Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function. Amanda L. Blasiusa, Katharina Brandla, Karine Crozata,1, Yu Xiaa, Kevin Khovanantha, Philippe Krebsa, Nora G. Smarta, Antonella Zampollib, Zaverio M. Ruggerib, and Bruce A. Beutlera,2 Departments of aGenetics and bMolecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Bruce A. Beutler, December 24, 2008 (sent for review December 21, 2008) The classical recessive coat color mutation misty (m) arose spon- some-related organelles (LROs) may be affected in other cell taneously on the DBA/J background and causes generalized hy- types. Such organelles include lysosomes, platelet dense gran- popigmentation and localized white-spotting in mice, with a lack ules, and cytotoxic granules of neutrophils, natural killer (NK) of pigment on the belly, tail tip, and paws. Here we describe cells, and T lymphocytes (7). moonlight (mnlt), a second hypopigmentation and white-spotting Members of the Rho family of GTPases, including Rhos, Racs mutation identified on the C57BL/6J background, which yields a and Cdc42, are well known for their ability to restructure the phenotypic copy of m/m coat color traits. We demonstrate that the actin cytoskeleton and for subsequent effects on multiple bio- 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result logical functions including cell migration, phagocytosis, vesicular from mutations that truncate the dedicator of cytokinesis 7 protein transport, apoptosis, and proliferation (8). These factors are (DOCK7), a widely expressed Rho family guanine nucleotide ex- implicated in keratinocyte cytophagocytosis of melanocyte den- change factor. Although Dock7 is transcribed at high levels in the drites, melanocyte dendrite formation, and melanosome trans- developing brain and has been implicated in both axon develop- port and exocytosis, all processes that are important for the ment and myelination by in vitro studies, we find no requirement deposition of melanin into the skin and growing hair shaft GENETICS for DOCK7 in neurobehavioral function in vivo. However, DOCK7 (9–13). Rho GTPases are active when bound to GTP, are has non-redundant role(s) related to the distribution and function inactive in their GDP-bound form, and are tightly regulated by of dermal and follicular melanocytes. a variety of factors including the guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP, the misty ͉ melanocyte ͉ coat color ͉ guanine nucleotide exchange factor ͉ GTPase-activating proteins (GAPs) that enhance the GTPase rho GTPase activity of Rho proteins, and the Rho guanine nucleotide- dissociation inhibitors (RhoGDIs) that sequester Rho GTPases elanoblasts arise from the neural crest during develop- in a GDP-bound state (8, 14). GEFs that activate Rho GTPases Mment, migrate laterally within the embryonic ectoderm, can be divided into 2 main groups: the classical GEFs containing and ultimately differentiate into pigment producing melanocytes the nucleotide-exchanging Dbl-homology (DH) domain, and the found in the skin and hair follicles during postnatal life. Some dedicator of cytokinesis (DOCK)180 superfamily (14). mutations are known to cause localized hypopigmentation We now describe moonlight (mnlt), a pigmentation variant that (white-spotting), while others cause generalized hypopigmenta- simultaneously displays both pigment dilution and white- tion (pigment dilution). White-spotting results from the absence spotting phenotypes. The mnlt phenotype consists of an overall of melanocytes in patches of skin or hair follicles, typically caused by defects in melanoblast ontogeny, proliferation, sur- dilution of coat color and variable amounts of white-spotting on vival, migration, or differentiation. Anatomically distinct forms the belly, tail tip, paws, and genitalia. These pigmentation of white-spotting include belly spots, dorsal spots, belting of the defects are similar to those found in the classical mutation misty caudal trunk, piebaldism, head spots, white tipped tail or digits, (m), which was discovered nearly 70 years ago (15). The gene and peppering (1). Spotting may occur as an isolated phenotype underlying the m/m phenotype has not previously been deter- but may be associated with other developmental errors, reflect- mined. Here, we demonstrate that mnlt and m are allelic and that ing the role of neural crest derivatives in many different organ both phenotypes arise from alterations of Dock7, which encodes systems. In humans, spotting mutations are sometimes associ- a Rho family GEF belonging to the DOCK180 protein family. In ated with various forms of Waardenburg Syndrome (WS), cell culture studies, DOCK7 has been shown to activate Racs and caused by mutations in many different genes and characterized Cdc42 and to promote axon formation as well as Schwann cell by localized hypopigmentation, deafness, and other developmen- migration (16, 17). Our findings indicate that DOCK7 plays an