt + adprhl1 CRISPR
Xla Wt + adprhl1 MO (Fig.1.C,D)
Xla Wt + adprhl1 MO (Fig. 2.C,D)
Xla Wt + adprhl1 MO (Fig.6.B,C)
Xla Wt + adprhl1 MO (Fig.S2.2B-2E)
Xla Wt + adprhl1 MO (Fig.1.E,F)
Xla Wt + adprhl1 MO (Fig.6.F,G)
Xla Wt + adprhl1 MO (Fig.6.H-K)
Xla Wt + adprhl1 MO (Fig.6.L,M)
Xla Wt + adprhl1 MO (Fig.7.Q-T)

	Fig 1. Cardiac adprhl1 expression and morpholino knockdown in Xenopus embryos.A, B: Comparison of adprh and adprhl1 mRNA expression. A stage 34 tadpole (left-lateral view, A) shows principal sites of adprh expression, with the position of detail images marked by white squares. Mucus producing small secretory epidermal cells (orange arrows) contain adprh from stage 28 (see Discussion 4.2) [47], detection in somites (white arrow) resolves towards hypaxial (ventral) muscle groups by stage 38, transient expression occurs in nephrostomes of the pronephros (cyan arrow), plus otic vesicle (green arrow), pharyngeal arches and the brain. A stage 40 tadpole (B, plus a stage 34 detail image) shows strong adprhl1 mRNA expression in the heart myocardium and also in the eyes within two forming muscle blocks, located medially (anterior) and at superior (upper) and inferior (lower) positions (white arrows). C-H: adprhl1 RNA-splice interfering MOs provide a defined activity and inert heart phenotype. C, D: Expression of actc1 (heart and skeletal muscle, C) and adprhl1 (D) mRNAs in stage 40 tadpoles after injection of 32 ng Adprhl1-e2i2MO at the one-cell stage. Impaired heart ventricle growth and a loss of adprhl1 mRNA signal is observed. Left-lateral view of tadpole and detail ventral view of heart region presented. E, F: Identical heart phenotype caused by injection of the distinct Adprhl1-i2e3MO morpholino. G, H: Normal ventricle size and adprhl1 signal in non-injected sibling tadpoles. Red arrows denote aberrant morphology. H, heart; A, atrium; V, ventricle. I: Transcript and protein composition for 40 kDa Adprhl1. Alignment of a RefSeq mRNA (NM_001093322.1) to the X. laevis adprhl1 S-homeologous locus showing exon and intron sizes. The lower drawing shows the contribution of each coding exon to the translated protein and highlights the position of the peptide antibody epitope (yellow rectangle) and of the conserved di-arginine sequence (red rectangle) mutated in this study. For reference, other sequences that reside within the ancestral active site are also marked (black). S1A Fig describes all the predicted transcripts from both S- and L-alleles while S16 Fig compares mammalian Adprhl1 mRNAs.
	Fig 2. Limited recovery of cardiac myofibril assembly in adprhl1 morpholino injected embryos by transgenic synthesis of recombinant 40 kDa Adprhl1 proteins.Experiments that combine adprhl1 MO knockdown with two distinct transgenes engineered to achieve adprhl1 over-expression. This is the concise version of S2 Fig. For brevity, Fig 2 presents only the high magnification (D and E) images that reveal ventricle wall myofibril patterns within the experimental hearts. The extended figure additionally includes the morphology of each ventricle, the extent of Adprhl1 protein production within, plus squares to locate the position of the myofibril images within each ventricle. A, B: Cardiomyocytes within the heart ventricle wall (anterior surface) of a stage 41 tadpole that was injected with the RNA-splice interfering MO, Adprhl1-e2i2MO, into dorsal (D-2/4) blastomeres. Additionally, it carried binary transgenes to over-express recombinant Adprhl1 protein, consisting of Tg[myl7:Gal4] driver and the Tg[UAS:human1-52-Xenopus53-354 adprhl1] responder. Scale bar = 5 μm (all panels). Fluorescence image (A) shows anti-Adprhl1 immunocytochemistry (green), anti-myosin (red) and DAPI-stained nuclei (blue). The second panel (B) displays a merge of myosin and phalloidin actin stain, with the phalloidin coloured green to evaluate signal overlap. C, D: Ventricle cardiomyocytes from a sibling tadpole that received the same Adprhl1-e2i2MO injection but carried only the UAS-responder transgene and hence did not produce excess recombinant Adprhl1. E, F: A double transgenic sibling that synthesized recombinant human-Xenopus hybrid Adprhl1 but was not injected with the MO. G, H: Ventricle cardiomyocytes from a second experiment, a stage 42 tadpole that was injected with Adprhl1-e2i2MO and carried the Tg[myl7:Gal4] driver but a different Tg[UAS:Xenopus adprhl1(silent 1-282bp)] responder transgene. This incorporates silent nucleotide changes (synonymous substitutions) to the cDNA sequence in order to partially evade endogenous translational regulation. I, J: Ventricle cardiomyocytes of a double transgenic, silent mutation, sibling tadpole that synthesized recombinant Xenopus Adprhl1 but was not injected with the MO. K, L: A non-injected sibling control harbouring only the silent mutation responder transgene that did not produce excess recombinant Adprhl1. Paired white arrowheads indicate Z-disc sarcomere positions, orange arrowheads denote non-striated filaments. V, ventricle; OT, outflow tract.
