Genetics of white color and iridophoroma in "Lemon Frost" leopard geckos

Abstract

Coloration patterns promote survival and reproductive success in the animal kingdom. Despite their importance, wide gaps exist in our understanding of the genetic and evolutionary mechanisms that underpin them. The leopard gecko, Eublepharis macularius, is a popular companion animal, and displays a variety of coloration patterns. We investigated a spontaneous semi-dominant mutation, known as "Lemon Frost", that causes extensive white color in leopard gecko skin. Although "Lemon Frost" individuals are aesthetically appealing, more than 80% of them develop tumors of white color (i.e., iridophoroma) 0.5 to 5 years after birth. To identify the gene that regulates white color and is likely also responsible for the iridophoroma, we genotyped 220 animals, including 33 homozygous mutants, with short-read sequencing. We used synteny, linkage analysis and homozygosity mapping to localize the mutation to a strong candidate gene, SPINT1, a tumor suppressor previously implicated in human skin cutaneous melanoma (SKCM) as well as in over-proliferation of epithelial cells in mice and zebrafish. Our work establishes the leopard gecko as a tractable genetic system and suggests that a tumor suppressor in melanocytes in humans can also suppress tumor development in iridophores in lizards.

Full text

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1
Genetics of white color and iridophoroma in “Lemon Frost” leopard geckos 2
3
Longhua Guo1,*, Joshua Bloom1, Steve Sykes2, Elaine Huang3, Zain Kashif1, Elise Pham1, 4
Katarina Ho1, Ana Alcaraz4, Xinshu Grace Xiao3, Sandra Duarte-Vogel5, Leonid Kruglyak1,* 5
6
1Department of Human Genetics, Department of Biological Chemistry, Howard Hughes Medical 7
Institute, University of California, Los Angeles, CA 90095, USA 8
2Geckos Etc. Herpetoculture, Rocklin, CA 95765, USA 9
3Department of Integrative Biology and Physiology, University of California, Los Angeles, CA 10
90095, USA 11
4College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91711, 12
USA 13
5Division of Laboratory Animal Medicine, David Geffen School of Medicine, University of 14
California, Los Angeles, CA 90095, USA 15
16
17
(*) To whom correspondence should be addressed: longhuaguo@mednet.ucla.edu; 18
lkruglyak@mednet.ucla.edu 19
20
Abstract 21
Coloration patterns promote survival and reproductive success in the animal kingdom. Despite 22
their importance, wide gaps exist in our understanding of the genetic and evolutionary 23
mechanisms that underpin them. The leopard gecko1, Eublepharis macularius, is a popular 24
companion animal, and displays a variety of coloration patterns. We investigated a spontaneous 25
semi-dominant mutation, known as “Lemon Frost”, that causes extensive white color in leopard 26
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2
gecko skin. Although “Lemon Frost” individuals are aesthetically appealing, more than 80% of 27
them develop tumors of white color (i.e., iridophoroma) 0.5 to 5 years after birth. To identify the 28
gene that regulates white color and is likely also responsible for the iridophoroma, we 29
genotyped 220 animals, including 33 homozygous mutants, with short-read sequencing. We 30
used synteny, linkage analysis and homozygosity mapping to localize the mutation to a strong 31
candidate gene, SPINT12,3, a tumor suppressor previously implicated in human skin cutaneous 32
melanoma (SKCM) as well as in over-proliferation of epithelial cells in mice and zebrafish4-16. 33
Our work establishes the leopard gecko as a tractable genetic system and suggests that a 34
tumor suppressor in melanocytes in humans can also suppress tumor development in 35
iridophores in lizards. 36
37
Introduction 38
Color-producing cells17-21 contribute to animal coloration and patterns. Some cells, such as 39
melanocytes, produce pigments chemically. Others, such as iridophores, produce colors 40
structurally by making crystal platelets22-25. Iridophores are not present in mammals, but are 41
widespread in insects, fish, birds, amphibians and reptiles. Different types of iridophores can 42
lead to different colors, including blue26,27 and white28. There have been few molecular genetic 43
analyses of the regulation of chromatophores in cells other than melanocytes. A recent study 44
found that endothelin signaling regulates iridophore development and proliferation in zebrafish29. 45
In mammals, this pathway is required for melanocyte development30, suggesting that signaling 46
pathways conserved in evolution can be adapted to regulate different types of chromatophores. 47
48
Many reptile species (e.g., geckos, chameleons, snakes) are bred in captivity as companion 49
animals, and breeders have established morphs with unique colors and patterns18. The 50
inheritance of different color morphs is usually carefully documented by breeders. The common 51
