Mice with mutations of 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 (7). moonlight (mnlt), a second hypopigmentation and white-spotting Members of the Rho family of , 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 , phagocytosis, vesicular from mutations that truncate the dedicator of cytokinesis 7 transport, , 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 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 (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 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 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 and characterized Cdc42 and to promote axon formation as well as 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 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). mnlt/mnlt mice display an genes within this region (22). High-quality coverage of 83.8% of overall lightened coat color and hypopigmentation of the distal all targeted nucleotide pairs was achieved in amplification extremities, particularly the paws, tail tip, and genitalia (Fig. 1). products from both wild-type and mnlt/mnlt templates on first The homozygotes often have belly spots as well. Although pass. No mutations were observed. However, in one candidate spotting is always present in the paws, other types of spotting are gene, Dock7, several contiguous exons could not be amplified or incompletely penetrant. It can be scored in the presence of the sequenced from the mnlt/mnlt template, although they were dominant Agouti allele derived from C3H/HeN mice, used in readily amplified and sequenced from the C57BL/6J template. mapping. Both male and female mnlt/mnlt mice are fertile, but We therefore sequenced Dock7 from C57BL/6J and mnlt/mnlt

140 100 100 120 90 90 80 100 80 70 70 80 60 60 50 60 50 Seconds 40 40 40 30 30 20 20 20 % Killing target cells target % Killing % Killing TAP1-/- cells TAP1-/- % Killing 10 10 0 0 0 C57BL/6J moonlight C57BL/6J moonlight C57BL/6J moonlight

Fig. 2. Moonlight bleeding time and cytotoxicity. Bleeding time for mnlt/mnlt and wild-type C57BL/6J mice. Data shown from one of two experiments with similar results, n ϭ 5(Left). NK cell cytotoxicity was assessed by in vivo killing of TAPϪ/Ϫ cells, mnlt/mnlt n ϭ 3, C57BL/6J n ϭ 6(Middle). Following immunization, in vivo killing was assayed on peptide pulsed target cells, mnlt/mnlt n ϭ 3, C57BL/6J n ϭ 6(Right). All panels depict means Ϯ SEMs.

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GGACGGTTGAAGACCAGTTGCAGAAACGAGGATTATAAAGTAATATGAATGCCCATTG C G R L K T S C R N E D Y K V I * GENETICS misty

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Fig. 3. Identification of the mnlt and m mutations. (A) The mnlt mutation was confined to chromosome 4 using a panel of 128 informative markers. The peak LOD score is 5.4 on chromosome 4. (B) Effect of the mnlt mutation on Dock7 mRNA and protein. Exons 22–28 are deleted, resulting in splicing of exon 21 directly to exon 29. Unbolded text indicates wild-type Dock7 sequence. An aberrant splice joins nt 2596 to nt 3519. Nucleotides of exon 29 are italicized, and the frame-shifted protein product is both highlighted and bolded. (C) Effect of the m mutation on Dock7 mRNA and protein. An anomalous insertion occurs within exon 18. Unbolded text indicates wild-type Dock7 sequence; following the insertion, aberrant nucleotides and the translation product are bolded and highlighted. For exon diagrams in (B) and (C), coding regions are shown in black; non-coding regions in gray.

