To The Editor
Over the last decade, large-scale, primarily ENU based, chemical mutagenesis projects
(Clark et al, 2004) inspired the development of high-throughput, broad coverage means
to analyze mutant mouse phenotypes. The dark skin mutations, GnaqMhdadsk1
, arose in just such an ENU mutagenesis screen. These dark skin mutations are due
to missense mutations in the Gnaq gene (Van Raamsdonk et al, 2004; Fitch et al, 2003).
A third dominant mutation, GnaqM1J
, arose an ENU mutagenesis screen using the C57BL/6J mouse strain at The Jackson Laboratory.
We report here the phenotype and molecular defect of this new allelic mutation.
deviant mice presented with abnormally pigmented tails inherited in a dominant manner
(Figure 1 A). Further observations indicated pigmentary changes throughout the skin
of deviant mice (Figure 1 Bi-iv). GnaqM1J
mice exhibited unusually dark pigmented tails (Figure 1 Bi), ears (Figure 1 Bii),
lower legs and foot pads, (Figure 1 Biii) and dorsal skin (Figure 1 Biv) as compared
to wild-type litter mates.
To define histological lesions, a complete set of tissues were harvested from 3 males
and 3 females, mutant and control, 12 week old mice and processed routinely. Paraffin
sections were stained with hematoxylin and eosin (H&E), and examined by an experienced
board certified veterinary anatomic pathologist (JPS) (Silva and Sundberg, 2012).
Histological analysis of tail skin taken from wild type C57BL/6J mice demonstrated
low levels of interfollicular epidermal pigment and dermal hyper pigmentation (Figure
2 A,B). By contrast, GnaqM1J
mutant mice tail sections exhibited high levels of dermal hyper pigmentation and interfollicular
epidermal pigmentation in similar regions of the tail (Figure 2 C,D). This was more
evident in cross sections of GnaqM1J
tail skin (Figure 2E, arrows), especially in oblique sections of the dermis (Figure
2 F). In addition, sections through ventral leg skin (Figure 2G,H) and foot pads with
eccrine glands (Figure 2I,J) revealed dermal hyper pigmentation, but no interfollicular
pigment in the epidermis. Also, wild-type dorsal skin sections and hair follicles
(Figure 2Ki-iii) exhibited no pigment evident in the dermal papilla or residual pigment
in the dermis below the hair follicle in the fibrous track of the former catagen follicle,
while dorsal skin sections from mutant mouse (Figure 2Li-iii) exhibit abnormal pigmentation
in the dermal papilla region extending into the underlying dermis in the fibrous streak
remaining at the end of catagen. Immunohistochemistry was also performed whereby paraffin
sections from mutant and control animals were stained with antibodies specific to
the following antigens: Smooth Muscle Actin, CD31, LYVE1, and VEGFA. The immunohistochemical
stains were used to examine vascular abnormalities in these mice, yet no differences
were observed between mutant and control mice (data not shown).
SNP mapping was performed to isolate the genomic region containing the mutant allele
by backcrossing the original C57BL6/J deviant mouse to the inbred mouse strain FVB/NJ.
Mapping studies revealed linkage to proximal mouse chromosome 19 near SNP marker 21,436,276
bp (GRCm38) with a logarithm of odds (LOD) score equal to 5.3. Targeted exome sequencing
of Gnaq identified a single base pair variant resulting in an A to G missense mutation
in exon 2 at 16,219,611 bp (Figure 1 C). This variant is predicted to result in a
Threonine to Alanine change at amino acid 54 (T54A). GNAQ consists of an alpha- helical
domain, three flexible switch domains and a GTPase domain (Kumar et al, 2009). The
T54A resides in the N-terminus of the alpha-helical domain. The “Sorting Tolerant
From Intolerant” (SIFT) algorithm identified the T54A variant as a deleterious mutation
in the helical domain of GNAQ (SIFT score 0.01), which may affect proper domain functioning.
(The Uniprot Consortium, 2014) (Figure 1 C). Previous ENU induced mutations GnaqMhdadsk1
, affected the C-terminus of the alpha-helical domain and GTPase-domain, respectively
(Van Raamsdonk et al 2004, Fitch et al, 2003). All three ENU-induced mutations, affecting
different areas of the GNAQ protein, resulted in similar, but not identical, pigmentary
changes to the skin of affected mice. Also, the differences in phenotype between the
previously described alleles (GnaqMhdadsk1
) and GnaqM1J
, may be due to genetic background of the mouse strain, whereby the previous alleles
were found on the C3HeB/FeJ strain, with a wild-type agouti locus (A/A), and the new
allele was found on the C57BL/6J background, with a mutant agouti allele (a/a) producing
only black pigment.
A multitude of signaling pathways operate in the skin, whose coordination is essential
for proper skin development and differentiation (Supplementary Figure 1 and 2). Yet,
the molecular mechanisms governing these processes are still largely unknown. We report
here a new, publicly available, ENU induced mutant (GnaqM1J
) that results in similar, but more extensive, skin hyperpigmentation defects as the
alleles (Fitch et al, 2003).
Early in mouse development migrating melanoblasts localize to the dermis and epidermis
(Fitch et al, 2003). Previously described mutations in Gnaq (GnaqMhdadsk1
) cause increases in the number of differentiated melanoblasts resulting in increased
pigmentation in the dermis of mutant mice (Fitch et al, 2003). Although GnaqM1J
is similar to the previous mutations, in that increased dermal pigmentation is observed
in adult mice, unlike the previous mutants, it results in both dermal and epidermal
hyperpigmentation in tail skin. In light of these mutations, we hypothesize that the
genes highlighted in Supplementary Figures 1 and 2 work in a coordinate manner to
direct development, localization, and differentiation of skin melanoblasts.
Studies using hairless mice with various degrees of cutaneous pigmentation suggested
that pigment may play a role in barrier function (Man et al, 2014). As such, mutations
in genes regulating skin pigmentation may seriously affect the function of the skin.
Gnaq can be tied to barrier formations by way of its interactions with other barrier
genes, like Cav1 (Supplementary Figure 2).
Mutations affecting skin color are infrequent in the mice, as most affect hair color
(Dadras et al, 2014; Sundberg and Silva, 2012). Having single gene mutations on the
inbred and very commonly used C57BL/6J background provides a new tool for refined
evaluation of pigment in the skin of mice.