Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7,8,9,10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR–Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.
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Endothelin-1 axes in the framework of predictive, preventive and personalised (3P) medicine
EPMA Journal Open Access 04 August 2021
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All data generated or analysed, and all methods used during this study are summarized in the Article (and its Supplementary Information). The raw data and images are available from the first and second authors upon reasonable request.
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We thank S. Miehls at the USGS Hammond Bay Biological Station and B. Laflamme at the Holyoke Dam for providing adult sea lampreys; B. Birsoy, J. Shi and M. Klymkowsky for assistance with X. laevis fertilizations; Z. Root for assistance with X. laevis and sea lamprey injections and husbandry; C. Altier for assistance with X. laevis husbandry; S. Schwikert for pro bono statistics consultation; C. Miller for his input on the manuscript; D. McCauley and J. York for providing HuC/D primary antibody and the IHC protocol; and R. Harland for providing secondary antibodies and staining advice for X. laevis. D.M.M., T.A.S., D.J., M.R., J.L.M. and M.V.C. were supported by National Science Foundation grants IOS 1656843, IOS 1257040 and IOS 0920751 to D.M.M. H.P.S. and A.W.H. were supported by the University of Colorado, Boulder Undergraduate Research Opportunities Program. D.J. was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 751066 and by the Scientific Grant Agency of the Slovak Republic VEGA grant no. 1/0415/17.
The authors declare no competing interests.
Peer review information Nature thanks Ingo Braasch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Petromyzon marinus wild-type and mutant larval alcian blue stained head skeletons and lecticanA expression.
Anterior to left in all panels. For detailed quantification information, see Supplementary Table 2 and Methods section ‘Statistics and reproducibility’. a, WT ventral view at st. T30. b, WT lateral view of the same specimen, with skeletal elements and cartilage types labelled in b′ and b″, respectively. In b′, regions of the oral skeleton are delineated. In b″, Epitrematic indicates the epitrematic processes on PAs 3 and 4, though these also exist on all branchial arches. Hypotrematic indicates the hypotrematic processes of PAs 3 and 4, though these also exist on all branchial arches. Hypobranchial indicates the hypobrancial cartilage connecting PAs 4, 5, and 6, but exists between all branchial arches. Subchordal indicates the subchordal cartilage connecting PAs 4, 5, and 6, but exists between all branchial arches. c–f, Alcian blue reveals Δednra, Δednrb, Δednra+b, ΔednA head skeleton phenotypes at st. T30 (n = 16/16, n = 5/18, n = 19/19, and n = 12/15 individuals for each gene, respectively). Red arrowheads highlight some regions where cartilages are missing or separated. g–k, lecticanA WT expression summary in P. marinus. This gene is homologous to gnathostome lectican genes (such as aggrecan and versican) and like those genes it is expressed in neural crest-derived mesenchyme before (for example, arrows in h and i) and during chondrogenesis (for example, expression in j and k). ll, lower lip; lmp, lateral mouth plate; mvs, medial velar skeleton; 1-9, pharyngeal arches (numbered individually); tr, trabecular; ul, upper lip. The scale bar in a is 500 μm and applies to images in a–f.
Extended Data Fig. 2 Petromyzon marinus Δednra phenotype and genotype summary.
For detailed quantification information, see Supplementary Tables 1, 2, 4, and Methods section ‘Statistics and reproducibility’. a, Phenotypic series of ednra sgRNA4 hypomorphs at st. T30 (n = 264/325 injected individuals displayed a similarly severe phenotype, here labelled ‘head skeleton + heart + pigment’). Left lateral views. Scale bar represents 500 μm. b, An example of a genotyped animal from injected with ednra sgRNA3 (n = 113/154 injected individuals displayed a similarly severe phenotype). Left lateral view. Scale bar represents 500 μm. Target site is shown in orange with a red PAM. Green nucleotide string indicates an insertion with sequence that is also observed at a nearby endogenous locus near the lesion on the reverse strand (underlined nucleotide string). This insertion is stacked inside of the lesion on the 5′ end. c, A staging series of Δednra illustrating the typical manifestation of the severe phenotype shown in a and b. Left lateral views in all panels. At st. T24, a slight heart oedema is usually apparent (arrow). From this stage, the reduction in head skeleton size becomes progressively more dramatic through development relative to WT (brackets mark the anterior and posterior boundaries of the skeletogenic mesenchyme). Scale bar represents 100 μm and applies to all images in this panel. d, A ventral view of the WT and Δednra heart, showing the most prominent aggregation of ectopic melanophores. Anterior to top, bracket indicates the heart in WT and the heart oedema in Δednra. e, Melanophore number is increased in the ventral fin fold in Δednra relative to WT (n = 5 WT versus n = 5 Δednra individuals, Student’s one-sided t-test P = 0.000255, Cohen’s d = −1.53, df = 13). Images at right illustrate the pigment cells counted in (green arrowheads). The median fin fold is outlined. f, External melanin cover is also increased in Δednra relative to WT (n = 5 WT versus n = 5 Δednra individuals, Student’s one-sided t-test P = 0.00749, Cohen’s d = −1.74, df = 9). Image at right is an example threshold image quantified in the analysis, see Methods. Box plots show all points and delineate all quartile thresholds; medians are indicated with a horizontal line.