cDNA amplified from the brain and observed a 922 bp deletion Neurobehavioral Analyses of Dock7mnlt/mnlt Mice. In vitro studies in the latter, encompassing bp 2,597 through 3,518 (with refer- have previously suggested that DOCK7 might be important for ence to the A of the start codon, GenBank accession no. the formation of (23). In addition, knockdown studies have NM࿝026082.4). Further sequencing at the genomic DNA level suggested that DOCK7 functions as an intracellular substrate for defined a 19,025 bp deletion encompassing bases 98,649,759 ErbB2, a plasma membrane tyrosine kinase, and is necessary for through 98,668,783 (NCBI mouse assembly version 37.1; sup- in vitro migration of Schwann cells and subsequent myelination porting information (SI) Fig. S1A). The boundaries of this of axons (17). Although these reports would suggest an essential deletion reside within introns 21 and 28, excising exons 22 role of DOCK7 in development and function of the nervous mnlt/mnlt m/m through 28 (of 48 total exons). The cDNA sequence is consistent system, neither Dock7 nor Dock7 mice show obvious mnlt/mnlt with splicing of exon 21 to 29, which results in a frameshift after neurological impairment. Dock7 mice cannot be distin- amino acid 865, with the incorporation of 30 aberrant amino guished from wild-type animals when measuring depressive acids followed by a premature stop codon (Fig. 3B). behavior in the tail suspension test (Fig. 4A), learning measured The subset of primers specific for Dock7 were subsequently by habituation of object exploration (Fig. 4B), working memory using the Y maze test (Fig. 4C), anxiety-like behavior in a used to amplify and sequence genomic DNA from m/m mice. An light/dark transfer test (Fig. 4D), or locomotor activity LTR retrotransposon insertion after nucleotide 2,045 (with (SI Text and Fig. S2). Female Dock7mnlt/mnlt mice show a trend reference to the A of the start codon, GenBank accession no. toward decreased social interaction, although the difference was ࿝ Dock7 m m NM 026082.4) was found to interrupt exon 18 of in / not significant and males were normal (Fig. 4E). Finally, mice (Fig. S1B). Following amino acid 682 of the DOCK7 Dock7mnlt/mnlt mice are neither blind nor deaf (data not shown). protein, ten aberrant amino acids are added, after which chain Overall, we find no clear evidence that behavior of Dock7mnlt/mnlt termination occurs (Fig. 3C). mice behavior differs from that of wild-type C57BL/6J mice and The 2,130 aa DOCK7 protein is a member of the DOCK180 conclude that neurological function is at least grossly normal in family, all members of which share 2 conserved domains, DHR-1 mice with early truncation of DOCK7. (DOCK homology region 1) or CZH-1 (CDM-zizimin homology 1) domain and the more C-terminal DHR-2 or CZH-2 domain. Discussion The deletion in mnlt/mnlt mice leads to chain termination after The DOCK180 proteins are guanine nucleotide exchange factors the DHR-1 domain. In m/m mice, chain termination occurs that act on Rho-family GTPases, a class of Ras-homolog small within the DHR-1 domain. The junctional sequences of GTPases of which numerous representatives are known in Dock7mnlt and Dock7m alleles have been deposited in GenBank mammals (8, 14). Rho GTPases are known to cycle between (accession numbers FJ590429 and FJ590430, respectively). GDP-associated and GTP-associated forms. The GTP-

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Fig. 4. Neurobehavioral studies of Dock7mnlt/mnlt mice. (A) Tail suspension test. Mice are suspended by the tail and monitored for movement. Shown is the cumulative time spent immobile over the course of the trial. (B) Habituation of object exploration. Mice are placed into a field containing 3 plastic objects. Contacts with each object are measured over 5 min of time. Contacts for the 3 objects are averaged for each mouse. Three consecutive trials are performed. (C) Y maze test. The percentage of spontaneous alterations is the number of consecutive triplets of different arm choices out of the total number of arm choices. (D) Light/dark transfer test. Total time spent in the brightly lit compartment was measured (Left) and the total number of transitions between light and dark compartments were counted (Right). (E) Social interaction test. Sociability is measured as the number of entries into a chamber containing a stranger mouse. Differences between Dock7mnlt/mnlt and C57BL/6J controls are not significant (females P ϭ 0.0619). Social novelty measures the number of entries into a chamber containing a stranger mouse as opposed to a chamber containing a familiar mouse. C57BL/6J is depicted by open bars; Dock7mnlt/mnlt is depicted by filled bars. Data are represented as means Ϯ SEMs, n ϭ 8.

