The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3

Abstract

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2’s exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

Introduction

Rodent mutations that affect either markings or spotting on the coat have contributed to our understanding of melanocyte differentiation and migration [1]. Some coat color mutant strains also serve as models for human diseases. For example, mice carrying the recessive mutation in the endothelin receptor B gene exhibit piebald (white spotting) and megacolon and serve as a model for Waardenburg syndrome type IV [2]. Dominant white spotting mice carry mutations in the Kit gene that encodes a receptor tyrosine kinase and serve as a model for piebaldism. Beige mice carry mutations in the Lyst gene and serves as a model for Chediak-Higashi syndrome. Thus, identifying genes responsible for coat color pigmentation contributes to greater understanding of physiological processes not only in pigmentation but in signal transduction, membrane trafficking, and development [3].

The Downunder rat is a fancy rat variety with characteristic markings on its belly. The ventral marking is a thick line running from the neck to the breech and is seen only when rats exhibit the hooded phenotype [4]. The Downunder pattern is inherited in a dominant fashion. In 2005, a fancy rat carrying the Downunder (Du) mutation was shipped to Japan from a US rat breeder to introduce it into laboratory strains [5]. By crossing this rat with laboratory rats, we generated a congenic strain carrying the Du mutation.

For this study, we first established the F344/Cg-Du congenic strain and examined eye anomalies in Du/+ rats as well as the nature of embryonic lethality among Du/Du embryos. In addition, we mapped the Du mutation to rat chromosome (Chr) 3. By haplotype mapping, we localized the Du locus to a 3.9-Mb region that includes two genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2).

Materials and Methods

Animals

A male fancy rat carrying the Du mutation was obtained from a rat breeder in Kansas City, MO, USA, in July 2005 [5]. TM/Kyo and WTC/Kyo rats were obtained from the National BioResource Project for the Rat (NBRP-Rat, Kyoto, Japan) [6]. F344/NSlc rats were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). All animal care and experimental procedures used were approved by the Animal Research Committees of Kyoto University and Tokyo University of Agriculture and carried out according to the Regulations on Animal Experimentation of both universities.

Congenic strain

The Du-carrying fancy rat was crossed with TM/Kyo rats (coat color genotypes in the Tyr⁺ and H loci were C/C and h/h, respectively), and F1 hybrids were obtained by caesarean section for microbiological cleaning. At the F3 generation, male rats were crossed with WTC/Kyo rats (c/c, h/h) to prevent sterility. After three additional inbreeding generations, sperm was collected and cryopreserved.

In 2009, rats carrying the Du mutation were rederived by intracytoplasmic sperm injection of F344/NSlc oocytes. Rats carrying the Du mutation were identified by the presence of the ventral marking. Because F344/NSlc rats (c/c, h/h) were albino, we selected colored Downunder rats and backcrossed them to F344/NSlc rats. After eight generations of backcrossing, we generated the F344.Cg-Du Tyr⁺/Kyo congenic strain.

Histopathology

Rats (10–12 weeks of age) were euthanized under isoflurane anesthesia. Brains and eyes were harvested, fixed in 10% neutral buffered formalin, and embedded in paraffin. Four micrometer-thick sections were prepared and stained with hematoxylin and eosin.

Examination of fetuses by cesarean section

Six Du/+ female rats were mated with Du/+ male rats. At embryonic day 20, fetuses were removed by cesarean section. The numbers of live fetuses and embryo-fetal deaths were counted. Embryo-fetal deaths were classified as either early death (implantation sites, resorbed embryos, and placental remnants) or late death (early macerated fetuses, late macerated fetuses, and dead fetuses). The number of implantations was calculated by summing the number of live fetuses and the number of embryo-fetal deaths.