Extended Data Fig. 3 Petromyzon marinus dlxA, -D, -B, hand, ID, lecticanA (lecA), myc, msxA, phox2, soxE1, soxE2, and twistA expression in Δednr lampreys at st. T26.5.
For detailed quantification information, see Supplementary Tables 1, 2, 4, and Methods section ‘Statistics and reproducibility’. a, b, Left lateral views in all panels. Treatment and gene assayed are indicated in the figure. Scale bar represents 100 μm and applies to all images (that is, all ISH images are to scale with each other). Gray and white dotted lines in f and g are examples of the head size and hand domain size measurements (respectively) used for the comparison in c. Δednra n = 7/21, n = 5/15, n = 2/5, and n = 0/8 individuals with perturbed dlxA, dlxD, dlxB and hand ISH signals, respectively. Δednra+b n = 20/24, n = 7/8, n = 4/6, and n = 0/9 individuals with perturbed dlxA, dlxD, dlxB, and hand ISH signals, respectively. Δednrb n = 0/8, n = 0/6, n = 0/5, and n = 0/7 individuals with perturbed dlxA, dlxD, dlxB, and hand ISH signals, respectively. c, Quantifying the X/Y lateral size of the hand ventral domain to head size ratio reveals a loosely supported trend upwards in Δednra+b, suggesting that the ventral hand domain is only nominally affected in these mutants (see Methods; left and right images of n = 6 WT and n = 8 Δednra+b individuals, Student’s two-sided t-test P = 0.0647, df = 13). Box plots show all points and delineate all quartile thresholds; medians are indicated with a horizonal line. d, Lateral views with anterior to left in all panels. Treatment and gene assayed are indicated in the figure. Overall, Δednra and Δednrb are most consistent in their gene expression patterns, despite all domains being shrunken in Δednra. Conversely, Δednra+b displays some disruptions in gene expression not observed in either single receptor perturbation (red arrowhead, white stars). ISH result numbers are as follows, all numbers indicate independent biological samples (individual lamprey larvae) that produced a reduced, discontiguous, or abrogated ISH signal: Δednra ID n = 0/5, lecA n = 16/16, myc n = 0/6, msxA n = 0/12, phox2 n = 0/8, soxE1 n = 0/4, soxE2 n = 15/21, twistA n = 0/6; Δednrb ID n = 0/10, lecA n = 4/8, myc n = 0/7, msxA n = 0/7, phox2 n = 0/8, soxE1 n = 0/7, soxE2 n = 4/8, twistA n = 0/9; Δednra+b ID n = 3/5, lecA n = 8/9, myc n = 0/4, msxA n = 0/8, phox2 n = 0/11, soxE1 n = 9/11, soxE2 n = 9/10, twistA n = 4/4.
Extended Data Fig. 4 Xenopus laevis Δednra and Δedn1 head skeleton defects and genotyping.
