Embryonic Expression of <i>Tbx15</i> Compared to <i>Agouti</i> in <i>a^t</i>/<i>a^t</i> Mice

<p>(A and C) <i>Tbx15</i>. (B and D) <i>Agouti</i>. At E12.5, expression of <i>Tbx15</i> in dorsal skin is approximately complementary to that of <i>Agouti</i> in ventral skin. At E14.5, the levels of expression for both genes are lower, but <i>Tbx15</i> expression has expanded ventrally and overlaps extensively with that of <i>Agouti</i>. In all four panels, arrows mark the approximate ventral limit of <i>Tbx15</i> and the approximate dorsal limit of <i>Agouti</i> (scale bars = 500 μm).</p

Embryonic Establishment of Dorsoventral Skin Patterning

<p>Pieces of skin from dorsal, flank, and ventral regions of <i>a^t/a</i> E12.5 embryos were transplanted into the testes of congenic animals as described in the text. Hair color of the grafts was examined 3 wk later. Grafts of ventral embryonic skin (<i>n</i> = 3) produced yellow hairs, dorsal embryonic skin (<i>n</i> = 4) produced black hairs, and flank embryonic skin produced mostly (13 out of 15) black and yellow hairs in distinct regions as shown. In parallel, in situ hybridization studies revealed that the embryonic flank contains the boundary of expression between <i>Agouti</i> and <i>Tbx15</i> (scale bars = 1 mm for hairs and 200 μm for in situ hybridization results).</p

The <i>de^H</i> Pigmentation Phenotype

<div><p>(A) 10-wk-old <i>de^H/de^H</i> and nonmutant animals on a <i>a^t</i> background. A thin stripe of yellow hair normally separates the dorsal black hairs from the ventral cream hairs. In <i>de^H</i>, the yellow stripe is extended dorsally, and the boundary between the yellow and the black hairs is fuzzier.</p> <p>(B) Skin slices taken from 1.5-mo-old <i>de^H/de^H</i> and nonmutant littermates (scale bar = 0.5 cm).</p> <p>(C) Proportion of total skin area as determined by observation of pelts taken from the interlimb region. The proportion occupied by the yellow lateral compartment (± SEM) differs between mutant and nonmutant littermate flanks (<i>p</i> < 0.0005, paired <i>t</i>-test, <i>n</i> = 6 pairs). There is also (data not shown) a small increase in the proportion of total skin area occupied by the ventral cream-colored compartment, 47.9 % in mutant compared to 37.8% in nonmutant (<i>p</i> < 0.005, paired <i>t</i>-test, <i>n</i> = 6 pairs).</p> <p>(D) On an <i>a^e/a^e</i> background, the extent of dorsal skin pigmentation is reduced in <i>de^H/de^H</i> neonates (P3.5).</p> <p>(E) Hair length in a representative pair of 1.5-mo-old <i>de^H/de^H</i> and nonmutant littermates, averaged over three skin slices at different rostrocaudal levels, and plotted as a function of the absolute distance from middorsum or the percentage of total slice length.</p></div

Dorsoventral Skin Characteristics

<div><p>(A) Skin slices from animals of different age and genotype demonstrate similar patterns of hair-length variation along the dorsoventral axis (scale bar = 1 cm).</p> <p>(B) Enlarged area from (A), demonstrating the transition in hair length and color in <i>a^t</i>/<i>a^t</i> mice (scale bar = 0.375 cm).</p> <p>(C) Proportional hair length for (A) plotted as a function of relative position along the dorsoventral axis.</p> <p>(D) Hair length plotted as a function of absolute position along the dorsoventral axis for 8-wk-old BA strain mice.</p> <p>(E) Proportion of zigzag hairs (± SEM) differs slightly between dorsum and ventrum of inbred mice (<i>p</i> < 0.0001, χ² test, <i>n</i> = 1,958, 1,477, 1,579, 1,502).</p> <p>(F) Differences in dorsal and ventral skin development at P4.5 (scale bar = 1 mm, upper; 200 μm, lower).</p> <p>(G) Differences in hair melanin content and DOPA staining for dorsum (d), flank (f), and ventrum (v) in <i>a^e</i>/<i>a^e</i> and <i>a^t</i>/<i>a^t</i> mice. The upper panel also demonstrates a cream-colored appearance of the <i>a^t</i>/<i>a^t</i> ventrum. The middle panel shows representative awls (scale bar = 100 μm). The lower panel shows DOPA-stained dermis (scale bar = 200 μm).</p></div