	Fig 3. Adprhl1 gRNAs—Position, sequence and activity in embryos.A: Diagram showing the hybridization position of gRNAs in relation to exons of the X. laevis adprhl1 locus. Separate gRNAs for S- and L-homeologous alleles were prepared if sequence differences existed between the two at the selected location. Arrows indicate the 5’-3’ direction of each gRNA. Red arrows denote gRNAs whose activities were further examined by sequencing mutant allele DNA. Black arrow gRNAs have their effects on embryo development presented in this figure while grey gRNAs feature in S8 Fig. The table lists the adprhl1-specific sequence of each gRNA along with its protospacer-associated motif (PAM). Mismatched bases of control gRNAs are coloured red. Mismatched 5’-bases added to enable gRNA transcription from plasmid template DNA are coloured blue. Note -e5-1 (S and L) targeting exon 5 gave poor synthesis yields (Materials and Methods 2.2). B: Effect of adprhl1 gRNAs plus Cas9 on embryo development. Parts-of-whole charts showing the frequency of stage 44 tadpole phenotypes that occurred after injection of gRNA along with Cas9 protein into one-cell stage embryos. Red rectangles surround charts for the principal gRNAs whose activities were also examined by DNA sequencing. Green rectangles denote control gRNAs and also a chart presenting the cumulative total for non-injected sibling tadpoles assessed from the experiments. The lower right chart shows the consequence of injecting a mixture of gRNAs to target adjacent intron 2—exon 3 regions of the adprhl1 gene (additional combinatorial gRNA experiments are shown in S8 Fig). Heart defects were detected at higher frequency using the gAdprhl1-e3-1 and in particular the -e6-1 gRNA. Images showing representative tadpoles after gAdprhl1-e6-1 mutation are shown in Fig 6.
	Fig 4. Targeting adprhl1 exon 6 causes near complete mutation and in-frame repair bias—S-homeolog DNA sequences.Sanger cloned DNA sequences of adprhl1 S-homeologous locus exon 6 after mutation by the gAdprhl1-e6-1 gRNA plus Cas9. Mutated sequences from the L-locus exon 6 are presented in S14 Fig. The gRNA position is shown by the red arrow placed above the expected sequence (top 2 rows, exon and genomic). Alignment of 251 (S-) DNA clones obtained from 16 tadpoles, with every sequence carrying a lesion at the gRNA binding site. Mutant nucleotide sequences are coloured red. A missense mutation is listed first, followed by deletions (red hyphens) and then insertions (red arrowheads). Frequently occurring sequences containing in-frame 3, 9 or 12 bp deletions are highlighted. The number of instances of each sequence is to the right, alongside its genotype score.
	Fig 5. Exon 6 classification of mutated adprhl1 sequences—small in-frame deletions responsible for heart defects.A: All cloned Sanger sequences grouped by heart morphology. Parts-of-whole charts record the frequency of exon 6 sequence genotype scores tallied for all embryos that received Cas9 and the gAdprhl1-e6-1 gRNA (far-left chart), or divided into two groups representing tadpoles with heart defects (centre-left chart) versus those whose hearts developed normally (centre-right chart). Sequences from non-injected control embryos were also compared (far-right chart). The total number of S- and L-locus sequences analysed is listed below each chart. The key to interpret the genotype score is included (below B). There was just one wild-type sequence detected among 500 examined and for this region of exon 6, there was a disproportionately high frequency of in-frame deletion mutations. B: Sequences and mutation details of individual embryos. Separate charts for 10 of the embryos, including 5 tadpoles with heart defects and 5 with normal heart morphology. Columns list the number of Sanger sequences and the number of distinct sequences (in square brackets) for each embryo, plus the presence of larger deletions (and their size) that would skip exon 6. Most importantly, the precise amino acid changes are given for all of the in-frame lesions. A standard nomenclature for protein sequence variations is used to describe the changes. Highlight colour matches the genotype score of the sequence. The colour of the small rectangle denotes a missense (teal), net deletion (blue) or net insertion (violet). Three specific amino acid deletions were common to many of the embryos: a single aa loss of Arg272, loss of 3 aa Arg271 to Gly273, or loss of 4 aa Gly270 to Gly273. Deletion of these amino acids must eliminate Adprhl1 protein function and thus be responsible for the cardiac malformations caused by the -e6-1 gRNA. The far-right column shows next generation sequence data (NGS, Materials and Methods 2.5) employed specifically to search for the presence of wild-type reads at the gRNA site. For the majority of injected embryos with a heart defect, increasing the depth of mutation analysis did not reveal any wild-type alleles. In contrast, wild-type alleles were found in each of the tadpoles with a normal heart. C: Structure model of X. laevis Adprhl1 protein backbone, oriented with the ancestral active site foremost [10]. The di-arginine (Arg271-Arg272 coloured red) sequence frequently deleted by the -e6-1 gRNA resides in a loop at the heart of the active site (Gly270, Gly273 yellow, His274 green). D: S-locus sequence at the -e6-1 gRNA site. Five rows represent common in-frame deletions of 3, 9, 9, 12 and 12 bp. Lines above and below the rows mark short direct repeats of nucleotide sequence utilized by the microhomology mediated end-joining DSB repair pathway.
	Fig 6. Onset of ventricle growth defects after mutation of adprhl1 exon 6.A, B: Expression of actc1 (heart and skeletal muscle, A) and adprhl1 (B) mRNAs in stage 34 tadpoles after adprhl1 exon 6 mutation using the gAdprhl1-e6-1 gRNA plus Cas9. Left-lateral view of tadpole and detail left, right and ventral views of heart region presented. C, D: Older stage 39 tadpoles that received the same exon 6 mutation. E, F: Sibling non-injected stage 34 tadpoles. G, H: Sibling non-injected stage 39 tadpoles. By stage 39, exon 6 mutated tadpoles show ventricle defects and early signs of cardiac oedema. One ventricle (C) is displaced towards the right side of the tadpole and has a malformed apex region. The second ventricle (D) is also displaced and has a faint adprhl1 signal, suggesting a thinner myocardial wall. Red arrows denote aberrant morphology. H, heart; L, R, V, left, right and ventral views.