For detailed quantification information, see Supplementary Tables 1, 2, 4, and Methods section ‘Statistics and reproducibility’. a–c, Δedn1.L+S (b) and Δednra.L+S (c) show hypomorphic head skeleton elements relative to WT (a) at st. N.F.48 (n = 20/47 and n = 37/71 injected individuals for each gene, respectively). Δedn1.L+S is typically less severe, with a discernible Meckel’s cartilage (labelled mc) present but fused to the palatoquadrate (pq), thus lacking a primary jaw joint (black arrowheads in a′, red arrowheads in b′). However in Δednra.L+S, Meckel’s cartilage is highly reduced, and frequently unrecognizable, only visible as a bump on the palatoquadrate in most cases. The infrarostral (ir) is always detectable, and no fusion of this element to Meckel’s cartilage was ever observed (that is, the intramandibular joint appears unaffected by a loss of edn1 or ednra, arrows in a′ and b′). The ceratohyal (ch) was highly reduced in both perturbations. The branchial arch skeleton (comprising pharyngeal arches 3-6), though slightly reduced, maintained its overall shape and structure more robustly relative to PA1 and PA2 derivatives (for example, Meckel’s and the ceratohyal). Ventral views with anterior to top in a–c, dissected views in a′ and b′, red dotted line in a′ and b′ indicates a cut made during dissection through the palatoquadrate. Scale bar in a represents 500 μm and applies to b and c. a′ and b′ are not to scale with each other. d–g, dlx3.S and hand2.L expression are reduced in ΔednraL+S (red arrowheads; outline) at st. N.F.33 (n = 3/7 and n = 5/8 injected individuals for each gene, respectively). Lateral views with anterior to left. Scale bar in d represents 100 μm and also applies to e–g, Gray and white dotted lines in f and g are examples of the head size and hand domain size measurements (respectively) graphed in h. h, Quantifying hand2.L ventral expression domain to head size ratio (see panels f and g for examples) reveals a significant decrease in the relative X/Y lateral size of the hand2.L domain (n = 8 and n = 16 left/right halves of 4 WT and 8 Δedn3 individuals, respectively, Student’s one-sided t-test P = 0.000803, Cohen’s d = 1.378, df = 11 [adjusted to match the number of animals]; see Methods). Box plots show all points and delineate all quartile thresholds; medians are indicated with a horizontal line. i–l, Genotyping examples of Δedn1.L+S (j, l [top]) and Δednra.L+S (k, l [bottom]) larvae. The alleles shown in l are derived from the animals pictured in j and k (for each gene, respectively). Target sites are shown in orange with a red PAM. i–k show ventral views with anterior to the left. Pink and purple nucleotide strings indicate inserted sequences that are also observed at the endogenous locus near the lesion on the forward strand (underlined nucleotide strings). Red nucleotide strings represent insertions without an obvious source. Insertions are stacked inside of each lesion on the 5′ end.
Extended Data Fig. 5 Petromyzon marinus Δednrb and Δednra+b phenotypes and genotyping.
For detailed quantification information, see Supplementary Tables 1, 2, 4, and Methods section ‘Statistics and reproducibility’. a, Left lateral images of Δednrb at st. T30. n = 40/42 and n = 177/403 injected individuals for sgRNA2 and sgRNA3, respectively. 100% mutant alleles were returned for the indicated individual. Target site for sgRNA3 is shown in yellow with a purple PAM. Four example alleles are shown from the indicated individual. An insertion from the reverse strand is shown in green; its endogenous ‘source’ is underlined. The insertion is stacked inside of the lesion on the 5′ end. Both scale bars represent 500 μm. b, Alcian blue staining reveals slight skeletal disruptions in Δednrb at st. T30 (red arrowheads) n = 5/18 Δednrb individuals. c, Genotyping examples of Δednra+b at st. T30 that were all found to harbour a majority of mutant alleles (>75%), n = 32/44 injected animals displayed a phenotype similar to those specimens pictured. A summary of the alleles found in the third individual are shown, which returned 100% mutant alleles. Target sites are shown in orange with a red PAM. Purple (forward) and green (reverse) nucleotide strings indicate insertions of sequences that are also observed at endogenous loci near the lesion (underlined nucleotide strings). Red nucleotide represents an insertion without a single obvious source. Insertions are stacked inside of each the lesions on the 5′ end. Scale bar in c represents 500 μm. d, HuC/D immunohistochemistry reveals only some slight defects in specific cranial