leopard gecko, Eublepharis macularius, is an especially attractive model to study the molecular 52
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regulation of coloration because dozens of color and pattern morphs have been established 53
over the past 30 years of selective breeding. These morphs either intensify a particular color 54
(Supplementary Figure1 A-I) or rearrange coloration patterns (Supplementary Figure1 J-L). A 55
draft leopard gecko genome assembly has been published, containing 2.02 Gb of sequence in 56
22,548 scaffolds, with 24,755 annotated protein-coding genes1. Embryonic development in ovo 57
and blastema-based tail regeneration have also been staged and documented in great detail31-33. 58
Here, we took advantage of these established resources and used quantitative genetics to gain 59
insight into the molecular regulation of white color in leopard geckos. 60
61
Results 62
63
The Lemon Frost allele is a spontaneous semidominant mutation 64
A spontaneous mutation occurred in a female hatchling from a cross between two wildtype 65
leopard geckos. This mutation increased the white color of the leopard gecko, resulting in 66
brightened white and yellow colors. This unique color morph was named Lemon Frost34 (Figure 67
1). A male leopard gecko carrying the lemon frost (lf) allele, Mr. Frosty (Figure 1B), was crossed 68
to 12 female leopard geckos of different genetic backgrounds. The F1 progeny, which were 69
heterozygous for the lf allele, were backcrossed to the same maternal lines or intercrossed to 70
establish a colony of more than 900 animals (Supplementary Figure 2). Homozygous F2 71
intercross progeny were named super Lemon Frost (Figure 1C). These homozygous mutants 72
have an accentuated color phenotype and thickened skin, which is most apparent in their 73
eyelids (Figure 1C, red arrow). Heterozygous Lemon Frost animals were also crossed to 74
another mutant, Blizzard, which is light yellow without other colors or patterns (Figure 1D). The 75
homozygous Blizzard progeny carrying the lf allele displayed excessive white color in their 76
heads and trunks, which brightened Blizzard’s yellow color (Figure 1E). The lf allele also 77
increased white color in the retina (Figure 1E). The segregation pattern of Lemon Frost in 78
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pedigrees is consistent with single-locus Mendelian inheritance (Figure 1F-H). The lf allele is 79
semidominant, as homozygous mutants have more pronounced phenotypes than do 80
heterozygotes (Figure 1B-C and F-H). 81
82
The lemon frost allele leads to iridophoroma, with potential metastasis in homozygous 83
animals 84
Heterozygous Lemon Frost mutants were recently reported to develop iridophoroma34, a tumor 85
of iridophores. Histopathological examination of the skin samples from homozygous mutants, 86
with accentuated phenotypes, showed large solid sheaths of round to polygonal neoplastic cells 87
that efface and expand the normal tissue architecture (Supplementary Figure 3). The cells have 88
abundant cytoplasm with bright brownish intracytoplasmic pigment. The nuclei are eccentric and 89
vary from round to fusiform. The white tumor masses stain dark with Hematoxylin and Eosin 90
(H&E), and remain brightly reflective under dark-field illumination (Supplementary Figure 4A,B), 91
consistent with their nature as iridophores26,35-38. Imaging with Transmission Electron 92
Microscopy (TEM) showed that the lf allele led to both increased numbers of neoplastic 93
iridophores and increased production of reflective platelets within each iridophore39 94
(Supplementary Figure 4C). In addition to skin, other affected organs in homozygous mutants 95
include liver, eye, and muscle. The interpretation of the widespread neoplastic nodules is that 96
the tumors are malignant iridophoroma. 97
98
More than 80% of both male and female animals carrying the lf allele developed white tumors 6 99
months to 5 years after birth. The tumors manifest as patches of white cells in the skin, which 100
are most evident on the ventral side of the animal (Figure 2A). The tumor skin can be severely 101
thickened and leathery (Figure 2B, Supplementary Figure 3). It is resistant to liquid nitrogen 102
freezing, or to Dounce homogenization, making RNA extraction infeasible. In severe cases in 103
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heterozygous mutants, the tumors develop into skin protrusions (Figure 2C, left), which contain 104
dense white masses (Figure 2C, right). Tumors cover a greater fraction of the skin of 105
homozygous mutants. Surprisingly, these tumors rarely develop into skin protrusions as in 106
heterozygous animals. Instead, they manifest as well-demarcated, white, thickened patches on 107
the ventral skin (Figure 2A), thickened layers of white masses all over the dorsal skin (Figure 108
2B), white, multifocal, variably sized, well-demarcated nodules in the liver, and patches of white 109
cells in the oral cavity (Figure 2D). 110