associated forms are generally coupled to effector proteins and membranous organelles (16, 26). The DHR2 domain inter- that are stimulated to induce polymerization of the actin acts with the Rho GTPase and is required for guanine nucleotide cytoskeleton or to maintain tubulin polymerization, which in exchange. DOCK7 has been shown to activate Rac and Cdc42 turn is required for higher-order processes such as migration, but not RhoA, through its DHR-2 domain (17). proliferation, adhesion, vesicular transport, secretion, main- In neuronal cell cultures, DOCK7 plays an important role in tenance of cell morphology, cytokinesis, ruffling, apoptosis, axon outgrowth, Schwann cell migration, and axon myelination phagocytosis, polarization, and cell survival (8). (16, 17). Rac activation by DOCK7 results in the inactivation of Hydrolysis of GTP to GDP occurs when a GTPase activating the microtubule destabilizing protein stathmin/Op18 and is protein interacts with the GTP-bound Rho-family GTPase. necessary for axon outgrowth (16, 27), a process that depends on Regeneration of the GTP-bound form of the protein depends specific localization of DOCK7 to the developing axon and is upon the action of a guanine-nucleotide exchange factor (GEF), likely mediated by the interaction of DOCK7 with PIP3 (16). exemplified by members of the DOCK180 superfamily. To date, Schwann cell migration appears to depend on the binding of this family consists of 11 members that have been divided into axonal neuregulin to ErbB receptors resulting in DOCK7 acti- 4 subfamilies on the basis of primary sequence alignment: vation and subsequent activation of both Rac1 and Cdc42 (17). DOCK-A, DOCK-B, DOCK-C, and DOCK-D (24–26). DOCK7 These results, along with the high expression of DOCK7 in the falls within the DOCK-C subfamily and is most closely related to developing brain (16), suggest that DOCK7 may play an impor- and . DOCK family members contain 2 defin- tant role in neurological functions. DOCK7 has also been found ing domains, DHR1 and DHR2. The DHR1 domain is a lipid to interact with the (TSC) protein, hamartin binding moiety predicted to allow interaction with phosphati- (28, 29). Tuberous sclerosis is a multisystem disorder character- dylinositol (PtdIns) (3, 5)-bisphosphate and PtdIns(3,4,5)P3 ized by tumor-like growths, and the TSC protein complex has (PIP3), produced by activated PI3 kinase (PI3K). The DHR1 been shown to have GTPase activity (29). domain may specify localization of DOCKs within the cell, thus Mutations in DOCK7 have not previously been reported in potentially limiting the activity of their target GTPases to any organism. In this paper, we demonstrate that m and mnlt are specific cellular substructures including the plasma membrane variant alleles of Dock7 that produce abnormal phenotypes in

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813208106 Blasius et al. Downloaded by guest on September 29, 2021 the homozygous state. Both alleles are predicted to eliminate mutations introduced by mutagenesis or spontaneous mutations. most of the DOCK7 sequence (68% and 59% of the polypeptide Alternatively, the genetic background on which the m and mnlt chain, respectively) by causing premature termination and to add mutations occurred might be permissive for the hemostatic and extraneous amino acids before termination. Although the mnlt nurturing phenotypes, respectively. Finally, cis-acting effects of phenotype was identified in an ENU mutagenesis screen, this either of these two mutations (a large insertion in the case of mutagen generally causes point mutations rather than large Dock7m and a large deletion in the case of Dock7mnlt) might elicit deletions. Thus, both Dock7mnlt and Dock7m presumably arose as phenotypic differences that are not related to the Dock7 locus spontaneous mutations. Homozygosity for either allele, or com- itself. pound heterozygosity for both alleles, causes generalized The phenotypes that we have ascribed with confidence to the reduction in pigmentation and white-spotting, suggesting non- m and mnlt mutations in Dock7 on the basis of complementation redundant function of DOCK7 in two processes related to analysis and interstrain comparisons are restricted to abnormal- pigmentation. We propose that the protein may be required for ities of pigmentation. We have been unable to detect a defect in vesicle transport or exocytosis and also for the formation, NK and T cell effector function in Dock7mnlt/mnlt mice, which also proliferation, migration, or survival of melanoblasts during show normal resistance to MCMV infection. MCMV suscepti- embryogenesis. Previous studies have shown that melanoblasts bility