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Fig. 1.
A single founder mutant embryo was raised to adulthood. (A) The founder mutant was photographed just as it was about to complete metamorphosis, which at this stage is indistinguishable from an albino froglet. (B) The founder mutant as an adult (a wildtype adult is shown in B′ for comparison). The white dotted square region is enlarged in the black dotted square (B′) where one can recognize that the formation of melanin is partially restored. (C) Eggs, which are less pigmented than wildtype (C′), were obtained from the adult in (B).
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Fig. 2.
Characteristic features of no privacy mutant. (A–F, E′) The same two sibling F2 embryos derived from an intercross of heterozygous F1 mutant carrier frogs (stock 534) were observed from stage 39/40 to stage 50 as indicated in the panels. (A–E) Side views of the mutant (top) and wildtype (bottom) during development. (E′) Ventrolateral view of the same embryos shown in (E), illustrating that the mutant (right) has a see-through belly due to lack of pigmented iridophores. Dorsal view (F, left: wildtype; right: mutant) and ventrolateral view (F′, mutant only) of the same embryos as seen in (A–E, E′) at stage 50. Dorsal (G) and lateral (G′) views of another mutant embryo and lateral view (H) of another wildtype embryo at stage 53, both of which are siblings of those above, where in white-dotted circles, the forelimbs, and in white-dotted ovals, the hindlimbs, are marked. The panel G′ was composed of two different focal planes of the same embryo.
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Fig. 3.
Appearance of visceral organs of no privacy and wildtype froglets. (A, B) Ventral view of outer surface of the peritoneum. (C, D) Ventral view of the liver (l), stomach (s), and intestine (i). (E, F) Dorsal view of the kidneys (k) and lungs (lu). (A, C, E) no privacy, (B, D, F) wildtype.
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Fig. 4.
Double mutant of nop and mip. (A) Phenotypically nop. (B) Phenotypically wildtype. (C) Phenotypically nop;mip double mutant. (D) Phenotypically mip. White arrows indicate normal gut-coiling pattern, which is clearly seen in nop background (A) but invisible in wildtype background (B). Red arrows indicate mirror-image gut-coiling pattern due to mip mutation, which is clearly seen in nop background (C) but not in wildtype background (D). The drawings in (A) and (C) indicate respectively wildtype gut coiling (right coil origin, counter-clockwise coil) pattern and mip gut coiling (left coil origin, clockwise coil) pattern.
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Fig. 5. Expression of markers for melanin-producing cells and neural crest cells. Gene expression in embryos at stage 33/34 (A, B) and stage 18 (C, D) was examined by whole-mount in situ hybridization with probes as indicated in the figures. Embryos were bleached after in situ hybridization in (A, B), whereas embryos in (C, D) were not bleached.
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Fig. 6.
Maternal effect of nop mutation leads to otolith defect. Dorsal views, at stage 46, of nop (A) and wildtype (B) embryos derived from the heterozygous mutant parents and nop (C) and wildtype (D) embryos derived from the same heterozygous male as used for (A) and (B) and homozygous female. Only when the nop mutant is derived from a homozygous female are otolith defects seen (white arrows in white-dotted circles) as compared to normal otoliths (black arrows in black-dotted circles). Variation of otolith defects (indicated by white arrows) are seen in nop (E–H) compared to the wildtype (I). (Br) Brain, (u) Utricle, (s) Saccule, (Ov) Otic vesicle.
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Fig. 7.
Examination of expression in nop of genes assayed in monitoring otolith patterning phenotypes in other X. tropicalis ear mutant. Expression was examined in embryos at stage 33/34 (A–D′) and stage 18 (E, F) by whole-mount in situ hybridization with probes as indicated in the figures. (A–D′) show only the otic vesicle region (each image is 200 µm×200 µm). None of the embryos shown were bleached following in situ hybridization.
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Fig. 8.
nop gene has a mutation in the hps6 gene. (A) Schematic representation of the X. tropicalis genome (v.9.0) containing the nop locus, showing the locations of critical SSLP markers to identify the nop locus. The red circled gene model denotes hps6 and the blue circled gene model denotes hps1. (B) Schematic representation of the BAC clone, ISB1-52K13, containing the hps6 gene. Wildtype and nop mutated sequence of the hps6 gene are shown below the BAC. The numbers are positions relative to the ORF start site as 1. The embryos show the phenotypes associated with BAC clone injection. The top embryo is an uninjected nop embryo; the middle embryo was rescued with a wildtype BAC injection; and the bottom embryo was not rescued when injected with a BAC containing the nop mutation.
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Fig. 9.
TALEN-mediated targeted mutagenesis of hps6 shows a phenotype indistinguishable from nop. (A) Comparison of nop lesion and mutations (δ8 and δ10) found in TALEN F1 frogs, where TALEN target sites are shown in orange on the wildtype sequence. The numbers indicate positions relative to the translation start site. (B) Dorsal (upper panels) and ventral (lower panels) views of tadpoles at stage 47/48 showing different pigmentation patterns due to different mutation backgrounds. Embryos marked by a, b, and c are sibling offspring of an F1 pair and d is from another F1 pair. Genotypes of those embryos are shown between dorsal and ventral panels. The descriptor “-†for tyr, refers to a 1-bp deletion mutant as previously described ( Nakajima et al., 2012). (C) Images of left eyes of embryos a–d shown in (B). (D) Images of right otic vesicles of embryos a–c shown in (B). Arrows indicate otoliths, where hps6 mutants (b and c) have defects as seen in nop ( Fig. 6F). A scale bar in image “a†of (C) is 0.1 mm and applies to all images in (C) and (D). We observed more than 20 embryos for hps6 single or hps6;tyr double mutants with consistent results, where the genotype for the hps6 mutation could be either δ8/δ10 or δ10/δ10.