	Fig 7. Impaired ventricle myofibril assembly caused by mutation of adprhl1 exon 6.A: Developing cardiac oedema typical of a stage 40 tadpole after injection of the gAdprhl1-e6-1 gRNA plus Cas9. Left-lateral view of tadpole and detail of heart region presented. The oedema increased embryo transparency so that the small ventricle became visible at an earlier stage compared to controls. B-D: Fluorescence images of the dissected heart ventricle placed with the anterior surface uppermost (B) and displaying merged signals (C, D) of phalloidin actin filaments (green), anti-myosin filaments (red) and DAPI nuclei (blue). The white square (B) denotes the position of the ventricular cardiomyocytes (C) and the white square (C) in turn marks the further magnified image (D). The ventricle is small compared to controls. Cardiomyocytes either have few assembled muscle filaments or contain disarrayed myofibrils with poorly defined sarcomeres (arrowhead, D). Scale bars = 100 μm (B), = 10 μm (C) and = 5 μm (D). E-H: Non-injected sibling control stage 40 tadpole and dissected cardiac ventricle. The cardiomyocytes of the ventricle wall assemble myofibrils that extend in a perpendicular to chamber direction (horizontal in the image, H). Discrete sarcomeres are visible (arrowhead, H). I: A typical stage 42 tadpole mutated with the -e6-1 gRNA and Cas9. The cardiac oedema and small ventricle are the only overt malformations. J-L: The dissected heart has the anterior ventricle surface uppermost, while its aberrant shape positions the outflow tract to the left of the atria after mounting (J)(in controls, the outflow is folded in front of the atria). There is mosaicism amongst the ventricular cardiomyocyte population (K, L), with round non-functional cells (asterisk *, L) and also elongated cells containing disarrayed myofibrils (arrowhead, L). M-P: Non-injected sibling control stage 42 tadpole and dissected ventricle. The tadpole epidermis is now transparent allowing simple assessment of cardiac morphology (M). Myofibrils are packed together (O) and Z-disc stripes are prominent (arrowhead, P). Q-T: For comparison, a stage 40 tadpole and a dissected heart ventricle obtained after injection of the RNA-splice interfering Adprhl1-e2i2MO morpholino at the one-cell stage. This tadpole was probed for myocardial myl7 mRNA and its epidermal pigment removed by bleaching. Left-lateral view and ventral detail (Q). The heart dissected from another MO injected tadpole is small (R), with myofibril disarray (S, arrowhead, T) that is comparable to the CRISPR targeted animals. Note, injection of the MO at the one-cell stage to match the CRISPR experiments yielded a slightly milder cardiac phenotype compared to the previous four-cell stage dorsal blastomere injections due to lower MO concentration apportioned to heart forming tissue (compare Fig 2C and 2D). Oed, oedema; H, heart; V, ventricle; OT, outflow tract; A, atria.
	Fig 8. Mice lacking Adprhl1 exons 3 and 4 are normal—they still produce 25 and 23 kDa ADPRHL1 proteins.A: Western blot detection of ADPRHL1 protein from individual adult mouse hearts carrying the Adprhl1em1(IMPC)H allele (em1). The 40 kDa ADPRHL1 protein was clearly lost from the em1 homozygote heart and no new species appeared in its place. Significantly though, two other major cardiac ADPRHL1 species, 25 and 23 kDa, were unaffected by the em1 deletion. Actin detection was used to normalize the samples. WT, wild type; Het, heterozygote; Hom, homozygote. Note that additional, fainter ADPRHL1 protein species were detected around 37 kDa (and 70 kDa—not shown), although their relative abundance was inconsistent in different western blot experiments. B: The contribution of coding exons to the 40 kDa ADPRHL1 protein, showing the position of 89 amino acids lost by em1 deletion (pink) relative to the peptide antibody epitope (yellow rectangle) and di-arginine sequence (red rectangle). C: Exploration of the potential composition of 25/23 kDa ADPRHL1 species. Exons 5–7 encode the C-terminal protein portion, providing 138 aa and 16 kDa to the open reading frame. A further 70 aa including a methionine to initiate translation would be needed to produce a 23 kDa protein. Blue lines signify the position of alternative in-frame methionines within the 40 kDa sequence.
	Fig 9. Questions regarding Adprhl1 (and Adprh) action.A-C: Structural model of human ADPRHL1 with protein backbone drawn as a tube (A) and magnified active site region (B) [10] that is based on the solved crystal structure of human ADPRH (3HFW) (C-active site shown only) [43]. Amino acids that are common to both proteins are coloured magenta (A, B). White arrows indicate the contribution made by each ADPRHL1 exon to the sequence, orange arrows highlight the positions encoded by the exon-2-3 and exon-4-5 boundaries, with yellow lines marking the remaining exon borders (A). The translated exon-2-3 and 4–5 boundaries reside close to each other and have parallel alignment. Thus a smaller 23 kDa ADPRHL1 form that lacks aa sequence from exons 3–4 could conceivably retain a similar protein fold. Select aa side chains within the active sites are shown as ball and sticks models. For active enzyme ADPRH, aspartates-55, 56, 302, 304 (Mg2+ coordination and catalysis), Cys129, Tyr263, serines-264, 269, 270 (substrate binding cleft) and the location of glycines-100, 127 are shown (C). A molecule of ADP occupies the active site, along with one of the two Mg2+ cations (green sphere) and a K+ ion (grey sphere) (C). For cardiac ADPRHL1, the corresponding active site residues are mostly changed. Asp57 (conserved and coloured red), Asn58, Glu302, Ala304 (no cation coordination), Phe130, Ser265, Glu266, arginines-271, 272 (changed substrate cleft), plus Asp100, Ser128 are shown (B). With this altered active site, ADP cannot be forcibly docked into the ADPRHL1 model. D, E: A deeper understanding of Adprhl1 action is required in order to describe how myofibrillogenesis is linked to chamber outgrowth in the