Genetic mapping

Thirty-nine progeny generated from the (Du/+ × WTC)F1 × WTC cross were used for rough mapping. They were genotyped for 25 SSLP markers on Chrs 1, 2, 3, 4, 8, 10, 12, 14, 16, 17, 18, 20, and X (Supplementary Table 1). For fine mapping, 35 F344/NSlc × (F344/NSlc × Du/+)F1 backcross progeny were produced. These animals were genotyped for 15 SSLP markers on Chr 3 (Supplementary Table 1).

Next-generation sequencing

A custom capture library was designed with a SureSelect Target Enrichment System (Agilent, Santa Clara, CA, USA). Probes were designed to capture DNA sequences from 24.2 Mb to 28.5 Mb on Chr 3 (USSC rn4, November 2004) that include the genes Arhgap15, Gtdc1, and Zeb2. To construct the library, genomic DNAs from F344.Cg-Du Tyr⁺/Kyo (N5) and F344/NSlc were used. A total of 3 µg input DNA per sample was used for SureSelect library preparation (Agilent) with the SureSelect XT HS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Version 1.6, October 2013) protocol.

Sequencing of SureSelect enriched libraries was performed on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA). Base calling and sample de-multiplexing were performed to generate paired FASTQ files for each sample. Adapter sequences were trimmed from raw reads. Reads were discarded when the proportion of bases with a quality score of 10 was less than 95% using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Filtered high-quality reads were mapped to the Brown Norway rat strain reference genome (Ensenbl Rat release 69 RGSC 3.4, Dec 2004) using Burrows-Wheeler Alignment tool v0.5.9 [7]. PCR duplicates were removed using Picard v1.93 (http://broadinstitute.github.io/picard). The cleaned alignment files were used to call single nucleotide polymorphisms (SNPs) with Genome Analysis Toolkit v1.6-9 [8], and the effects of variations were annotated using SnpEff v3.5e [9].

Sanger sequencing

Genomic DNA was isolated from the spleen by standard phenol-chloroform extraction and dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The exons and intron-exon boundaries of Gtdc1 and Zeb2 were amplified with specific primer sets (Supplementary Table 2) and KAPA2G™ Robust HotStart ReadyMix with dye (Merck KGaA, Darmstadt, Germany). Direct sequencing was performed by Macrogen Japan Corp. (Tokyo, Japan).

Quantitative PCR

Total RNA was isolated from rat brains using a FastGene™ RNA Premium Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan). Complementary DNA (cDNA) was synthetized using FastGene™ Scriptase II (NIPPON Genetics Co., Ltd.). Quantitative reverse transcription PCR was performed using a Thermal Cycler Dice Real Time system (Takara Bio Inc., Shiga, Japan) with a KAPA SYBR Fast qPCR Kit (Roche Diagnostics K.K., Tokyo, Japan). The primer sequences for quantifying cDNA were as follows: 5′-ATACCAGCGGAAACAAGGATTTCA-3′ and 5′-CAGGAATCGGAGTCTGTCAAGTCA-3′ for Zeb2, and 5′-GTTGTCATCTCAACAGCCAAGCA-3′ and 5′-CAAGTCTTTGGGACAGAGTGGGTAA-3′ for Gtdc1. By monitoring the amplification curves of a test sample and reference samples that contained between 10¹ and 10⁶ molecules of the mRNA of interest, the number of target mRNA molecules in the test sample was determined and normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA. The primer sequences of Gapdh were as follows: 5′-GGCACAGTCAAGGCTGAGAATG-3′and 5′-ATGGTGGTGAAGACGCCAGTA-3′.

Results

F344.Cg-Du Tyr⁺/Kyo congenic strain

F344.Cg-Du Tyr⁺/Kyo congenic heterozygous rats (Du/+) exhibited the characteristic ventral marking, very similar to the original Du-carrying rats. The dorsal marking, which was caused by the hooded mutation, of the congenic Du/+ rats was wider than that of their wild-type littermates (Fig. 1).

Fig. 1.

Ventral and dorsal markings on F344.Cg-Du Tyr⁺/Kyo congenic rats. Top: Du/+ heterozygous rat. Bottom: wild-type (+/+) littermate. Ventral markings were prominent on Du/+ rats. Dorsal markings were wider than on wild-type rats. Both rats were homozygous for the hooded (h) mutation.