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Fig. 10.
Further characterization of a later generation of nop. (A, B) A different pigmentation mutation additively affects the nop pigmentation phenotype. A double mutant for nop and an axanthic gray color mutant line ICB (A) and a nop single mutant (B) (which could be heterozygous or wild-type at the ICB locus) in stock 614. We obtained offspring of this population and compared eye size with wildtype. (C) Illustration of the axis and boundary used for eye diameter measurement. (D) Box-and-whisker plot representing the distribution of eye size in wildtype (+/+) and nop (−/−) tadpoles. The middle line shows the median value, with the box representing the 25th and 75th percentiles and the whiskers extending to the minimum and maximum observed values. The asterisk (*) indicates a statistically significant difference between two populations. Statistical comparison of wildtype (n=10, mean=338 µm, SD=18 µm) and nop (n=10, mean=322 µm, SD=11 µm) eye diameters yields a p-value of 0.03 (two-tailed t-test).
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Fig. 11.
Evaluation of eye size and RPE morphology in a nop line with a relatively normal morphology. Embryos from the line described in Fig. 4 were used for these studies. (A) Box-and-whisker plot representing the distribution of eye diameter of tadpoles displaying the wildtype (n=14, mean=357 µm, SD=19 µm) or nop (n=16, mean=354 µm, SD=15 µm) pigmentation phenotype (regardless of whether the heterotaxy phenotype is mip or wildtype). The middle line shows the median value, with the box representing the 25th and 75th percentiles and the whiskers extending to the minimum and maximum observed values. Statistical comparison of the two populations results in a p-value of 0.58 (two-tailed t-test), suggesting no statistically significant difference between the two populations. Heads from wildtype (B) and nop (C) stage 47 embryos were fixed, embedded in plastic and then sections prepared to compare the morphology of the RPE in the wildtype and mutant. One can see that the eye size overall is essentially the same in wildtype and mutant, as shown statistically in (A), and that the morphology of the lens (L) and neural retina (NR) appears unchanged in the mutant. The pigment in the retinal pigment epithelium (RPE) appears reduced and disorganized in the mutant, and in high magnification images (B′) and (C′) it is clear that while overall pigmentation is reduced in the mutant there are also clumps of melanophores, or multi-melanophores, (white arrowheads) distributed throughout the RPE.
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Fig. SI. Variation of eye defects seen in nop in the early generation of the line. (A-C, D) Wildtype eyes, (A'-C', E, F) nop eyes. At stage 42, eyes of nop developed normally except for hypopigmentation of the nop RPE (A') compared to wildtype (A). (B', C', F) Examples of severe defects seen in some nop animals. (E) nop froglet having relatively normal looking eyes. Histology (A-C, A'-C') was performed as described (Nakayama et al., 2015).
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Fig. S2 Gynogenetic centromere linkage for low-resolution linkage assignment
Genomic DNAs from 17 gynogenetic nop mutant (lanes: M) or wildtype (lanes: W) embryos from a mutant carrier female were pooled and subjected to SSLP (Simple Sequence Length Polymorphism) analysis. The centromeric SSLP markers specific to each chromosome (Chr) were identified in advance by using haploid embryos from the same female (lanes: SSLP) as described below. In Chr7 lanes, one SSLP (smaller bands) was specifically detected in pooled genomic DNAs of gynogenetic diploid mutants (red box). Although Chr8 also showed apparent linkage (red star), Chr7 appeared most promising and we investigated that further, confirming with additional markers a narrow window with the variant allele.
Note: The centromeric markers used for Chr1, 9, and 10 were described previously (Khokha et al., 2009). For other chromosomes, since the markers described previously (Khokha et al., 2009) did not show polymorphisms, we searched the database (http://tropmap.biology.uh.edu/) for other markers that were genetically located close to previously known markers (respectively for Chr2-8, 0.32 cM, 0.027 cM, 0.104 cM, 0.129 cM, 0.028 cM, 0.131 cM, 0.227 cM apart from) and tested for polymorphisms. As a result, we used the following markers, Chr2 (042F05); Chr3 (119A02); Chr4 (097A03); Chr5 (074D05); Chr6 (073D02); Chr7 (017F12); Chr8 (045E03). Among those, after we identified the nop mutation responsible for mutant phenotype, we found that 119A02 had been incorrectly assigned to Chr3 in the database. This seemed to happen because the database was based on an earlier generation of genome assembly, which, retrospectively, was found to contain misassembled scaffolds. The result with the Chr3 marker (119A02) therefore became inconclusive, but does not affect other results throughout the text since we could confirm the mutation lesion was on Chr7 by more refined mapping methods.
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