ganglia, namely the opV and epibranchial ganglia (n1-5). The white dotted boxes in the top left lateral images are shown in greater detail below, as indicated for each treatment. Abbreviations: all, anterior lateral line; g/all, geniculate/anterior lateral line (fused); mmV, maxillomandibular trigeminal; n1-5, nodose 1-5; opV, ophthalmic trigeminal; p, petrosal; pll, posterior lateral line. Scale bar represents 20 μm and applies to both the WT and Δednra+b enlargements. n = 6/6 Δednra+b individuals showed a similar phenotype. e, foxD-A (left lateral, st T24) and soxB1b (dorsal view, anterior to right) ISHs show DRGs still express these genes at larval stages in Δednra+b (arrowheads). n = 0/14 and n = 0/9 Δednra+b individuals showed missing or discontiguous ISH signal for foxD-A and soxB1b, respectively. f, A size analysis of WT (n = 4) versus Δednra+b (n = 6) individuals’ left side cranial ganglia confirmed a decrease in the lateral surface area of the opV (Student’s one-sided t-test P = 0.00762, Cohen’s d = 1.42, df = 9) and n1-5 (Student’s one-sided t-test P = 0.00120, Cohen’s d = 1.625, df = 9), but not the mmV (Student’s one-sided t-test P = 0.276, df = 9), g/all (Student’s one-sided t-test P = 0.189, df = 9), p (Student’s one-sided t-test P = 0.289, df = 9), nor the pll (Student’s one-sided t-test P = 0.212, df = 9). g, Counting the number of anterior dorsal root ganglia (as visualized by HuC/D IHC, from the first somite to the anterior boundary of the yolk) in WT (n = 7) versus Δednra+b (n = 7) individuals revealed no significant change in their number (Student’s one-sided t-test P = 0.129, df = 13). Box plots show all points and delineate all quartile thresholds; medians are indicated with a horizontal line.
Extended Data Fig. 6 Petromyzon marinus Δdlx genotyping post-ISH and alcian blue staining.
For detailed quantification information, see Supplementary Tables 2, 4, and Methods section ‘Statistics and reproducibility’. a, Examples of post-ISH genotyping for each dlx locus targeted for mutagenesis at st. T26.5. ΔdlxA n = 16/51, ΔdlxD n = 16/50, ΔdlxA+C+D n = 14/17 individuals produced perturbed lecticanA (lecA) ISH signals (as pictured). Gene, target site, and measured frequency of indel alleles are all indicated in the figure for each individual. Forward strand target sites are shown in yellow with a purple PAM, reverse strand target sites are shown in orange with a red PAM. Purple and green nucleotide strings represent insertions that reflect endogenous sequence in the forward and reverse orientation, respectively, near the lesion (underlined nucleotide strings). Red nucleotide strings represent insertions without an obvious source. Insertions are stacked inside of each the lesions on the 5′ end. All ISH images are to scale with each other. b, Alcian blue staining at st. T30 reveals truncations and gaps in the intermediate head skeleton (arrowheads), and occasional fusions of branchial arches (arrows) in Δdlx lampreys. ΔdlxA n = 4/61, ΔdlxD n = 5/34, ΔdlxA+C+D n = 11/35 individuals displayed missing, discontiguous, and/or fused cartilages in the pharynx (as pictured). Scale bar in b represents 100 μm.
Extended Data Fig. 7 Xenopus laevis Δedn3 pigmentation phenotype and genotype summary.
For detailed quantification information, see Supplementary Tables 1, 4, and Methods section ‘Statistics and reproducibility’. a–h, Δedn3.L+S (a, c, e–h) have reduced neural crest-derived pigment cells (including at least melanophores and iridophores) relative to WT (b, d), n = 31/71 injected individuals displayed a > 50% reduction in pigmentation, or in the case of a, e–g, The uninjected half of the specimen (these animals were injected unilaterally at the 2 blastomere stage, a′ shows the lineage tracer GFP [coinjected as mRNA], a″ shows an overlay of a and a′), n = 27/69 animals displayed a > 50% reduction in pigment on the injected half of the animal. As expected, pigmentation loss in the eye was never observed (because eye pigmentation is not derived from the neural crest), and the black coloration of the claws always remains, which is also observed in Xenopus tyrosinase mutants (Yonglong Chen, personal communication). All images show dorsal views, anterior to top in a, d–h, and to the right in b and c. Scale bar in a represents 1mm and applies to a′ and a″. Scale bar in b represents 5 mm and applies to c. Scale bar in d also represents 5 mm and applies to e–h. i, genotyping of a leucistic tadpole revealed a high rate of mutant alleles across both the ‘long’ and ‘short’ homeologues. Target sites are shown in yellow with a purple PAM site.