Eye anomalies

Some Du/+ rats exhibited eye anomalies that presented as microphthalmia and anophthalmia (Fig. 2A). We examined a total of 70 Du/+ rats, of which 16 (22.9%) exhibited such eye anomalies. Of these 16 animals, three had anomalies in their right eye, nine in their left eye, and four had anomalies in both eyes (Fig. 2B). Eye anomalies were observed more frequently in the left eye but this difference was not statistically significant (P=0.083). Anophthalmia was accompanied by loss of the optic nerve (Fig. 2A). Histologically, the anophthalmia showed the complete loss of the eye structures, including lens, cornea, retina, and uvea (Fig. 2C). In severe microphthalmia cases, there was only a small mass of pigmented intraocular remnant/vestigial tissue resembling ciliary processes (Fig. 2C), while the ocular adnexa, inclufing Harderian glands, lacrimal glands, eyelid, conjunctiva, and extraocular muscles, were almost normal. Such anomalies were not observed in wild-type littermates. These findings indicate that the Du mutation also caused the eye anomalies with incomplete penetrance.

Fig. 2.

Eye anomalies in F344.Cg-Du Tyr⁺/Kyo congenic rats. A. Upper: Dorsal view of the head of a Du/+ rat with an eye anomaly. The left (L) eye is absent. Lower: Ventral view of the brain harvested from the rat in the upper panel. The optic nerve from the right (R) eye is intact (arrowhead), but that from the left (L) eye (white arrowhead) is absent. B. Number of rats exhibiting eye anomalies. Out of 70 rats examined, 16 (22.9%) exhibited eye anomalies. The observed eye anomalies consisted of anophthalmia and microphthalmia. Two Du/+ rats exhibited anophthalmia in the right eye and microphthalmia in their left eye; the bar representing these rats contains hatched lines. C. Microscopic images of normal eye, anophthalmia (shown in panel A), and microphthalmia cases. The normal eye consists of an intact lens, retina, ciliary body (Ci), iris, and cornea (Cor). The eye with microphthalmia lacks a lens, cornea, and retina, but a pigmented intraocular vestigial mass (arrow) is present. In the anophthalmia case, these intrinsic structures of the eye are completely absent. The ocular adnexa, including Harderian glands (Hg), eyelid, conjunctiva (Con), and muscle (M), are normal in both microphthalmia and anophthalmia. Car: cartilage. Bars: 200 µm.

Embryonic lethality

To examine the incidence and stage of embryonic lethality, we crossed Du/+ females with Du/+ males. We observed 37 live fetuses and 16 embryo-fetal deaths from six pregnant Du/+ rats. The proportion of embryo-fetal deaths relative to the number of implantations was 30.2%, compared with 25% embryo-fetal death when homozygous lethality occurred in Du/Du embryos (P=0.78). Reasons for embryonic deaths included implantation site (0.5 ± 1.2; average ± SD), resorbed embryo (2.0 ± 1.7), and placental remnant (0.2 ± 0.4; Table 1). This finding suggests that Du/Du embryos died in the early stage of organogenesis (gestation days 10 to 12) [10].

Table 1. Number of dead embryos and fetuses observed in Du/+ females mated with Du/+ males at E20.

Stage	Number of fetuses	Mean ± SD
Viable	37	6.2 ± 2.9
Embryonic death (total)	16	2.7 ± 1.9
Implantation site	3	0.5 ± 1.2
Resorbed embryo	12	2.0 ± 1.7
Placental remnant	1	0.2 ± 0.4
Early macerated fetuses	0	0.0 ± 0.0
Late macerated fetuses	0	0.0 ± 0.0
Dead fetuses	0	0.0 ± 0.0

Genetic mapping of Du mutation

We observed 21 Downunder (Du/+) and 18 non-Downunder (+/+) rats in (Du/+ × WTC)F1 × WTC/Kyo backcross progeny (n=39), indicating that Du is a single dominant mutation (P=0.63). The initial screening revealed that Du was significantly linked with D3Mgh16 (P<0.001) and D3Mgh19 (P<0.001). To map Du to Chr 3, we further performed a linkage analysis using F344/NSlc × (F344/NSlc × Du/+)F1 backcross progeny (n=35) and thereby mapped Du to between D3Rat229 and D3Rat190. Du showed no recombination with D3Rat49 (27.5 Mb), D3Got21 (27.6 Mb), and D3Got18 (27.9 Mb; Fig. 3A).