embryo. This diagram illustrates several questions that will need to be addressed:? -1: What is the precise composition of the smaller 23 kDa Adprhl1 protein? The mouse em1 allele has already provided some information. It does not contain exons 3–4 encoded sequence but will include exons 5–6 sequence recognised by the peptide antibody.? -2: What is the composition of the additional 25 kDa mouse Adprhl1 protein? It is likely to be related to the 23 kDa species.? -3: Which sequences localize Adprhl1 to Z-disc/actin filament barbed end boundaries and what myofibril components does it associate directly with? The localization is observed when using an N-terminal epitope tag but not with the exons 5-6-specific antibody. The epitope for the peptide antibody is possibly obscured when Adprhl1 associates with myofibrils.? -4: Might Adprhl1 cooperate with the chaperones and co-chaperones that fold and assemble actin filaments? GimC and TRiC are among factors known to contribute to actin dynamics. There are many additional components of the functional sarcomere unit of myofibrils that Adprhl1 could interact with.? -5: Does Adprhl1 retain binding activity for a post-translational modification that is related to ADP-ribose? For example, an ADP-ribose modification can be partially degraded to a smaller phospho-ribose group by a pyrophosphatase or phosphodiesterase reaction.? -6: Could the substrate binding clefts of Adprhl1 and Adprh also be targets for ADP-ribosylation? Comparison of the di-arginine versus di-serine residues present in the active sites of the two proteins. Di-arginine is common among verified sites for ADP-ribosylation on arginine side chains. Moreover, serine is another acceptor site for ADP-ribosylation that can be hydrolysed by the action of Adprhl2.? -7: Does the active enzyme Adprh contain sequences that provide specificity for particular target proteins, in addition to the binding and hydrolysis of ADP-ribosylated arginine? In the illustration, Adprh is depicted acting on ADP-ribosylated actin, one of a number of known (arginine acceptor) targets of bacterial toxin ADP-ribosyltransferases. Could domains adjacent to the active site help stabilize the interaction with particular modified target proteins. Might this be the activity that is common to both Adprh and Adprhl1.? -8: Could smaller protein forms of Adprh also exist, for example in skeletal muscle? There could yet be analogy between 23 kDa Adprhl1 action in cardiac muscle and Adprh in skeletal muscle. However, evidence against this comparison would be that exon structure is not conserved between the two family members.
	S1 Fig. Xenopus laevis adprhl1 transcripts and expression detected by exon-specific probes. A: All X. laevis adprhl1 transcript alignments showing exon and intron sizes [26]. A single RefSeq mRNA aligns to the S-locus (NM_001093322.1, plus rna29777 that includes longer untranslated regions) while the L-locus is represented by four predicted transcripts. The L-transcript (XM_018247162.1) that closely matches the S-allele was cloned from cardiac cDNA and its sequence verified. Both have been used as hybridization probes in situ (B, C, M, N). The core coding sequence, comprising exons 1 through to 7, encodes a 40 kDa Adprhl1 protein. The precise composition of the prevalent 23 kDa Adprhl1 species also found in Xenopus hearts is unknown, although some progress mapping smaller proteins in mouse is presented (Results 3.11, Fig 8, Discussion 4.3). Current vertebrate gene alignments of adprhl1 include predicted longer transcripts containing an additional 3’-exon 8, which has been assigned to the X. laevis L-allele (XM_018247161.1, plus rna10651). Red arrows mark the position of two distinct exon 8 hybridization probes. The translated sequence from the L-p1 region is conserved with mammals whereas L-p2 is divergent. B-N: Adjacent pairs of exons (1–7) produce strong signals but exon 8 probes do not. A stage 36 tadpole (left-lateral view, B) and detail images of hearts (C-N) showing adprhl1 mRNA expression detected with region-specific antisense probes. The probe size (lower left, bases) and substrate incubation time (right, 7 or 25 hours) for the colour reaction is listed on each panel. Probes covering most of the coding sequence synthesized from either S- or L-locus cDNAs will detect the combined expression from all alleles (B, C, M, N). Smaller S-allele probes that correspond to pairs of adjacent exons between 1–7 (D-I) each produce strong heart signals equivalent to that observed using the larger 789 base coding fragment (B, C). In contrast to exons 1–7, the two L-p1 and L-p2 probes detect little cardiac expression from the putative exon 8, despite prolonged substrate incubation (J, K, L-sense probe control). O-U: Tested individually, exons 1–7 produce weak signals due to their short length. A probe size greater than 200 bases would be a minimum requirement for successful hybridization in situ. V: The contribution of each coding exon to the 40 kDa Adprhl1 protein. The positions of the peptide antibody epitope (yellow rectangle), conserved di-arginine sequence (red) and the two ancestral cation coordination sites (black) are marked. All in-frame methionines are shown underneath and mostly match the mouse ADPRHL1 protein (Fig 8C). https://doi.org/10.1371/journal.pone.0235433.s001