Extended Data Fig. 8 Xenopus laevis Δedn3 peripheral nervous system in larvae and subadult frogs.
For detailed quantification information, see Supplementary Tables 1, 2, 4, and Methods section ‘Statistics and reproducibility’. a, b, phox2a expression at st. N.F.41 in WT (a) and Δedn3 (b) larvae. Δedn3 larvae have no consistent defects or reductions in the epibranchial ganglia (arrows) or presumptive enteric nervous system precursors (regions within dashed boxes are shown enlarged and with enhanced contrast in a′ and b′), n = 0/4 Δedn3 individuals displayed a severely reduced ISH signal. Scale bar in a represents 200 μm and applies to b. c, d, Neurofilament immunohistochemistry at st. N.F.48 on WT (c) Δedn3 (d) larvae. Despite severe reductions in pigment cells on the gut and in the skin, Δedn3 larvae show no obvious defects in the cranial nerves (CNs), dorsal root ganglia (DRG), posterior lateral line (PLL), or vagal nerve (VN) n = 0/8 Δedn3 individuals displayed missing or overtly mis-patterned cranial nerves. Scale bar in c represents 500 μm and applies to d. e, Despite heavy pigmentation loss, Δedn3 tadpoles show no significant change in the number of dorsal root ganglia present at st. N.F.48 (as visualized by DAPI counterstain on Neurofilament IHC specimens, see Methods), n = 6 and n = 8 left/right halves of 3 WT and 4 Δedn3 individuals, respectively, Student’s one-sided t-test P = 0.381, df = 6 (adjusted to match the number of animals). f–p, Images of dissected and prepared subadult frog guts. f, A brightfield image of a dissected WT frog gut, illustrating the approximate locations of the assays pictured in panels g–p (boxes not to scale). A white dotted line indicates the boundary between the small and large intestine. Scale bar represents 1 mm. g, h, Cross sections of small intestines dissected from WT (g, g′) and Δedn3 (h, h′) subadult frog guts. g′ and h′ show HNK-1 immunohistochemistry. Though they lack pigmentation within the mucosa (compare g to h, lumen labelled lu), Δedn3 frogs have no overt defects in the myenteric (my) or submucosal (sm) plexuses of the small intestine (compare g′ to h′) n = 0/4 tissue pieces derived from 4 subadult frogs showed HNK-1 signal reduction, while n = 4/4 showed a reduction in pigmentation. Scale bar in g represents 100 μm and applies to h. i, j, Brightfield images of the external surface of the dissected large intestine of WT (h) and Δedn3 (i) subadult frogs. Δedn3 frogs lack pigmentation on this part of the gut (both melanophores and iridophores), n = 4/4 subadult frog guts displayed a >50% reduction in pigmentation. Anterior to top. Scale bar in i represents 200 μm and applies to j. k–n, Optical Z plane sections on flat-mounted large intestines from WT (k, m) and Δedn3 (l, n) frogs after HNK-1 immunohistochemistry, visualized at the plane of the myenteric (k, l) and submucosal (m, n) plexuses. The myenteric plexuses of Δedn3 frogs is largely normal (compare k to l), while the submucosal plexuses lack the ganglia seen in WT frogs (arrowheads, compare m to n), n = 4/4 subadult frogs displayed regions with low submucosal plexus density, see panel q for quantification. Scale bar in k represents 50 μm and applies to k–n. o, p, Haematoxylin and eosin (H&E) staining on transverse 7 μm-thick paraffin sections of the large intestine of WT (o) and Δedn3 (p) frogs. Δedn3 frogs have an excess of goblet cells (arrowheads) compared to WT as seen in mammalian Edn3/Ednrb mutants39, n = 3/3 subadult frogs showed dense increases in goblet cells. Lumen to top. q, Comparing n = 4 WT and n = 7 pieces of dissected lower intestines from 3 and 4 WT and Δedn3 frogs, respectively, reveals a significant reduction in the number of submucosal ganglia per mm2 in Δedn3 (Student’s one-sided t-test P = 0.00646, Cohen’s d = 1.47, df = 6 [adjusted to match the number of animals]). Box plots show all points and delineate all quartile thresholds; medians are indicated with a horizontal line.