Fig. 3.

Genetic mapping of the Du locus. A. Left: Genetic linkage map that includes the Du locus. Right: Haplotypes observed among backcross progeny carrying the recombinant chromosome between D3Rat53 and D3Got25. Filled boxes represent heterozygous genotypes, whereas open boxes represent homozygous genotypes. The Du locus was mapped between D3Ra229 and D3Rat190. B. Physical map of the Du locus. Top: Physical positions of genetic markers used to define the Du locus and numbers of single nucleotide polymorphisms (SNPs) that were identified by next-generation sequencing (NGS). Bottom: Genotypes of the F344 and F344.Cg-Du congenic strains. The F344.Cg-Du strain exhibited a heterozygous genotype, highlighted in grey, for the rs106540905, D3-Du-SNP10, and D3-Du-SNP02 SNP markers. Genotypes of SNP markers are represented by nucleotides. Asterisks indicate no nucleotide at the D3-Du-SNP02 SNP marker; the F344 genome has no nucleotides and the Du allele has an “A” nucleotide at the SNP. We mapped the Du locus to between rs197659597 and D3Rat190, which includes the genes Gtdc1 and Zeb2. F/F: homozygous for the F344 allele in SSLP markers.

To develop SNP markers and identify variations specific to the Du allele, we performed next-generation sequencing (NGS) of a 4.21-Mb region within the Du locus. The targeted region spans nucleotides 27,944,460 to 32,154,450 of the Chr 3 reference genome (mRatBN7.2). We identified 5,439 variations within the targeted region (Fig. 3B). Representative SNP markers that can be used for haplotype mapping with congenic rats were summarized in Supplementary Table 3. At the N19 generation, a 3.9-Mb region defined by rs197659597 and D3Rat190 segregated with the Downunder phenotype, which demonstrated that the Du locus lies within this 3.9-Mb region (Fig. 3B). Two genes, Gtdc1 and Zeb2, were mapped to the Du locus. No variation specific to the Du allele was found in any of the exons or splice sites of these genes, which was verified by Sanger sequencing.

Gene expression analysis

To find more potential candidate genes, we determined Gtdc1 and Zeb2 transcript levels in Du/+ rat brains. The relative expression level of Gtdc1 in Du/+ rats did not differ from that of wild-type rats (mean ± SD; 3.93 × 10⁻² ± 3.45 × 10⁻² vs. 1.42 × 10⁻² ± 0.82 × 10⁻², P=0.0700). In contrast, Zeb2 levels were significantly reduced (mean ± SD; 6.35 × 10⁻² ± 1.84 × 10⁻² vs. 3.19 × 10⁻² ± 2.44 × 10⁻², P=0.0156; Fig. 4). This finding suggests that Zeb2 is the likely candidate gene for the Du mutation.

Fig. 4.

Relative Gtdc1 and Zeb2 mRNA levels in Du/+ rats. Relative Gtdc1 and Zeb2 mRNA levels in wild-type (open bars) and Du/+ (filled bars) rats (n=3, each). Zeb2 mRNA was significantly reduced in Du/+ rat brains compared with wild-type rat brains. *P<0.02.

Discussion

In this study, we established the F344.Cg-Du congenic line, discovered eye anomalies in Du/+ rats, determined the developmental stage at which Du/Du embryos die, and localized the Du locus to a 3.9-Mb region on rat Chr 3. The Du locus includes Gtdc1 and Zeb2. Additionally, we found that Zeb2 mRNA levels are significantly lower in Du/+ rats compared with wild-type rats.