	S2 Fig. Limited recovery of cardiac myofibril assembly in adprhl1 morpholino injected embryos by transgenic synthesis of recombinant 40 kDa Adprhl1 proteins. Experiments that combine adprhl1 MO knockdown with two distinct transgenes engineered to achieve adprhl1 over-expression. This is the extended version of Fig 2. It additionally shows the morphology of the experimental heart ventricles and the extent of Adprhl1 protein production within. The extra panels locate the position within each ventricle wall of the high magnification images (that are also presented in Fig 2) that reveal myofibril patterns found in sample cardiomyocytes. 1A-E: A stage 41 tadpole and its dissected heart ventricle that was injected with the RNA-splice interfering MO, Adprhl1-e2i2MO, into dorsal (D-2/4) blastomeres. Additionally, it carried binary transgenes to over-express recombinant Adprhl1 protein, consisting of Tg[myl7:Gal4] driver and the Tg[UAS:human1-52-Xenopus53-354 adprhl1] responder. Left lateral view of head and trunk (A), while the dissected heart was placed with the anterior surface uppermost (B). The white square (B) denotes the position of a detail image of the ventricle (C) and the white square (C) in turn marks the position of further magnified images (D, E). Scale bars = 100 μm (B), = 10 μm (C) and = 5 μm (D, E). Fluorescence images (B-D) show anti-Adprhl1 immunocytochemistry (green), anti-myosin (red) and DAPI-stained nuclei (blue, D). The final panel (E) displays a merge of myosin and phalloidin actin stain, with the phalloidin coloured green to evaluate signal overlap. 2A-E: A sibling tadpole that received the same Adprhl1-e2i2MO injection but carried only the UAS-responder transgene and hence did not produce excess recombinant Adprhl1. 3A-E: A double transgenic sibling that synthesized recombinant human-Xenopus hybrid Adprhl1 but was not injected with the MO. 4A-E: From a second experiment, a stage 42 tadpole that was injected with Adprhl1-e2i2MO and carried the Tg[myl7:Gal4] driver but a different Tg[UAS:Xenopus adprhl1(silent 1-282bp)] responder transgene. This incorporates silent nucleotide changes (synonymous substitutions) to the cDNA sequence in order to partially evade endogenous translational regulation. The heart had a strong MO defect on the anterior ventricle surface, but lower MO concentration and incomplete phenotype towards its right side (4B). 5A-E: A double transgenic, silent mutation, sibling tadpole that synthesized recombinant Xenopus Adprhl1 but was not injected with the MO. 6A-E: A non-injected sibling control harbouring only the silent mutation responder transgene that did not produce excess recombinant Adprhl1. Paired white arrowheads indicate Z-disc sarcomere positions, orange arrowheads denote non-striated filaments. V, ventricle; OT, outflow tract. https://doi.org/10.1371/journal.pone.0235433.s002
	S3 Fig. Over-expression of recombinant 40 kDa Adprhl1 does not yield extra 23 kDa Adprhl1. Western blots of transgenic tadpole hearts that carried stable lines of the Tg[myl7:Gal4] driver and one of a series of Tg[UAS:adprhl1] responders designed to over-express variants of 40 kDa Adprhl1 protein. Stage 43–44 heart extracts were probed with Adprhl1 antibody, with Actin detection used to normalize the samples. Hum corresponds to a Tg[UAS:human ADPRHL1] responder that induces large-scale synthesis of human-species ADPRHL1 protein in tadpole hearts. This transgene cDNA sequence is sufficiently different from Xenopus to evade the endogenous translational control mechanism that normally limits the production of Adprhl1. Hyb corresponds to the Tg[UAS:hum1-52-Xen53-354 adprhl1] responder that over-synthesizes a human-Xenopus hybrid form of Adprhl1 that also escapes translational control. Xen denotes a Tg[UAS:Xenopus adprhl1] transgene. Using unmodified Xenopus adprhl1 cDNA, transgene mRNA transcription is activated but no additional recombinant protein accumulates [10]. WT equates to control (wild type) hearts. On a separate gel with a higher signal exposure, Xen(silent) denotes the Tg[UAS:Xenopus adprhl1(silent 1-282bp)] transgene containing silent nucleotide changes to the 5’-Xenopus cDNA. This transgene partially evaded endogenous control and recombinant Adprhl1 accumulated in a fraction of the cardiomyocytes up to stage 42. However, there is a technical barrier to performing western blot analysis of transgenic hearts at these early stages. The time required to identify double-positive embryos by the transgenes’ marker eye fluorescence and subsequent sample preparation in the numbers necessary to obtain a signal is prohibitive. The silent mutation transgene gave a transient protein induction [10] and no additional Adprhl1 was detected in this sample prepared from stage 44 hearts. In hearts that synthesized excess 40 kDa hybrid Adprhl1, there was no commensurate increase in the abundance of the 23 kDa protein detected by the Adprhl1 antibody. It suggests the 23 kDa Adprhl1 species is not a processed fragment of the full length protein. Of course, this conclusion depends on the hybrid and natural Xenopus forms of Adprhl1 behaving equivalently. There are just 21 amino acid differences between the two variants. https://doi.org/10.1371/journal.pone.0235433.s003
	S4 Fig. Excess Adprhl1 production does not trigger cell proliferation, nor cause cell death. Tadpole heart ventricles with transgenic over-expression of 40 kDa Adprhl1 protein combined with markers of cell division (A-D) and cell death (E-R). The cardiac Tg[myl7:Gal4] driver and Tg[UAS:Xenopus adprhl1(silent 1-282bp)] responder transgenes were utilized. A-D: Two hearts showing anti-Adprhl1 (green), mitosis marker anti-phospho-Histone H3 (red) and phalloidin actin stain (magenta). White squares (A, C) denote the position of detail images (B, D). Cells undergoing mitosis were readily detected within all regions of embryonic stage 40–41 hearts. There was no correlation between excess Adprhl1 production and cell division. Mitotic cells usually had no Adprhl1 signal (B) but occasionally did contain Adprhl1 protein (D). Note, a second P-H3-positive cell (A) near to the featured cell lay deeper within the myocardial wall so was not detected by the thin optical section of the high magnification image (B). Scale bars = 100 μm (A, C), = 10 μm (B, D). E-I: Heart showing anti-Adprhl1 (green, E, F, H), ApopTag® TUNEL reaction stain (red, G, I), phalloidin (magenta, E, F, H) and DAPI (blue, F, H, I). The white square on the heart (Inset, E) locates images within the ventricle (F, G). Similarly, the squares (F, G) mark the position of detail images (H, I). The ApopTag® stain detects fragmented DNA of dying cells, caused by