111
Linkage and association analysis in a breeding pedigree 112
To identify the genetic locus that regulates white color and tumor growth in Lemon Frost 113
mutants, we used restriction site-associated DNA sequencing (RAD-Seq) to genotype 188 114
animals from the breeding pedigree (Figure 3, Supplementary Figure 2), including 33 super 115
Lemon Frost (lf/lf), 116 Lemon Frost (lf/+), and 39 wild-type (+/+) individuals. We identified a 116
total of 14,857 variants covering 2,595 scaffolds of the genome assembly. To map the Lemon 117
Frost locus, we tested the effect of allelic dosage at each marker on white coloration of the 118
geckos in a standard semi-dominant association mapping framework, accounting for population 119
structure through the use of marker-based relatedness. We used a p-value threshold of 7.09e-5 120
(Methods) to control the false positive rate at 1%. Forty-eight markers on 31 scaffolds were 121
significantly associated with white coloration (Supplemental Table). The top two association 122
signals corresponded to scaffolds 6052 and 996. 123
124
Synteny analysis and homozygosity mapping 125
Because the gecko genome assembly is highly fragmented, we used synteny to examine 126
whether the 31 scaffolds associated with coloration belong to a single genomic interval. We 127
compared the gecko scaffolds to homologous regions of the most closely related species with 128
chromosome-scale genome assemblies: chicken40 and human41. We found that 17 out of 22 129
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scaffolds that have synteny information (including scaffolds 6052 and 996) correspond to one 130
region on chicken chromosome 5 and human chromosome 15 (Figure 3A-C, Supplemental 131
Table). The 28 markers on these 17 scaffolds are in linkage disequilibrium (Figure 3B), which 132
decays with distance when markers are ordered by synteny (Figure 3B). These results indicate 133
that a single genomic region is associated with the Lemon Frost phenotype, as expected for a 134
new mutation with a Mendelian segregation pattern. 135
136
To narrow down the location of the causal gene within this genomic region, we used whole 137
genome sequencing and homozygosity mapping. We pooled DNA from 25 super Lemon Frost 138
genomes (lf/lf), 63 Lemon Frost genomes (lf/+), and 71 wildtype geckos (+/+) and sequenced 139
each pool to 30x coverage. We reasoned that the lf mutation in Mr. Frosty and its flanking 140
variants should form a haplotype that would be found in the super Lemon Frost pool with 100% 141
frequency, in the Lemon Frost pool with 50% frequency, and would not be seen in the wildtype 142
pool. We scanned the genome in 10 kb windows and measured the fraction of heterozygous 143
variants from Mr. Frosty that followed this expected pattern in the pools. This statistic was 144
highest for a window on scaffold 996 (Supplementary Table, Methods), the main candidate 145
scaffold from statistical mapping. The expected frequency pattern was observed for 20 of 22 146
variants in this window (630-640kb on scaffold 996). Four of the top six intervals fall in the 147
region from 570kb to 640kb on scaffold 996, with the signal decaying with distance away from 148
this region (Figure 3D,E). The linkage between this region and Lemon Frost was replicated in an 149
independent 3-generation backcross between Mr. Frosty and a Sunburst Tangerine morph 150
(Figure 4). These results indicate that scaffold 996 contains the Lemon Frost mutation. 151
152
SPINT1 is a strong candidate gene for the Lemon Frost phenotype 153
The genomic interval spanning positions 570kb-640kb on scaffold 996 contains a single gene, 154
SPINT1. SPINT1 (serine peptidase inhibitor, Kunitz type 1), also known as hepatocyte growth 155
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factor activator inhibitor type 1 (HAI-1), is a transmembrane serine protease inhibitor expressed 156
mainly in epithelial cells2,3,16. It is the only gene in the larger associated region reported to be a 157
suppressor of epithelial cell tumors in model organisms and in humans2,4-14,42. Because the 158
breeding and transmission data indicate that the lf allele arose from a single spontaneous 159
mutation, we reasoned that a mutation disrupting SPINT1 causes the over-proliferation of white-160
colored skin cells in Lemon Frost geckos. 161
162
The Lemon Frost SPINT1 allele differs from the reference genome assembly at two positions in 163
the exons, as well as at 147 positions in the introns and the 3’UTR (Supplemental Table). This 164
large number of variants is a consequence of differences in genetic background between Mr. 165
Frosty’s parents and the non-Lemon Frost individual used to generate the reference, and makes 166
it challenging to identify the causal mutation. Both differences in the coding sequence of 167
SPINT1 are synonymous. Notable differences in non-coding regions include 7 large 168