Gtdc1 encodes glycosyltransferase-like domain-containing 1 which was cloned from a fetal brain cDNA library by a high throughput DNA sequencing analysis [11]. Glycosyltransferase catalyzes the synthesis of carbohydrate portions of glycoproteins, glycolipids, and proteoglycans, but GTDC1 function remains unknown. Gtdc1 transcripts are expressed at relatively high levels in the adult lung, spleen, testis, and peripheral blood leukocytes [11]. The GTDC1 gene was reportedly disrupted in a human patient with signs of neurodevelopmental disorders, while gtdc1 knockdown in zebrafish led to impaired CNS development [12]. Gtdc1-modified mice have not been developed, and no information on Gtdc1 involvement in pigmentation is currently available.

Zeb2 encodes a transcription factor called zinc finger E-box binding homeobox 2. In humans, mutations in the ZEB2 gene are known to cause Mowat-Wilson Syndrome (MOWS; OMIM #235730), an autosomal dominant complex disorder [13, 14]. MOWS patients exhibit typical facial dimorphism but suffer from severe intellectual disability and delayed motor development. Additionally, anomalies are often observed in the corpus callosum or the hippocampus, if not both. Other anomalies have been observed in the urogenital tract, eye, ear, and heart. MOWS patients also often suffer from Hirschsprung disease (HSCR), which affects the gastrointestinal tract [15,16,17].

Homozygous Zeb2-knockout (KO) mice are embryonic lethal; they are severely retarded in their growth by E9.5 before dying [18]. Heterozygous Zeb2-KO mice exhibit craniofacial abnormalities and corpus callosum formation defect but not abnormal pigmentation [19]. Moreover, melanocyte-specific Zeb2 deletion resulted in the congenital loss of hair pigmentation in mice, which suggests an essential role for Zeb2 in pigmentation [20]. Additionally, we detected a significantly lower level of Zeb2 transcripts in Du/+ rats. Taken together, these observations suggest that Zeb2 is a strong candidate gene for the Du mutation.

We did not find any variations specific to the Du allele in any of the exons or intron-exon boundaries of Zeb2, which suggests that the causative variation may be found in a regulatory region (usually located upstream of a gene) of Zeb2. The Du locus includes at least a 3.0-Mb upstream region of Zeb2, and we identified an ample number of variations in the Du locus by NGS. In a follow-up study, we will sort through these variations using bioinformatics analyses that can predict enhancers and regulatory regions. Alternatively, the causative variation may be an insertion of DNA such as transposable elements. To find such variations, sequencing of large DNA fragments derived from the Du locus is necessary, and may be accomplished by either constructing a BAC library [21] or nanopore DNA sequencing [22].

The ventral marking of Du/+ rats closely resembles to the marking of black-and-tan (a^t/a^t) mice in which a sharp dorso-ventral boundary is observed [23]. Formation of the boundary in a^t/a^t mice is modified by Tbx15 [24]. These findings suggest that the pigmentation of the ventral coat differs from that of the dorsal coat and that the pigmentation area is regulated by genetic factors. Thus, we speculate that the downunder marking may result from the visualization of the ventral-specific pigmentation in rats and that the Du mutation, possibly the Zeb2 downregulation, may interact with genes involved in the formation of the dorso-ventral boundary.

Lastly, we found that the Du mutation usually manifests phenotypically in hooded (h/h) rats that exhibit white markings on their ventral and lateral trunk. The hooded phenotype is unique to rats and is prevalent among most rat strains [25], which enabled us to follow the Du mutation. Thus, the availability of different species of model animals is crucial to identifying novel gene functions.

In summary, in addition to ventral markings, the Du mutation is associated with eye anomalies and early post-gestation lethality. The Du locus is located within a 3.9-Mb genomic region on Chr 3, and Zeb2 is the most likely candidate gene.