either apoptosis or necrotic destruction. The images focus on a cardiomyocyte on the ventricle anterior surface with excessive accumulation of Adprhl1 and a round appearance. Nevertheless, no ApopTag® signal was observed for this cell (G, I) nor indeed any Adprhl1-positive cells screened across 11 transgenic hearts. Scale bars = 100 μm (E, N), = 10 μm (F, G, J, K, O, P), = 5 μm (H, I, L, M, Q, R). J-M: Programmed cell death within the heart is a rare event during ventricle chamber outgrowth stages. Comparable images of a ventricle from a sibling transgenic tadpole (L has DAPI only). To prove the TUNEL reaction worked in situ, towards the apex, a solitary apoptotic cell was detected in an area without excessive Adprhl1 production (K, M). A typical condensed nucleus was observed with a fragmented DAPI stain (L) and intact ApopTag® signal (K, M). N-R: A further positive control for the ApopTag® detection method utilized a different responder transgene to induce necrotic cell death of the cardiomyocytes. The Tg[UAS:M2(H37A)] comprises part of a system for controllable genetic cell-ablation, producing the toxic viral ion channel M2(H37A) [55]. The resulting small malformed heart was mounted with left side uppermost, with outflow tract to the left and ventricle to the right (inset, N). The images focus on the dying ventricle (O, P), in which numerous clusters of dots were present, positive for both ApopTag® fragmented DNA and DAPI (P-R). The scattered signals indicated cardiomyocytes had ruptured and that their cellular contents and degrading DNA had dispersed within the myocardium. V, ventricle; OT, outflow tract; A, apoptotic cell remnant; N, necrotic cell death fragments. https://doi.org/10.1371/journal.pone.0235433.s004
	S5 Fig. Adprhl1 morpholinos—Position, sequence and activity in embryos. A: Diagram showing the hybridization position of MOs mapped to the first three exons of the S- and L-homeologous loci for X. laevis adprhl1. Morpholinos targeting potential translation initiation sites in adprhl1 mRNA are shown by red arrows, with the corresponding methionine from the Adprhl1 protein sequence printed above. In addition to the 5'-most ATG predicted as the start for 40 kDa translation, there are five internal ATG sequences within the same reading frame. Morpholinos that target RNA-splicing [10] are coloured violet. B: Table containing the MO sequences and their complementarity to the S- and L-homeologs of adprhl1. Asterisks (*) denote sequence variability within X. laevis (see Supplementary Methods 5.11 in S1 Data). Deliberately mismatched bases within control MOs are coloured blue. C: List showing the potential for hybridization to other members of the ADP-ribosylhydrolase gene family. Only MOs-S6 and L6 that target methionine-162 are noteworthy as they could cross-react with the four adprh loci that are present in Xenopus (adprh.S, adprh.L, LOC495095, adprh-like.2.1.S). D: The translation inhibition MOs produce varied effects on embryo development. Parts-of-whole charts showing the frequency of tadpole phenotypes assessed at stage 44 after 32 and 16 ng MO injection at the one-cell stage (percentage values listed for 32 ng injection). The number of independent experiments and total number of embryos assessed is given under each chart. Heart defects were observed for the MOs that interfere with adprhl1 RNA-splicing and for translation inhibiting MOs-L2 and S6. However, all three MOs designed to the most 5’-ATG (Met1), S1a, S1b and L1, caused unforeseen severe tail defects (after delayed blastopore closure at gastrulation). Morpholinos-3, 4 and 5 had no effect on embryo development. For MO-4 and 5, experiments are presented where a mixture of MOs targeting both S- and L-alleles was injected. Images showing representative embryos for active MOs are shown in S6 Fig. For the translation inhibiting MOs-L2 and S6 that did yield heart defects, both exhibited flaws that limit the MOs usefulness. Adprhl1-ATGMO2(L2) produced inert hearts with small ventricles and was designed to an internal ATG within exon 1 whose translation product would begin at Met26 (S6H–S6K Fig). However, this methionine is only found in the L-homeolog, with the MO being a poor match to the corresponding S-allele sequence. Thus S-allele function should be unaffected. Adprhl1-ATGMO6(S6) targeted an exon 3 sequence and potential translation from Met162 that was briefly considered as the start for the 23 kDa Adprhl1 protein. MO-S6 injection caused tadpole hearts that could contract but had failure of ventricle outgrowth (S6L and S6M Fig). Unfortunately, only the MO designed to the S-homeolog produced a heart phenotype whereas a preparation of -ATGMO6(L6) corresponding to the closely related L-allele sequence yielded distinct early developmental defects that precluded analysis of the heart (D). https://doi.org/10.1371/journal.pone.0235433.s005
	S6 Fig. Adprhl1 morpholinos—Contrast between RNA-splicing versus translation inhibition. A: The diagram showing hybridization positions of MOs mapped to the first three exons of the S- and L-homeologous loci for X. laevis adprhl1. B, C: Reproduced for comparison, panels from Fig 1 featuring Adprhl1-e2i2MO RNA-splice interfering MO. Expression of actc1 (heart and skeletal muscle, B) and adprhl1 (C) mRNAs in stage 40 tadpoles after injection of 32 ng -e2i2MO. Impaired heart chamber growth and a loss of adprhl1 mRNA signal is observed. Left-lateral view of tadpole and detail ventral view of heart region presented. D, E: Normal ventricle size and adprhl1 signal in non-injected sibling tadpoles. F-O: Morpholinos designed to inhibit Adprhl1 protein translation produce varied effects on embryo development. Targeting distinct (but same reading frame) ATG-translation initiation sequences can yield malformations, but each exhibits a flaw that limits the MOs usefulness (see S5 Fig legend for details). F, G: Three overlapping MOs designed to the 5’-most AUG of adprhl1 mRNA each cause a tail growth defect, overshadowing any phenotype they might cause in the heart. Stage 39 tadpoles resulting from Adprhl1-ATGMO1(S1b) injection are shown. H-K: Adprhl1-ATGMO2(L2) injection results in inert hearts with small ventricles. Two severities of phenotype at stage 39 are shown, mildly affected tadpoles with small ventricles (H, J) and also details from tadpoles with a complete loss of ventricle growth (I, K). L, M: Adprhl1-ATGMO6(S6) gives some hearts that contract but have loss of chamber growth. N, O: Normal ventricle size and adprhl1 signal in non-injected stage 39 sibling tadpoles. Red arrows denote aberrant morphology. H, heart; T, tail. https://doi.org/10.1371/journal.pone.0235433.s006