insertion/deletions (indels) in the introns and a 13-nucleotide insertion in the 3’UTR 169
(CAAGTGTATGTAT). Indels in introns and promoters of SPINT1 have been reported to lead to 170
loss of SPINT1 function in fish and mice8,9,6. 171
172
Sequencing of RNA extracted from normal gecko skin and from skin peripheral to tumors in 173
homozygous mutants confirmed that SPINT1 is expressed in this tissue (Supplemental Figure 174
5). However, we did not observe a significant difference between homozygous mutants and 175
wildtype geckos in SPINT1 mRNA levels or splicing patterns. This result suggests that the 176
putative causal mutation in SPINT1 may alter translation or protein activity, rather than 177
transcription. Alternatively, the mutation might reduce SPINT1 expression only in tumors, which 178
are refractory to RNA extraction as noted above. 179
180
Discussion 181
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Several lines of evidence support our hypothesis that a defect in SPINT1 causes iridophoroma 182
in Lemon Frost geckos. First, SPINT1 function is dosage-dependent, consistent with our 183
observation that Lemon Frost is a semi-dominant phenotype. In humans, carcinoma tissues in 184
vivo and carcinoma-derived cell lines in vitro have reduced SPINT1 on the cell membrane15,43 185
through enhanced shedding of the extracellular domain or decreased mRNA or protein 186
expression. Reduced expression of SPINT1 has been associated with a negative prognosis of 187
human Skin Cutaneous Melanoma (SKCM)4 and pancreatic ductal adenocarcinoma13. 188
Knockdown of SPINT1 expression by siRNA in cancer cell lines led to increased invasion or 189
metastasis14,15,44. Second, loss of SPINT1 function in fish and mice leads to tumor formation in 190
epithelial cells. In mice, homozygous deletion of SPINT1 leads to disrupted placental basement 191
membranes and embryonic lethality9,11. Rescued mosaic animals developed scaly skin with 192
hyperkeratinization12. Intestine-specific deletion of SPINT1 leads to increased tumor growth of 193
intestine epithelium10. Increased expression of SPINT1 in the skin abrogated matriptase-194
induced spontaneous skin squamous cell carcinoma45. In zebrafish, reduced expression led to 195
hyperproliferation of basal keratinocytes8 and enhanced proliferation of epithelial cells6. 196
Furthermore, SPINT1 deficiency was used to establish a disease model for Skin Cutaneous 197
Melanoma (SKCM) in zebrafish4. In all three studies in zebrafish, skin inflammation was 198
observed. Third, insertions in introns8,9 and promoters6 have caused loss of SPINT1 function. 199
Together with our genetic localization of the lf locus to SPINT1, these lines of evidence make 200
this gene a very strong candidate for the Lemon Frost phenotype. 201
202
Molecular genetics in reptiles is not well established due to long reproductive cycles and 203
challenges in laboratory breeding. Early work focused on careful documentation of patterns of 204
inheritance18,46. Molecular studies have examined sequence variants in a candidate 205
pigmentation gene, melanocortin-1 receptor, and their association with melanic or blanched 206
phenotypes in different species and ecological niches47-54. Recently, CRISPR-Cas9-mediated 207
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gene editing was successfully used to mutate the tyrosinase gene in the lizard Anolis sagrei55. 208
Although this species is only distantly related to the leopard gecko, this advance offers promise 209
that targeted studies of the role of SPINT1 mutations in the Lemon Frost phenotype will become 210
possible. 211
212
White iridophoroma is common in many reptile species56, including green iguanas57, captive 213
snakes58, bearded dragons59 and veiled chameleons60. The genetic causes of this phenotype in 214
these species are unknown. Most of our knowledge about molecular and cellular regulation of 215
iridophores derives from work in zebrafish27-29,61-71. Interestingly, few cases of iridophoroma 216
have been reported in zebrafish72. We found that an evolutionarily conserved gene, SPINT1, 217
regulates the proliferation of white iridophores in the leopard gecko. The tumor suppressor 218
function of SPINT1 establishes a link between iridophoroma and regulation of white coloration in 219
reptiles. Our work suggests that cancer genes can play as important a role in iridophores as 220
they do in melanocytes and melanoma73, and that Lemon Frost leopard geckos can serve as a 221
disease model to study Skin Cutaneous Melanoma. 222
223
Methods 224
225
Gecko maintenance and experimental procedures 226
227
All activities involving animals included in this manuscript were approved by the University of 228
California, Los Angeles (UCLA) Institutional Animal Care and Use Committee. Leopard geckos 229
were acquired from a commercial breeder. Housing conditions at UCLA included: room 230
temperature of 70-80 F, cage temperature of 72-95 F, room relative humidity between 30-60%, 231
