The G-netics of dark skin

Several mutant strains of mice have dark skin pigmentation due to an aberrant accumulation of pigment-producing melanocytes in the dermal layer of the skin. A new study shows that three such strains carry activating mutations in the genes encoding the G-protein subunits Gαq or Gα11, resulting in more pigment cell precursors and an excess of dermally retained pigment cells at birth.

Mutations that affect mouse pigmentation have been studied for over a century. But despite this long history, large-scale mutagenesis programs^1,2 continue to identify new genes involved in this process. A series of ten dominant dark skin (Dsk) mutations recently described by Greg Barsh and colleagues³ can be classified into two categories, depending on whether dermal or epidermal pigmentation is increased. On page 961 of this issue, Van Raamsdonk et al.⁴ report that three of the four Dsk mutants with increased dermal pigmentation carry activating mutations in Gnaq and Gna11. These genes encode functionally related Gα subunits that mediate the first step in transducing signals from G protein–coupled receptors, identifying a key role for these genes in regulating skin color.

The road less traveled

Melanoblasts, the precursors of the pigment-producing cells of the skin and hair, arise during development from a migratory cell population called the neural crest. Some of the key molecules required in the developmental path from neural crest to melanoblast include the transcription factors Pax3, Sox10 and Mitf and the cell-surface receptors Kit and Ednrb⁵. As the two layers of the skin develop, melanoblasts enter the dermis and then migrate from the dermis to the epidermis through the basement membrane that separates them. Then, as hair follicles develop, melanoblasts undergo a further migration into the hair follicles, leaving the epidermis mostly devoid of pigment cells. In certain places where hair is sparse, such as the tail and external ears, melanocytes persist in the epidermis so that these structures become pigmented.

The developmental events regulating these migratory processes must be tightly controlled, but little is known about how this is achieved. One study suggested the cadherins have a role in this process: as dermal melanoblasts move through the basement membrane, they turn on E-cadherin, which is then downregulated and replaced by P-cadherin during migration into the hair follicles⁶. But our understanding of these regulatory events remains rudimentary.

Mutant G proteins

The new work from Greg Barsh and colleagues provides interesting insights into the regulation of melanoblast fate in the early embryo. As a starting point, Van Raamsdonk et al. adopted a positional cloning strategy to identify the genes underlying the Dsk1, Dsk7 and Dsk10 loci. They found that all three phenotypes were caused by mutations in genes encoding functionally related G-protein subunits: Dsk1 and Dsk10 are missense mutations in Gnaq, whereas Dsk7 is a missense mutation in Gna11.

These observations led to an obvious question: do these mutations produce a loss or gain of function in the resulting G proteins? To address this point, the authors crossed the mutations onto Gnaq- or Gna11-null mutant backgrounds and showed that the Dsk phenotypes could be suppressed by loss of function of the other allele. A Dsk mutation in Gna11 could also be suppressed by loss of Gnaq function, and also vice versa, to some extent. These results indicate that the newly characterized Dsk mutations are hypermorphic, gain-of-function alleles that lead to an overstimulation of the signal transduction pathway. Furthermore, the dark skin phenotype showed a dose response to these hypermorphic alleles: as the number of Dsk alleles increased, the dermis became progressively darker (Fig. 1). In addition, the two Gα subunits seem to be interchangeable: the phenotype of a mouse with two hypermorphic Gna11 alleles was similar to that of a mouse with one hypermorphic Gna11 allele and one hypermorphic Gnaq allele.

Figure 1: Photographs illustrating the dark skin phenotype of Dsk7 mice.

Photo courtesy of Greg Barsh

The top panels show (from left to right) footpads of wild-type, Gna11^Dsk7/+ and Gna11^Dsk7/Desk7 mice. The dermal pigmentation increases with increasing copies of the mutated allele. The bottom panel shows mice homozygous with respect to the hypomorphic mutation Kit^W-v. The mouse on the left is Gna11^+/+, and the one on the right is Gna11^Dsk7/+. The dark skin on the ears of the Dsk7 mouse shows that activated G-protein signaling can partially rescue the pigmentary defects caused by a reduction in Kit activity.

Unexpectedly, the overactive G proteins encoded by the Dsk alleles do not seem to have a strong adverse effect on fitness. With increasing mutant dose, mice became proportionately smaller, such that a mouse with four mutant copies is ∼25% underweight, but otherwise appears normal. This was somewhat unexpected, as transgenic mice with increased Gnaq expression in the heart have cardiac hypertrophy⁷.

What is the developmental basis for the increase in dermal pigmentation? Using a reporter gene⁸, the authors followed melanoblast development in Dsk mutant embryos and found an early excess of melanoblasts. But the number of melanoblasts that ultimately reached the epidermis and hair follicles was not affected by the mutations. It seems, then, that the migration of melanoblasts across the skin basement membrane is precisely regulated, such that only the correct number reaches the epidermis. In the mutants, however, the excess melanoblasts resulting from overstimulation of G-protein signaling are retained in the dermis, resulting in the dark skin phenotype.

Receptor coupling

Are these mutant Gα subunits constitutively active in isolation, or do they require coupling to receptor? The latter possibility seems more likely a priori, given the relatively mild phenotype of Dsk mice. Ednrb is a G protein–coupled receptor required for early melanoblast development⁹ and is known to couple to several Gα subunits, including Gα11 and Gαq (ref. 10). Van Raamsdonk et al. show that the virtual absence of melanocytes in Ednrb mice was not rescued by the Dsk mutations. In contrast, the Dsk mutations did partially rescue the pigmentary defects resulting from loss of Pax3 or Kit (Fig. 1).

The time at which Dsk embryos have increased numbers of melanoblasts correlates well with the crucial period during which Ednrb is required for their development⁹. Therefore, the Dsk mutations probably exert their effects through coupling of Gα11 and Gαq to this receptor. But the mutations have not been shown to act autonomously in melanoblasts. It is possible that the Gα proteins are active in neighboring dermal cells, which then signal to the melanoblasts to affect their behavior. Indeed, two dark skin mutations that cause increased epidermal pigmentation are due to changes in genes encoding keratin and EGF-receptor, resulting in hyperkeratosis and thickening of the epidermis, and in these instances, hyperpigmentation is a secondary effect³. This caveat aside, melanoblasts from the mutant mice may be extremely valuable in dissecting the signaling pathways downstream of Ednrb. If the proliferative effects of the receptor are indeed mediated by Gα11 and Gαq, IP3 is probably the intracellular messenger, leading to calcium release and activation of the MAPK/ERK pathway through Src¹⁰.

Despite decades of research on coat-color genes, mutagenic screens continue to identify new dominant mutations that affect pigmentation. Recessive screens will probably identify many more, particularly if we can identify those lethal mutations that affect the early stages of neural crest development. Few such mutations have been described, and there is a rich vein to be mined.