	S7 Fig Activity of distinct Cas9 RNAs and protein for tyrosinase gene knockout in X. laevis embryos. A: Comparing the coding sequence of four distinct Cas9 RNAs and the primary sequence of a commercial Cas9 protein preparation. The deduced Cas9 amino acid sequence originating from Streptococcus pyogenes is identical for all examples. Nevertheless, three shades of blue were used to depict Cas9 because the nucleotide sequences utilized differ due to distinct codon usage. Coding sequences were further modified by inclusion of epitope and purification tags (3xFLAG-green, V5-yellow, 6xHis-grey), nuclear localization signals (SV40-red, nucleoplasmin-magenta), self-cleaving 2A peptide (brown) and orange fluorescent protein reporter (orange). As an example of their size, the hSpCas9 protein is 1423 amino acids long, composed of N-terminal methionine, 22 aa 3xFLAG, 17 aa SV40-NLS, 1367 aa Cas9 (without N-term Met) and 16 aa nucleoplasmin-NLS sequences. B: Table showing the frequency of albino tadpoles obtained after disruption of the tyrosinase gene by injection of each Cas9 reagent together with tyr gRNAs into one-cell stage embryos (Materials and Methods 2.3). Parts-of-whole charts assigned the resulting stage 42 tadpoles to five albino phenotype classes that described the extent of pigmentation-loss and thus completeness of the gene knockout. C: Tadpoles representing the range of pigmentation-loss phenotypes observed after tyrosinase knockout. Left-lateral views, anterior half of tadpoles presented. Definitions of the five albino phenotype classes were comparable to those used by Guo et al [30]. D: Charts from a single experiment using Cas9 protein showing how the efficiency of tyrosinase knockout reduced as the time point of injection (minutes post-fertilization) increased. Beyond 90 minutes, embryos had reached the two-cell stage and thus injection of the same total mass of reagents was divided between both blastomeres. Based on this timed series, an upper limit of 60 minutes post-fertilization was set for all Cas9 injections at the one-cell stage.
	S8 Fig. Adprhl1 gRNAs—Full list of gRNA experiments and embryo phenotype frequencies. Parts-of-whole charts showing the frequency of stage 44 tadpole phenotypes that occurred after injection of adprhl1 gRNAs along with Cas9 protein into one-cell stage embryos. The number of independent experiments and total number of embryos assessed is given under each chart. Red rectangles surround graphs for gRNAs whose activities were also examined by DNA sequencing. Green rectangles denote control gRNAs and also a graph presenting the cumulative total for non-injected sibling tadpoles assayed in the experiments. The highest frequencies of heart defects were detected using the gAdprhl1-e3-1 and in particular the -e6-1 gRNA. The lower seven panels show the consequence of combining gRNAs that hybridize to two genomic regions of adprhl1 into a single injection. None of these combinatorial gRNA experiments increased heart defect frequencies beyond that obtained by gAdprhl1-e6-1 alone. Nonetheless, sequencing data was analysed for experiments where exons 3 and 4 were targeted at neighbouring positions to determine if resulting lesions contained deletions between the DSB sites (see S12 Fig). https://doi.org/10.1371/journal.pone.0235433.s008
	S10 Fig. Exon 3 classification of mutated adprhl1 sequences—Missense mutations are likely to retain function. A: All DNA sequences grouped by gRNAs injection. Each DNA sequence of adprhl1 exon 3 obtained after gRNA plus Cas9-mediated mutation was assigned a genotype score (a number code), based on the size of amino acid sequence modification it encoded. Parts-of-whole charts record the frequency of these genotype scores tallied for all embryos that received the -e3-1(S+L) gRNA (left chart), an embryo injected with a combination of neighbouring -e3-1(S+L) and -i2-2L gRNAs (centre chart), and a non-injected control embryo (right chart). The total number of S- and L-locus sequences analysed is listed below each chart. The key to interpret the genotype score is also included (far right). The inclusion of the -i2-2L gRNA induced larger deletions at the L-locus that would lead to exon 3 skipping, as shown by the increased proportion of score 02 sequences. B: Sequences and mutation details of individual embryos. Separate charts for 11 of the embryos, including 5 tadpoles that developed heart defects, 5 that developed normally and the one non-injected sibling. Columns list the number of sequences and the number of distinct sequences (in square brackets) for each embryo, plus the presence of larger deletions (and their size) that skip exon 3 and score 02. More importantly, for those embryos harbouring subtle mutations that scored 04 or 05, the precise amino acid change is also given. A standard nomenclature for protein sequence variations is used to describe the changes, with a simplified summary (highlighted) underneath stating whether the mutation caused a missense, deletion or aa insertion modification. Highlight colour matches the genotype score of the sequence. The rectangle border colour denotes a missense (teal), net deletion (blue) or net insertion (violet). At this position within exon 3, wherever missense mutations were found (eg p.(Lys145Ile)), the tadpole had preserved a normal heart morphology.