and a 12:12 hours light cycle. A heating pad was provided at one side of the cage to establish a 232
temperature gradient. Animals were singly housed in polycarbonate cages with cardboard lines 233
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(TechboardⓇ) at the bottom, water was provided in bowls inside the cage, and PVC pipe pieces 234
and plastic plants were offered as environmental enrichment. Geckos were fed 2-6 fresh 235
crickets and 2-4 mealworms three times per week. 236
237
Geckos were euthanized with an intracoelomic injection of sodium pentobarbital (EuthasolⓇ) at 238
a dose of 100-200 mg/Kg. Immediately after euthanasia, a necropsy was performed, including 239
external examination, body and organ weighing, gross assessment of normal and abnormal 240
tissues, and tissue collection for histopathology processing and assessment. Normal and 241
abnormal tissues were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with 242
H&E for pathologic evaluation. 243
244
Phenotyping 245
246
Lemon Frost and super Lemon Frost phenotypes were determined according to a list of rules, 247
based on increased white color of the body, eye, and belly compared to normal wildtype animals 248
(http://www.geckosetc.com/lemon_frost_info.html). Pictures were taken for each animal to 249
document the phenotype. 250
251
Genotyping 252
253
Genomic DNA was extracted from fresh tail tips with Easy-DNA gDNA purification kit (K180001, 254
ThermoFisher), or from the saliva with PERFORMAgene (PG-100, DNAgenotek). Genomic 255
DNA extracted from saliva was further purified with ethanol precipitation before genotyping 256
assays. DNA libraries for whole genome sequencing were prepared with Nextera DNA Library 257
Prep Kit (FC-121-1031, Illumina). Libraries for RADseq were prepared according to the 258
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procedures of Adapterama III74 with few modifications. Libraries were sequenced on a HiSeq 259
3000 (Illumina). 260
261
Only scaffolds larger than 5kb in the draft genome assembly were used as a reference. RADseq 262
reads and Whole Genome Sequencing (WGS) reads were aligned to the leopard gecko draft 263
genome1 with bwa mem75. Variants for WGS were identified with GATK76. Variants for RADseq 264
were identified with Stacks77,78. All variants were filtered with VCFtools79. Only high-quality 265
variants were used in homozygosity mapping or statistical mapping (DP>=30, GQ>=30). 266
267
Transcriptome sequencing 268
269
Skin tissue samples around 6mm in diameter were taken from the ventral side of the geckos 270
after anesthetization with 1-5% isoflurane. As tumor tissues are refractory to RNA extraction, 271
flanking tumor-free tissue samples were taken for homozygous Lemon Frost animals. All 272
samples were homogenized with TissueRuptor in buffer RLT immediately after collection. 273
Lysates were immediately frozen on dry ice until all tissues were collected from animals. Then 274
all lysates were centrifuged for 5 minutes at 13,000 rpm to remove debris. Supernatants were 275
taken to fresh tubes, and mRNA was extracted according to the procedures of RNeasy Fibrous 276
Tissue Mini Kit (74704, QIAGEN). 277
278
Libraries of extracted mRNA were prepared with RNA HyperPrep kit (KAPA) and sequenced on 279
a HiSeq 3000 (Illumina). RNA-seq reads were mapped to the leopard gecko draft genome1 280
using HISAT2 with default parameters. Identification of alternative and differential splicing 281
events was performed using JuncBase80. Gene expression was compared using Sleuth81 after 282
RNA transcript abundance was quantified using Kallisto82. 283
284
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Pathology 285
286
Complete postmortem examination was performed, and representative tissue samples were 287
obtained. All tissues obtained at necropsy were preserved in 10% neutral-buffered formalin 288
solution for up to 5 days before being processed and embedded in paraffin. All tissues were 289
sectioned at 5 μm, and routinely stained with Hematoxylin and Eosin. 290
291
Statistical Mapping 292
293
Biallelic markers with minor allele frequency of less than 5% and with fewer than 10 individuals 294
called as homozygous for both the reference and alternative alleles were excluded from 295
mapping and kinship matrix construction. A kinship matrix was calculated using the function 296
A.mat with default parameters from the rrBLUP83 R package. Phenotype was encoded as 0 for 297
wild type, 1 for Lemon Frost, and 2 for super Lemon Frost. Association statistics between this 298
phenotype vector and marker genotypes were computed using the function gwas2 in the NAM84 299
R package using a linear mixed model with a random effect of kinship to control for population 300
structure. The effective number of tests was computed to be 141.1 based on the procedure of 301
Galwey et al85. A family-wise error rate significance threshold was calculated as 0.01/141.1 or 302