tal anomalies including defects of hematopoiesis, megacolon, or important role in pigmentation and may be involved in mela- neurological, cardiac, and craniofacial abnormalities (2). nocyte ontogeny and function. Pigment dilution occurs when melanocytes are present but are unable to produce or export melanin in normal quantities. This may be due to impaired synthesis of melanin or to defects of Author contributions: A.L.B., K.B., P.K., and B.A.B. designed research; A.L.B., K.B., K.C., Y.X., K.K., P.K., and A.Z. performed research; K.C. and Z.M.R. contributed new reagents/analytic melanosomal trafficking and/or exocytosis (3). Various forms tools; A.L.B., Y.X., K.K., P.K., A.Z., Z.M.R., and B.A.B. analyzed data; and A.L.B., N.G.S., and of oculocutaneous albinism, including different varieties of B.A.B. wrote the paper. Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syn- The authors declare no conflict of interest. drome (CHS), and Griscelli syndrome result from mutations that 1Present Address: Centre d’Immunologie de Marseille-Luminy, Universite´ delaMe´ diterra- cause aberrant melanin synthesis, cargo loading, trafficking, or ne´e, 13288 Marseille, France. secretion (4–6). Associated defects of immunological, neuro- 2To whom correspondence should be addressed. E-mail: [email protected]. logical, or hemostatic function may be observed with such This article contains supporting information online at www.pnas.org/cgi/content/full/ mutations because the formation or intracellular transport of 0813208106/DCSupplemental. homologous membrane-delimited organelles known as lyso- © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813208106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 females are poor mothers and their pups invariably require foster A care to reach weaning age. Many mutations resulting in hypopigmentation are due to generalized defects in granule function, affecting both melano- somes and other LROs. In particular, the related platelet dense granules may be abnormally formed or secreted, resulting in a bleeding diathesis. Furthermore, immune cells, including NK cells and T cells, use cytotoxic granules for killing target cells. To test LRO function in mnlt/mnlt mice, we assayed bleeding time, but found no defect (Fig. 2). Furthermore, we tested cytotoxic efficiency of NK cells and T cells against target cells using an in vivo assay (Fig. 2). Again, we found no evidence of a generalized LRO anomaly. Further evidence of normal NK cell function comes from the normal resistance of mnlt/mnlt mice to mouse B D cytomegalovirus (MCMV) in an in vivo test for susceptibility (18). At 105 pfu of MCMV per mouse, signs of sickness were observed in BALB/c mice by day 3 and 50% mortality was observed on day 6, whereas C57BL/6J and mnlt/mnlt mice appeared healthy up to 8 days after inoculation (data not shown). E In addition, viral clearance by mnlt/mnlt and C57BL/6J mice was comparable when splenic viral titers were measured at day 5 post infection (data not shown). Mapping and Identification of the mnlt Mutation. mnlt/mnlt mice C F (C57BL/6J background) were outcrossed to C3H/HeN mice and the F1 progeny were backcrossed to the mutant stock or inter- crossed. F2 mice were scored based on pigmentation. On 24 meioses the mutation was mapped to chromosome 4 (peak LOD score of 5.4; Fig. 3A). Fine mapping on a total of 57 meioses established a 12.4 Mb critical region delimited by D4Mit301 (88.7 Mb) and D4Mit176 (101.2 Mb). This region overlapped with the previously published critical region for m, a spontane- ous mutation that arose on the DBA/J background. The m phenotype is similar to that of mnlt, featuring generalized Fig. 1. Moonlight coat color phenotype. (A) mnlt/mnlt mice have an overall reduction in pigmentation (mnlt/mnlt, Left; C57BL/6J, Right). mnlt/mnlt mice hypopigmentation, white digits, a belly spot, and white tail tip additionally display (B) a white tail tip (mnlt/mnlt, Top; C57BL/6J, Bottom), (C) (15, 19). Although subsequent studies mapped m to chromosome paws, (D and E) genitalia (D) mnlt/mnlt ;(E) C57BL/6J and (F) belly spot. 4 (20, 21), the causative mutation was never identified. To determine whether m and mnlt might be allelic mutations, homozygotes of the 2 strains were crossed. Complementation Results was not observed, suggesting that mnlt and m represent 2 alleles Moonlight Phenotype. N-ethyl-N-nitrosourea (ENU) mutagenesis of the same gene. of male C57BL/6J mice and subsequent breeding to the third The mnlt critical region contained a total of 218 annotated (G3) generation allowed production of mice with homozygous genes, derived from an ‘‘OR’’ search of NCBI, OTTMUSG, germline mutations. A coat color mutant was identified in the G3 ENSMUSG, and GENESCAN. Primers were designed for PCR population and shows strict recessive inheritance. The pheno- amplification and sequencing of 1483 potential coding exons of type was named moonlight (mnlt).