often reveals subtle NK cell defects (37). Additionally, from Dock7m/m mice are defective in proliferation and differen- Dock7mnlt/mnlt mice do not appear to have a bleeding diathesis, tiation (30), suggesting that DOCK7 plays an important role in which is often caused by defects in platelet dense granules (4, 7). some aspect of melanoblast and/or melanocyte function. We therefore conclude that if DOCK7 is required for LRO DOCK180 family members function in migration, prolifera- function in mice; this requirement is not widely generalized with tion, and survival of cells (14, 25), and a defect in any of these respect to cell lineage. in melanoblasts could be responsible for the spotting pattern observed. The pigment dilution phenotype suggests a melano- Materials and Methods cyte defect in the formation or trafficking of melanosomes or Mice. m/m mice (C57BLKS background) were purchased from Taconic Farms. their exocytosis to keratinocytes of the hair shaft. The activation C57BL/6J and C3H/HeN mice were bred at The Scripps Research Institute. of Rac and Cdc42, but not RhoA, by DOCK7 suggests a possible C57BL/6J mice were mutated with ENU as previously described (38, 39) and mechanism for the pigmentation phenotypes displayed by mnlt/mnlt variants were observed among the G3 population and expanded m/m mnlt/mnlt to form a stock based on their visible phenotype. Mice were maintained Dock7 and Dock7 mice. Both Rho and Rac family GENETICS members contribute to the formation of melanocyte dendrites under standard housing conditions, and all procedures were approved and that are necessary for the transfer of melanin from melanocytes performed according to institutional guidelines for animal care. The Dock7mnlt/mnlt stock was transferred to the Mutant Mouse Regional Resource to adjacent keratinocytes (12, 13). Indeed, numerous studies Centers for distribution (stock no. 030498-UCD). indicate that dendrite formation in neural cells is controlled by a balance between Rac and Rho activities, with Rho activation Mapping and DNA Sequencing. Homozygous Dock7mnlt/mnlt males were out- promoting neurite retraction and Rac activation promoting crossed to C3H/HeN females. F1 hybrid mice were subsequently backcrossed to neurite outgrowth (13, 31, 32, 33). A similar balance between the mutant stock or intercrossed. F2 progeny were phenotyped and linkage Rac and Rho activity in dendrite formation has been described was measured using 128 microsatellite markers. The critical region was in melanocytes, wherein high cAMP levels inhibit Rho activity masked with RepeatMasker, and optimized primers were designed to amplify while promoting Rac activity and dendrite formation. Cdc42 and sequence all annotated coding regions and splice junctions. Amplification activity has also been implicated in neurite outgrowth, and is of genomic template from C57BL/6J and mnlt/mnlt mice was performed using suggested to be important for melanocyte function. Additionally, two Beckman FX robots, which also cleaned, measured, and diluted the all three of these GTPases appear to play important roles in products, adding the correct sequencing primers to each. Sequencing was performed using an ABI 3730XL DNA analyzer. Trace alignments were made keratinocyte cytophagocytosis, a process in which the keratino- using the programs phred and Phrap, and analysis performed with the pro- cytes in the skin and hair follicles phagocytose a melanocyte gram consed. High quality coverage was defined as a phred score Ն30 for any dendrite, leading to a phagolysosome from which melanin bp within the target. granules disperse throughout the cytoplasm. A number of publications have suggested that DOCK7 might Bleeding Time. While a mouse is restrained, the tail tip is cut 3 mm from the be involved in fundamental neurodevelopmental processes such end. The tail is submerged in saline solution heated to 37 °C. Blood flow is as axon formation and myelination (16, 17). However, neither observed while holding the tail down and the time is recorded until bleeding Dock7mnlt nor Dock7m alleles have a profound effect on nervous ceases. system development or function. We cannot formally exclude the possibility that certain subtle aspects of behavior are abnormal Cytotoxicity and MCMV Resistance. For the in vivo NK cell cytotoxicity assay, in these mice, nor can we be certain that the fragmentary splenocyte suspensions from C57BL/6J controls and NK target, TAP1-deficient proteins encoded by these alleles are functionally null. However, mice were labeled with a low and high concentration of carboxyfluorescein we tentatively conclude that if DOCK7 contributes to the succinimidyl ester (CFSE) dye, respectively. The two populations were mixed at a 1:1 ratio and a total of 4 ϫ 106 cells were injected i.v. into recipient mice. formation of neural or glial elements of the central or peripheral Recipients were bled two days after injection, and peripheral blood mono- nervous system, its function as such is redundant. The closely nuclear cells (PBMCs) were analyzed for CFSE staining by flow cytometry. related protein DOCK6 has also been shown to interact with Killing of targets in TAP1-deficient recipients was used as a control to calculate Racs, as well as Cdc42, and to function in neurite growth (35). 