p<7.09e-5. 303
304
Homozygosity Mapping 305
306
Pooled animals and Mr. Frosty were sequenced to ~30x coverage on a HiSeq 3000 (Illumina). 307
Variants were identified with GATK and filtered with VCFtools. Biallelic heterozygous variants 308
from Mr. Frosty, including indels, were used as markers to localize the Lemon Frost mutation. 309
Allele ratios (AF) were calculated by dividing the read count of alternative alleles by the sum of 310
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the counts of reference alleles and alternative alleles. Variants closely linked to the Lemon Frost 311
mutation are expected to have AF between 0.4 and 0.6 in the Lemon Frost pool and in Mr. 312
Frosty, AF > 0.85 in the super Lemon Frost pool, and AF < 0.15 in the wildtype pool. The 313
number of variants meeting these criteria was counted for every 10kb genome interval. The 314
fraction of such variants among all variants heterozygous in Mr. Frosty within the interval was 315
then calculated. Intervals with fewer than 5 variants were excluded because they could not 316
provide statistically meaningful results. 317
318
Transmission Electron Microscopy 319
320
Dissected skin tissues were fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M sodium 321
cacodylate buffer overnight at 4 °C. After being washed in PBS, samples were post-fixed in 1% 322
osmium tetroxide in 0.1M sodium cacodylate, and dehydrated through a graded series of 323
ethanol concentrations. After infiltration with Eponate 12 resin, the samples were embedded in 324
fresh Eponate 12 resin and polymerized at 60°C for 48 hours. Ultrathin sections of 70 nm 325
thickness were prepared, placed on formvar-coated copper grids, and stained with uranyl 326
acetate and Reynolds’ lead citrate. The grids were examined using a JEOL 100CX transmission 327
electron microscope at 60 kV, and images were captured by an AMT digital camera (Advanced 328
Microscopy Techniques Corporation, model XR611). 329
330
Acknowledgement 331
We thank Aaron Miller, Jasmine Gonzalez, Kendall Placido and James Walter for their 332
assistance in gecko DNA collection and phenotyping. We thank members of the Kruglyak lab for 333
helpful feedbacks on the project, and Giancarlo Bruni, Stefan Zdraljevic, Eyal Ben-David and 334
Olga Schubert for helpful comments on the manuscript. We thank Chunni Zhu (Electron 335
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Microscopy Core Facility, UCLA Brain Research Institute) for her assistance in TEM sample 336
processing and imaging. We thank Jonathan Eggenschwiler for helpful discussions. 337
338
This work is supported by the Howard Hughes Medical Institute (LK) and the Helen Hay 339
Whitney Foundation (LG). 340
341
Figure legends 342
343
Fig 1 The Lemon Frost mutant of the common leopard gecko, Eublepharis macularius. (A) 344
wild type; (B) heterozygous mutant; (C) homozygous mutant, with red arrow pointing to the eye 345
lid; (D) blizzard mutant with minimal color; (E) Lemon Frost mutation (lf) on the blizzard 346
background; (F-H) segregation of the lf allele. Lemon Frost (LF) denotes heterozygotes for the 347
mutation; super LF denotes homozygotes for the mutation. All proportions are consistent with 348
expectations for single-locus Mendelian inheritance (chi-square test p > 0.1). 349
350
Fig 2 Tumor growth and metastasis in the Lemon Frost mutant. Designations are 351
homozygous mutant (lf/lf); heterozygous mutant (lf/+); wild type (+/+). (A) tumors in ventral skin; 352
(B) thick layers of white tumor cells (lf/lf) vs. normal white cells (+/+); (C) outgrowth of white 353
tumor cells (lf/+); (D) metastasis of white tumor cells in the liver and oral cavity. Red arrows: 354
white colored tumor cells. Arrowhead in B: normal white cells. 355
356
Fig 3 Localization of the Lemon Frost mutation. (A) p-value for association with white color 357
and (B) linkage disequilibrium for 28 markers syntenic to chicken chromosome 15 (red, ordered 358
by synteny), 4 markers syntenic to chromosome 5 (cyan), and 16 markers without synteny 359
information (green). (C) A schematic of the region showing synteny and gene annotation. (D) 360
Fraction of markers showing expected allele frequency pattern in pools, plotted for 10kb 361
windows along scaffold 996. The four windows with the highest fraction are marked by asterisks 362
and span the location of the gene SPINT1. Windows with fewer than 5 variants were not plotted 363
(dashed red lines). (E) Genome-wide distribution of the fraction of markers showing expected 364
allele frequency pattern in pools for all 10 kb windows. The 4 highest windows on scaffold 996 365
(red arrows) marked in D are among the 6 highest windows in the entire genome. 366
367
Fig 4 The lemon frost allele in a backcross. (A) We genotyped 7 progeny with the Lemon 368
Frost phenotype and 6 wild type progeny from the third generation of a backcross of Mr. Frosty 369