	S10 Fig. Exon 3 classification of mutated adprhl1 sequences—Missense mutations are likely to retain function. [continued] C: Structure model of X. laevis Adprhl1 protein backbone, oriented with the ancestral active site cleft to the left and facing away (Arg271-Arg272 coloured red) [10]. Exon 3 encodes a whole number of codons, contributing 41 amino acids arranged as two antiparallel α-helices. The extended loop mutated by the -e3-1(S+L) gRNA connects the two helices together and resides on the opposite face to the active site (coloured yellow and magenta). Within exon 3, the consequence of in-frame, small amino acid deletions or insertions could not be defined with certainty. Nonetheless, triplet amino acid deletions were found in the normal cohort and may have protected the heart in two tadpoles (see tadpole#20170329014), which suggested these alleles were functional. Conversely, small insertions were biased towards animals with defective hearts, including examples where just a single amino acid was inserted between residues Lys145 and Pro146. Just one mutation gave apparently conflicting results. A 21 bp deletion resulting in a loss of 7 amino acids (p.(Met141_Gly147del)) was present in the heart defect group but also as the sole score 04–05 mutation detected in a normal tadpole (see tadpoles#20170406007, #20170406003). The model of Adprhl1 structure provided some context for the observed sequence modifications. It is plausible that a three amino acid deletion could be accommodated without loss of structural integrity (C). https://doi.org/10.1371/journal.pone.0235433.s010
	S13 Fig. Exon 4 classification of mutated adprhl1 sequences—Incomplete mutation rarely causes heart defects. A: All DNA sequences grouped by gRNA injection. Parts-of-whole charts record the frequency of sequence genotype scores tallied for all embryos that received Cas9 and gRNAs targeting adprhl1 exon 4. The gAdprhl1-e4-1 gRNA was injected individually (far-left chart) and in combination with the neighbouring -e4-2(S+L) (centre-left chart) or -e4-3(S+L) gRNAs (centre-right chart), while sequences from non-injected control embryos were also compared (far-right chart). The total number of S- and L-locus sequences analysed is listed below each chart. The key to interpret the genotype score is also included. Mutation at exon 4 was extensive but incomplete and some wild-type sequences persisted for all combinations of gRNAs tested. B: Sequences and mutation details of individual embryos. Separate charts for 8 typical embryos with normal heart formation, including two representing each gRNA mixture plus sibling controls. Columns list the number of sequences and the number of distinct sequences (in square brackets) for each embryo, plus the presence of larger deletions (and their size) that skip exon 4 and genotype score 02. For smaller in-frame lesions, the precise amino acid change is also given. For these attempts to disrupt exon 4, most tadpoles retained a small proportion of wild-type sequence alleles. Of two presented here with 100% mutation, the reason their hearts were unaffected could not be determined conclusively. Nonetheless, one featured a subtle missense substitution while the second contained a deletion of 3 amino acids that would shorten an α-helix. They were also both notable for harbouring alleles likely to skip exon 4 from the mature mRNA https://doi.org/10.1371/journal.pone.0235433.s013.
	S13 Fig. Exon 4 classification of mutated adprhl1 sequences—Incomplete mutation rarely causes heart defects. [continued] C: Structure model of X. laevis Adprhl1 protein backbone, oriented with the ancestral active site cleft to the left and facing away (Arg271-Arg272 coloured red). Exon 4 encodes a whole number of codons, contributing 47 amino acids. The start and end points of exon 4-derived amino acid sequence lie in close proximity within the model. Mutations induced by the three exon 4 gRNA positions are dispersed (Gly172 green, Gln191 blue, His 213 magenta), but non reside on the same face as the active site. https://doi.org/10.1371/journal.pone.0235433.s013
	S15 Fig. Range of ventricle phenotype severities observed after mutation of adprhl1 exon 6. After adprhl1 mutation, tadpole heart phenotype was assessed at stage 44 and animals with aberrant morphologies were divided into two severity classes based on whether the ventricle was able to contract or not. The two examples here represent extremes from the range of hearts considered abnormal, from the most severely affected inert ventricle (A-H) to the mildest malformation observed in the beating ventricle class (I-P). It should be noted that most of the abnormal hearts were of an intermediate severity, were assigned to the beating group and resembled the earlier stage 40–42 examples shown in the principal figure (Fig 7), particularly with regard to the mosaicism found amongst the ventricular cardiomyocytes. A: Strong phenotype. Cardiac oedema of a stage 44 tadpole after injection of the gAdprhl1-e6-1 gRNA plus Cas9. Right-lateral view of tadpole and left detail of small inert heart presented. Aside from the heart, there are no other discernible defects. Axial structures are straight and gut looping has commenced. B, C: Fluorescence images of the dissected heart placed with anterior-left surface uppermost showing phalloidin actin filament stain (green) scanned at the level of the ventricle myocardial wall (B) and a slice through the lumen (C) located 10 μm deeper. Ventricle growth has completely failed and no trabeculae ridges have formed at the inner surface of the chamber. D-G: Abnormal cardiomyocytes (D) with merged signals for phalloidin actin (green), anti-myosin (red) and DAPI nuclei (blue) from the region of ventricle wall framed by the white square (B). The white square (D) in turn marks the further magnified images (E-G) that show separate myosin and actin signals in addition to the channel merge. H: Muscle filaments inside a single cardiomyocyte identified by the open arrowhead (E-H). The cardiomyocytes vary with regard to the composition of their myofibril structure. Many retain a round shape and contain clusters of short, thin muscle filaments. These have primitive striated patterns to the myosin filaments but the actin strands are poorly defined with little periodicity (open arrowhead, E-H). Where cardiomyocytes have assembled longer myofibrils, the sarcomere spacing appears abnormal (filled arrowhead, E-G, also see below). Scale bars = 100 μm (B), = 10 μm (D) and = 5 μm (E). I: Mildest malformed phenotype after adprhl1 mutation. Left-lateral view of stage 44 tadpole and ventral view of its small heart. J, K: The dissected heart ventricle with anterior surface uppermost scanned at the level of the myocardial wall (J) and a slice through the lumen (K) 8 μm deeper. The ventricle is small with a thin myocardial wall, but trabeculae ridges have formed correctly inside. L-P: Cardiomyocytes of the ventricle (L-O) and a myofibril detail within a single cell (P). A few round cells are present with prominent red myosin stain (open arrowhead, L) but most have produced myofibrils (filled arrowhead, M-P). At high magnification, the periodicity of the actin filaments in particular appears equally spaced rather than the characteristic striated pattern, with no brighter actin stripe indicative of a Z-disc (P). Q-X: Comparable images from a sibling non-injected control stage 44 tadpole. There is no oedema (Q), while the ventricle has the normal packing of elongated cardiomyocytes in the chamber wall (R, T) and parallel (base to apex) alignment of deep lying trabeculae (S). Within the myocardial wall, myofibrils extend predominantly in a perpendicular (to chamber) direction and their sarcomere repeats are mature with a clear Z-disc actin stripe (filled arrowhead, U-X). Oed, oedema; V, ventricle; OT, outflow tract; A, atria. https://doi.org/10.1371/journal.pone.0235433.s015

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