0% killing and normalize all measurements. Moreover, mutations in DOCK8 have been found in patients For the in vivo cytotoxic T (CTL) cytotoxicity assay, mice were with mental retardation and developmental disabilities (36). The immunized i.p with 107 ␥-irradiated (1500 Rad) Ad5E1-MEC fibroblasts (mu- participation of other GEFs and other Rho family GTPases in rine embryo cells expressing human adenovirus type 5 early region 1) (40). neurodevelopment may compensate for DOCK7 deficiency. Eight days after immunization, mice were injected with a total of 4 ϫ 106 cells m/m of a 1:1 ratio of naive C57BL/6J splenocytes (CFSE low) and C57BL/6J spleno- Not all of the phenotypic anomalies observed in Dock7 and b Dock7mnlt/mnlt mice are shared. For instance, Dock7mnlt/mnlt mice cytes pulsed with an Ad5E1-MEC derived, H2-D restricted peptide (VNIRNC- m/m CYI; CFSE high). Recipient mice were bled two days after injection, and PBMCs do not show the bleeding phenotype seen in Dock7 animals were analyzed for CFSE staining by flow cytometry. mnlt/mnlt (30). On the other hand, Dock7 females are unable to MCMV stock (Smith strain) was prepared and titered as previously de- m/m raise their offspring, while Dock7 mice are adequate mothers. scribed (18, 41). In vivo susceptibility to the MCMV virus was tested as previ- We note that neither of these accessory phenotypes was mapped ously described (18). Briefly, MCMV-susceptible BALB/c mice were used as to the Dock7 locus and therefore could result from other positive controls for susceptibility, while MCMV-resistant C57BL/6J mice were

Blasius et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 29, 2021 used as negative controls. Mice were inoculated with 105 pfu MCMV via i.p. each measuring 20 ϫ 40.5 ϫ 22 cm, separated by a clear wall with an opening injection and monitored for signs of illness over 8 days. Viral titers were at floor level. Each outer chamber contains a small, round wire cage (Galaxy determined within the spleen at day 5 post infection as previously de- Cup, Spectrum Diversified Designs, Inc.). Mice are habituated to the apparatus scribed (42). for 5 min. A stranger mouse of the same sex is placed in one of the wire cages. Sociability is measured as the time spent in the chamber with the stranger Behavioral Assays. In the light/dark transfer test, a Plexiglas box is divided into mouse over 5 min. For the social novelty test, a second, unfamiliar mouse is a dark side (10 lux, 14.5 ϫ 27 ϫ 26.5 cm) and a brightly lit side (670 lux, 28.5 ϫ placed into the previously empty cage. Time spent in the chamber with the 27 ϫ 26.5 cm). The divider contains a 7.5 ϫ 7.5 cm opening at floor level. Mice unfamiliar mouse is recorded over 5 min. are placed in the dark compartment to start. Both the time in light and number For the tail suspension test, each mouse is suspended by its tail using of transitions are evaluated for 5 min. adhesive tape on a metal bar located 30 cm above a flat surface. Immobility is During the Y maze test, mice are placed in a Y maze (3 arms) and recorded quantified as the duration of time that no whole body movement occurs. Mice by video camera for 5 min. The total number of arm entries and the order of are observed for a total of 6 min. entries are evaluated. In the habituation of object exploration assay, 3 plastic objects are placed ACKNOWLEDGMENTS. A.L.B was supported by NIH Training Grant-T32 in each of 3 corners of an open field. A mouse is allowed to explore the area AI07244. K.B. was supported by the Alexander von Humboldt Foundation for 5 min. Three such sessions are completed for each mouse with a separation through a Feodor Lynen postdoctoral fellowship. P.K. was supported by a long of 5 min between sessions. Exploration or contacts are defined as the mouse term fellowship from European Molecular Biology Organization. This work approaching the object nose-first within 2–4 cm. was supported by NIH grants GM067759 and AI070167, and BAA Contract Social interaction is assessed in a Plexiglas container that has 3 chambers, HHSN272200700038C.