to the Sunburst line for markers in the SPINT1 region and observed a consistent inheritance 370
pattern. (B) Sequencing chromatogram of a heterozygous animal (lf/+) at an insertion marker. 371
(C) Sequencing chromatogram of a homozygous animal (+/+) at the same insertion marker. 372
373
SupFig 1 Coloration and pattern diversity of the common leopard gecko, Eublepharis 374 macularius. (A) wild type; (B) black night; (C) variant of black night; (D) granite snow; (E) gem 375
snow; (F) white knight; (G) sunburst tangerine; (H-I) variants of sunburst tangerine; (J) red 376
stripes; (K) bold stripes; (L) rainbow. 377
378
SupFig 2 Breeding pedigree of the Lemon Frost mutation. Mr. Frosty, the original carrier of 379
the spontaneous Lemon Frost mutation, was bred to 12 female geckos from different genetic 380
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15
backgrounds. F1s carrying the lf allele were bred among themselves or back to their female 381
parent, producing the second generation of animals heterozygous or homozygous for the lf 382
allele. Blue: lf/lf; green: lf/+; red:+/+. Dashed line: same individual/line. 383
384
SupFig 3 Histopathology of skin tumors. (A) Thick layers of white tumor tissue (star) 385
infiltrating white skin (arrow). (B) Skin biopsies organized and fixed in a paper roll for sectioning. 386
(C) H&E staining of the skin sections. Arrow: skin; star: infiltrated tumor mass. (D) H&E staining 387
of the skin sections showing normal skin cells and neoplastic cells (star). Neoplastic cells have 388
eccentric and condensed nuclei. 389
390
SupFig 4 Potential metastasis of iridophoroma. (A) In normal skin, cell nuclei are oval and 391
perpendicular to the skin surface. In Lemon Frost skin, cell nuclei are flat, elongated and parallel 392
to the skin, reminiscent of epithelial-to-mesenchymal transition. (B) Iridophoroma in the liver, 393
stained dark in H&E sections. In dark field imaging, iridophores are bright white. Such 394
iridophores invade blood vessels in the tissue (red arrows). (C) In TEM imaging, white tumor 395
skins in super LF are filled with abundant iridophores with excessive brightly reflective crystals 396
(Tumor). In normal skin, iridophores are much fewer and have less crystals (Normal). 397
398
SupFig 5 SPINT1 expression in gecko skin. SPINT1 mRNA reads from transcriptome 399
sequencing were aligned to the genome and visualized in IGV. Top 3 rows show samples from 400
homozygous mutants. Bottom 3 rows show samples from wild type geckos. Skin tissue adjacent 401
to the tumors was used in the mutants. Peaks mark SPINT1 exons. The last exon on the right is 402
transcribed together with the 3’UTR. 403
404
405
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Lemon Frost (LF) x Wild Type (WT)
LF WT
(33 25):
F1 LF x WT
LF WT
(55 72):
F1 LF x F1 LF
super LF WT
(7 7): :1 2
LF
D
E
F G H
A
+/+ lf/+ lf/lf
+/+; blizzard/blizzard
lf/+; blizzard/blizzard
Fig 1 The Lemon Frost mutant of the common leopard gecko,
Eublepharis macularius. (A) wild type; (B) heterozygous mutant; (C)
homozygous mutant, with red arrow pointing to the eye lid; (D) blizzard
mutant with minimal color; (E) Lemon Frost mutation (lf) on the blizzard
background; (F-H) segregation of the lf allele. Lemon Frost (LF)
denotes heterozygotes for the mutation; super LF denotes
homozygotes for the mutation. All proportions are consistent with
epectations for single-locus Mendelian inheritance (chi-square test p >
0.1).
B C
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+/+ +/lf lf/lf
+/+
lf/lf lf/lf +/+
lf/lf +/+
A
BC
D
Fig  Tumor groth and metastasis in the Lemon Frost mutant.
Designations are homozygous mutant (lflf); heterozygous mutant (lf); Ḁ̀
wild type (). (A) tumors in entral skin; (B) thick layers of white tumor
cells (lflf) s. normal white cells (); (C) outgrowth of white tumor cells
(lf); (D) metastasis of white tumor cells in the lier and oral caity. ed
arrows white colored tumor cells. Arrowhead in B normal white cells.
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0.00
0.25
0.50
0.75
1.00
markers
A B
C
Fig  Localiation of the Lemon Frost mutation. (A) p-alue for association with white color ̀
and (B) linkage disequilibrium for  markers syntenic to chicken chromosome 1 (red, ordered ̀
by synteny),  markers syntenic to chromosome  (cyan), and 1 markers without synteny
information (green). (C) A schematic of the region showing synteny and gene annotation. (D)
Fraction of markers showing epected allele frequency pattern in pools, plotted for 10kb windows
along scaffold 99. The four windows with the highest fraction ̀ are marked by asterisks and span
the location of the gene SPT1. indows with fewer than  ariants were not plotted (dashed
red lines). (E) Genome-wide distribution of the fraction of markers showing epected allele ̀
frequency pattern in pools for all 10 kb windows. The  highest windows on scaffold 99 (red ̀
arrows) marked in D are among the  highest windows in the entire genome.