Model for Acquisition of Dorsoventral Patterning in the Trunk and the Role of <i>Tbx15</i>

<div><p>(A) A tricolor pigmentation pattern is generated by the combination of distinct mechanisms that affect distribution of <i>Agouti</i> mRNA and histochemical staining for melanocytes; effects of the latter mechanism by itself are evident in <i>a^e</i>/<i>a^e</i> mice (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020003#pbio-0020003-g001" target="_blank">Figure 1</a>). In <i>a^t/a^t</i> mice, reduced hair melanocyte activity and high levels of <i>Agouti</i> mRNA in the ventrum lead to a cream color; as melanocyte activity gradually increases towards the dorsum, a lateral stripe is apparent on the flank. The distributions of <i>Agouti</i> mRNA and histochemical staining for melanocytes are both affected by <i>Tbx15</i> and are externally evident by a widening of the lateral stripe and an increased proportion of total skin occupied by the cream-colored area.</p> <p>(B) The lateral yellow stripe in <i>a^t/a^t</i> mice lies at the same level as the limb dorsoventral boundary. As described in the text, we propose that distinct dorsoventral compartments in ectoderm of the trunk provide an instructional cue to the mesoderm, leading to expression of <i>Tbx15</i> in dorsal trunk mesenchyme and acquisition of dorsal dermis character. In the absence of <i>Tbx15</i>, dorsal mesenchyme assumes ventral characteristics instead.</p></div

Effect of <i>de^H</i> on <i>Agouti</i> Expression

<div><p>Comparable sections from <i>a^t/a^t</i>; <i>de^H</i>/<i>de^H</i> and <i>a^t/a^t</i>; +/+ littermates.</p> <p>(A) At E14.5, <i>de^H</i>/<i>de^H</i> embryos have a smaller body cavity and loose skin within which <i>Agouti</i> expression appears to be shifted dorsally, as marked by arrows (scale bars = 500 μm).</p> <p>(B) At P4.5, <i>Agouti</i> expression in both dorsal and ventral skin is similar in <i>de^H</i>/<i>de^H</i> compared to nonmutant, but in the midflank region, there is increased <i>Agouti</i> expression in <i>de^H</i>/<i>de^H</i>, especially in the upper dermis (scale bars = 200 μm). Sections shown are representative of two mutant and two nonmutant samples examined at each time.</p></div

Developmental Expression of <i>Tbx15</i>

<div><p>(A) At E12.5, transverse sections at different levels show expression in head mesenchyme (a and b); myotome, occipital, and periocular mesenchyme (b); palatal shelf, cervical sclerotome, and nasal cartilage (c); maxillary and mandibular processes (d); limbs (e); and myotome and lateral mesenchyme (e and f) (scale bars = 500 μm).</p> <p>(B) Transverse sections through the flank at different times show expression in lateral mesenchyme (E11.5), expanding dorsally at E12.5, and both ventrally and dorsally at E13.5, detectable in loose mesenchyme underlying the dermis and the abdominal and subcutaneous muscles (scale bar = 500 μm). At P3.5, <i>Tbx15</i> is expressed in the entire dermis and is most strongly expressed in dermal sheaths (scale bar = 200 μm).</p></div

Molecular Genetics of <i>de^H</i> and <i>Tbx15</i>

<div><p>(A) Genetic and physical map, as described in the text. Markers M1 to M3 are SSCP markers generated from a BAC contig of the region; marker M4 is STS 16.MMHAP32FLF1 and was also used as an SSCP marker. M2 and M3, which flank the <i>Tbx15</i> and <i>M6pr-ps</i> on the UCSC genome browser map and lie 634 kb apart, were nonrecombinant with <i>de^H</i> in 2340 meioses.</p> <p>(B) The <i>de^H</i> mutation is a deletion that starts in <i>Tbx15</i> intron 1 and ends in the <i>M6pr-ps</i>.</p> <p>(C) Sequence of deletion breakpoints.</p> <p>(D) Diagram of <i>Tbx15^LacZ</i> allele constructed by gene targeting. As described in the text, this allele is predicted to give rise to a protein truncated after approximately 154 codons and is lacking critical residues of the T box. Heterozygotes for the targeted allele exhibit normal size, morphology, and hair-color patterns, but homozygotes and <i>Tbx15^LacZ</i>/<i>de^H</i> compound heterozygotes are identical to <i>de^H</i> homozygotes.</p></div