A Rac1-Independent Role for P-Rex1 in Melanoblasts

TO THE EDITOR Given the recent discovery of RAC1activating mutations in melanoma, and our finding that PIP3-dependent Racexchanger 1 (PREX1) is overexpressed and drives metastasis in this cancer, an important question is to establish whether the functions of P-Rex1 are mediated specifically by Rac alone (Lindsay et al., 2011; Berger et al., 2012). Here we describe a Rac1-independent in vivo role for P-Rex1 through identification and characterization of a mouse coat color phenotype. P-Rex1 is a guaninenucleotide exchange factor (GEF) for Rac, whose primary cell function is induction of actin-mediated membrane ruffling and lamellipodia formation at the leading edge of cell migration (Welch et al., 2002; Hill et al., 2005; Barber et al., 2007). To investigate this question we decided to examine the role of Rac1 and P-Rex1 in melanoblast development. Previously, we reported a ‘‘white belly’’ phenotype of mice with Prex1 deletion (Lindsay et al., 2011). Impaired melanoblast migration was mostly responsible for this phenotype, with melanoblasts lacking at the most distal points of migration (belly and paws). Constitutive deletion of Rac1 is embryonically lethal, but a coat color defect of mice with melanocyte-specific RAC1 abrogation (Tyr::Cre Rac1) has also been described; these mice have a larger belly spot on their ventral side, suggesting that alternative Rho-GTPases can be activated to enable melanoblast migration to the perimeter of the Tyr::Cre Rac white belly (Sugihara et al., 1998; Li et al., 2011). A role for Rac1 in proliferation was also observed, as there was a marked reduction of melanoblast numbers in this phenotype. In line with these previous studies, and because mice with melanocyte-specific RAC1 abrogation require euthanization shortly after birth because of neurological problems, we used the same embryonic melanoblast reporter models to assess the downstream effects of P-Rex1 in vivo (Mackenzie et al., 1997; Mort et al., 2010; Li et al., 2011). Melanocyte-specific reporter mouse strains employed were Tyr::Cre Z/EG, which drives green fluorescent protein expression in the melanoblast lineage, and DCT::bgalactosidase (otherwise referred to as DCT-lacZ). First, we hypothesized that, if the effects of P-Rex1 were mediated exclusively via Rac1, double mutant Tyr::Cre Rac1; P-Rex1 / mice would exhibit the same coat color phenotype as Tyr::Cre Rac mice alone. However, Tyr::Cre Rac; P-Rex1 / mice display a dramatic alteration in coat color phenotype from Tyr::Cre Rac mice (n1⁄4 7; Figure 1a). The ventral and dorsal coats of these mice are almost entirely white, with hypo-pigmented limbs and tail. Graying pigmented areas were only observed in the head coat. We concluded from this experiment that P-Rex1 and Rac1 together constitute fundamental signaling components of the mouse coat color phenotype, with minimal rescue of melanoblast development conferred by other GEFs or RhoGTPases. It was also clear that P-Rex1 must be able to exert phenotypic effects other than via Rac1. To explore the Rac1-independent effects of P-Rex1 further, we crossed Tyr::Cre Rac; P-Rex1 / mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Lindsay et al., 2011). Relative to Tyr::Cre Racmice or P-Rex1 / embryos alone, Tyr::Cre Rac; P-Rex1 / Accepted article preview online 30 July 2014; published online 11 September 2014 Abbreviations: GEF, guanine-nucleotide exchange factor; OHT, 4-hydroxytamoxifen; PREX1, PIP3dependent Rac-exchanger 1 CR Lindsay et al. Role of Rac1 and P-Rex1 in Melanoblast Development