D
3
4
5
6
7
markers
-log10(pval)
LD
Chicken (Chr5)
Gecko (scaffold996)
570kb 640kb
0.25
0.50
0.75
Scaffold996
ratio (candidates/background)
30kb
930kb
630-640kb
570-580kb
660kb
570-580kb
570-580kb
SPINT1
***
*
30 40
E
Frequency
0.0 0.2 0.4 0.6 0.8 1.0
0 10 20
scaffold 996
Marker ratio (candidates/background)
220kb
260kb
290kb 340kb
360kb 420kb
440kb
910kb
880kb
790kb
710kb
500kb
520kb
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Mr. Frosty (lf/+) Sunburst (+/+)
16-3652 (lf/+) Sunburst(+/+)
17-9094 (lf/+) Sunburst(+/+)
18-9835 (lf/+) Sunburst(+/+)
LF (lf/+) non-LF (+/+)
n=7 n=6
A
B C
A C A C A C A A A G G G A C A C A C A A A G G G
The insertion:
A T A C A T A C A C T T
Fig The ̀ lem rs allele in a backcross. (A) e genotyped  progeny with the
Lemon Frost phenotype and  wild type progeny from the third generation of a backcross
of Mr. Frosty to the Sunburst line for markers in the SPT1 region and obsered a ̀
consistent inheritance pattern. (B) Sequencing chromatogram of a heterozygous animal
(lf) at an insertion marker. (C) Sequencing chromatogram of a homozygous animal ()
at the same insertion marker.
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A B C
D E F
G H I
J K L
SupFig 1 Coloration and pattern diversity of the common leopard gecko, Eublepharis
macularius. (A) wild type; (B) black night; (C) variant of black night; (D) granite snow; (E)
gem snow; (F) white knight; (G) sunburst tangerine; (H-I) variants of sunburst tangerine; (J)
red stripes; (K) bold stripes; (L) rainbow.
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20 43 29 41 31 21 42 22 44 28 45 36 30
451 21 142 26 30 2940 773 24 39 24 259 29 289 24 566 24 3064 1610 24 37 2940 23 29 24 1761 22 31 28 3064 3651 25 38 4601 21 24 5047 25 21 2024 32 24 2369 24 2942 2624 24 2942 35 27 6672 35 22 2943 24 5056 24 3652 28 5939 7597 5046 4607 24 36 5045 34 33 30 5753 24 40
619 4822 4823 7608 2712 6049 6050 6594 7609 3529 6609 6610 7792 8652 8653 6601 236 2526 11029 551 5580 11981 9020 1535 2574 2575 17−7597 562 506 3441 5597 7160 9025 12828 1972 4872 4916 7576 6353 3362 4812 6097 6576 7646 7647 6068 6556 7620 7621 8656 6089 6570 6583 8640 8641 7205 7180 7204 8253 9038 4031 7666 5593 7209 441 2398 2399 2593 2594 3434 5590 5591 5628 6180 9717 9745 10997 10998 11520 3440 11071 7172 4193 4194 4911 5576 8251 9073 4029 973117−5047 1238 3026 5572 5574 7704 13312 5037 5018 5571 7669 7670 9754 7584 9006 9021 9022 9687 11295 13113 13620 69 3023 4869 9008 9009 12796104494 1311 7176 11690 4862 5041 5613 5697 7163 7164 7198 9011 11699 12853 2529 6175 9674 9675 9785 11016 12793 13313 7598 10461 4898 9681 7129 7590 9013 13325 12849 3447 6185 6186 7673 7674 3458 3459 7189 7190 7578 9023
Mr. Frosty
SupFig 2 Breeding pedigree of the Lemon Frost mutation. Mr. Frosty, the original carrier of the
spontaneous Lemon Frost mutation, was bred to 12 female geckos from different genetic backgrounds. F1s
carrying the lf allele were bred among themselves or back to their female parent, producing the second
generation of animals heterozygous or homozygous for the lf allele. Blue: lf/lf; green: lf/+; red:+/+. Dashed line:
same individual/line.
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*
*
*
*
SupFig 3 Histopathology of skin tumors. (A) Thick layers of white tumor tissue (star)
infiltrating white skin (arrow). (B) Skin biopsies organized and fixed in a paper roll for
sectioning. (C) H&E staining of the skin sections. Arrow: skin; star: infiltrated tumor mass.
(D) H&E staining of the skin sections showing normal skin cells and neoplastic cells (star).
Neoplastic cells have eccentric and condensed nuclei.
A B
C
D
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Bright Field Dark Field
upFig  otential metastasis of iridophoroma. (A) n normal skin, cell nuclei are oal and
perpendicular to the skin surface. n Lemon Frost skin, cell nuclei are flat, elongated and
parallel to the skin, reminiscent of epithelial-to-mesenchymal transition. (B) ridophoroma in ̀
the lier, stained dark in HE sections. n dark field imaging, iridophores are bright white.
Such iridophores inade blood essels in the tissue (red arrows). (C) n TEM imaging, white ̀
tumor skins in super LF are filled with abundant iridophores with ecessie brightly reflectie
crystals (Tumor). n normal skin, iridophores are much fewer and hae less crystals (ormal).
A
B
CTumor Normal
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upFig  T1 epression in gecko skin. SPT1 mA reads from transcriptome sequencing were aligned to the genome
and isualized in G. Top  rows show samples from homozygous mutants. Bottom rows show samples from wild type geckos. ̀
Skin tissue adacent to the tumors was used in the mutants. Peaks mark SPT1 eons. The last eon on the right is transcribed
together with the T.
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