Extended Data Fig. 9 Petromyzon marinus ΔednA and ΔednE phenotype and genotype summary.
For detailed quantification information, see Supplementary Tables 1, 4, and Methods section ‘Statistics and reproducibility’. a–d, Examples of genotyped material and the loci returned for each specimen. Gene, target site, and measured frequency of indel alleles are all indicated in the figure for each individual. ednA sgRNA1 n = 22/67, ednA sgRNA3 n = 29/38, ednE sgRNA2 n = 70/73, ednE sgRNA3 n = 38/51 injected individuals showed similarly severe phenotypes as those pictured for each gene. Forward strand target sites are shown in yellow with a purple PAM, reverse strand target sites are shown in orange with a red PAM. Green nucleotide string indicates an insertion that is observed at the endogenous locus in the forward orientation near the lesion (underlined nucleotide string). Red nucleotide is an insertion without a single obvious source. Insertions are stacked inside of each the lesions on the 5′ end. Scale bars represent 500 μm. e, ednA, -C, and -E sgRNAs were coinjected, and the material was subjected to soxE2 ISH at st. T26.5.
Extended Data Fig. 10 ednr and edn synteny and phylogeny.
a, Synteny of ednr genes. All ednr loci are shown for chicken (G. gallus), human (H. sapiens), and sea lamprey (P. marinus). Red and blue boxes indicate genes or groups of genes that were only observed at the ednra or ednrb loci, respectively, across species. Chicken and human information is after Braasch and Schartl5. Sea lamprey genomic information is derived from the germline genome27. b, Amino acid tree made using the Maximum Likelihood method on a ClustalW alignment, derived from a subset of sequences used in Square et al.21. Bootstrap scores (n = 100) are indicated at each node. See Supplementary Table 5 for accession numbers. c, Synteny analysis of edn loci and their surrounding hivep and phactr genes. Most vertebrate edn genes are located between hivep and phactr paralogues, which were previously used by Braasch et al.6 to determine the relationships of jawed vertebrate edn genes. We found that sea lamprey edn genes are also associated with hivep and/or phactr genes in most cases, and used the sequence similarity of their predicted gene products to infer the phylogeny of the edn locus. d, e, Hivep (b) and Phactr (c) phylogenies were created by applying the Maximum Likelihood tree building method to ClustalW alignments of amino acid sequences. Concatenated Hivep and Phactr sequences were also used to generate a phylogenetic tree but the results were largely the same as the Phactr tree, but with lower confidence values at some nodes. Bootstrap scores (n = 100) are indicated at each node. See Supplementary Table 5 for sequence information and accession numbers.
Extended Data Fig. 11 Phenotypes of larvae injected with 22 different negative control sgRNAs.
Embryos injected with an untargeted negative control sgRNA + Cas9 had normal morphology at st. T30. Embryos injected with 21 different sgRNAs targeting other developmental regulators expressed in the larval lamprey head resulted in a range of different phenotypes at st. T30 and st. T26.5. Heart oedema was not a feature of the mutant phenotype of most sgRNAs. See figure for larvae numbers. These results confirm that the phenotypes described by this work are not non-specific side effects of the CRISPR/Cas9 method in the sea lamprey.
Supplementary Table 1 | sgRNA and genotyping primer sequences and frequencies of severe mutant phenotypes for genes mutagenized in this study.
Supplementary Table 2 | Numbers of sgRNA/Cas9-injected embryos and larvae displaying abnormalities revealed by ISH, IHC, and histological staining.
Supplementary Table 3 | Statistical analyses of mutant phenotypes observed in embryos and larvae injected with different sgRNAs.
Supplementary Table 4 | Numbers of Indel alleles sequenced in genotyped severe mutant individuals.
Supplementary Table 5 | GenBank accession numbers and UCSC Petromyzon marinus Genome Browser intervals used to derive amino acid sequences used for phylogenetic analyses.
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Square, T.A., Jandzik, D., Massey, J.L. et al. Evolution of the endothelin pathway drove neural crest cell diversification. Nature 585, 563–568 (2020). https://doi.org/10.1038/s41586-020-2720-z
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