1. Baxter LL, Hou L, Loftus SK, Pavan WJ (2004) Spotlight on spotted mice: A review of 25. Cote J-F, Vuori K (2007) GEF what? Dock180 and related proteins help Rac to polarize white spotting mouse mutants and associated human pigmentation disorders. Pigm cells in new ways. Trends Cell Biol 17:383–393. Cell Res 17:215–224. 26. Cote J-F, Motoyama AB, Bush JA, Vuori K (2005) A novel and evolutionarily conserved 2. Tachibana M, Kobayashi Y, Matsushima Y (2003) Mouse models for four types of PtdIns(3,4,5)P3-binding domain is necessary for DOCK180 signalling. Nat Cell Biol Waardenburg syndrome. Pigm Cell Res 16:448–454. 7:797–807. 3. Bennett DC, Lamoreux ML (2003) The color loci of mice—a genetic century. Pigm Cell 27. Wittmann T, Bokoch GM, Waterman-Storer CM (2004) Regulation of microtubule Res 16:333–344. destabilizing activity of Op18/stathmin downstream of Rac1. J Biol Chem 279:6196– 4. Di Pietro SM, Dell’angelica EC (2005) The cell biology of Hermansky-Pudlak syndrome: 6203. Recent advances. Traffic 6:525–533. 28. Nellist M, Burgers PC, van den Ouweland AM, Halley DJ, Luider TM (2005) Phosphor- 5. Shiflett SL, Kaplan J, Ward DM (2002) Chediak-Higashi Syndrome: A rare disorder of ylation and binding partner analysis of the TSC1-TSC2 complex. Biochem Biophys Res lysosomes and lysosome related organelles. Pigm Cell Res 15:251–257. Commun 333:818–826. 6. Menasche G, et al. (2000) Mutations in RAB27A cause Griscelli syndrome associated 29. Rosner M, Hanneder M, Siegel N, Valli A, Hengstschlager M (2008) The mTOR pathway with haemophagocytic syndrome. Nat Genet 25:173–176. and its role in human genetic diseases. Mutat Res 658:234–246. 7. Wei ML (2006) Hermansky-Pudlak syndrome: A disease of protein trafficking and 30. Sviderskaya EV, Novak EK, Swank RT, Bennett DC (1998) The murine misty mutation: organelle function. Pigm Cell Res 19:19–42. Phenotypic effects on melanocytes, platelets and brown fat. Genetics 148:381–390. 8. Jaffe AB, Hall A (2005) Rho GTPases: Biochemistry and biology. Annu Rev Cell Dev Biol 21:247–269. 31. Yasui H et al (2001) Differential responses to nerve growth factor and epidermal 9. Van Den Bossche K, Naeyaert J-M, Lambert J (2006) The quest for the mechanism of growth factor in neurite outgrowth of PC12 cells are determined by Rac1 activation melanin transfer. Traffic 7:769–778. systems. J Biol Chem 276:15298–15305. 10. Scott G, et al. (2003) The proteinase-activated -2 mediates phagocytosis in a 32. Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG (1999) Rac Rho-dependent manner in human keratinocytes. J Invest Dermatol 121:529–541. downregulates Rho activity: reciprocal balance between both GTPases determines 11. Scott G, Leopardi S (2003) The cAMP signaling pathway has opposing effects on Rac and cellular morphology and migratory behavior. J Cell Biol 147:1009–1022. Rho in B16F10 cells: Implications for dendrite formation in melanocytic cells. Pigm Cell 33. Jalink K et al (1994) Inhibition of lysophosphatidate- and thrombin-induced neurite Res 16:139–148. retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding 12. Cardinali G, et al. (2005) Keratinocyte growth factor promotes melanosome transfer to protein Rho. J Cell Biol 126:801–810. keratinocytes. J Invest Dermatol 125:1190–1199. 34. Brown MD, Cornejo BJ, Kuhn TB, Bamburg JR (2000) Cdc42 stimulates neurite out- 13. Scott G (2002) Rac and rho: The story behind melanocyte dendrite formation. Pigm Cell growth and formation of growth cone filopodia and lamellipodia. J Neurobiol 43:352– Res 15:322–330. 364. 14. Rossman KL, Der CJ, Sondek J (2005) GEF means go: Turning on RHO GTPases with 35. Miyamoto Y, Yamauchi J, Sanbe A, Tanoue A (2007) Dock6, a Dock-C subfamily guanine guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol 6:167–180. nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite 15. Woolley GW (1941) ‘‘Misty,’’ a new coat color dilution in the mouse, Mus musculus. Am outgrowth. Exp Cell Res 313:791–804. Nat 75:507–508. 36. Griggs BL, Ladd S, Saul RA, DuPont BR, Srivastava AK (2008) Dedicator of cytokinesis 8 16. Watabe-Uchida M, John KA, Janas JA, Newey SE, Van AL (2006) The Rac activator is disrupted in two patients with mental retardation and developmental disabilities. DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18. Genomics 91:195–202. 51:727–739. 37. Beutler B et al (2007) Genetic analysis of resistance to viral infection. Nat Rev Immunol 17. Yamauchi J, Miyamoto Y, Chan JR, Tanoue A (2008) ErbB2 directly activates the 7:753–766. exchange factor Dock7 to promote Schwann cell migration. J Cell Biol 181:351–365. 38. Hoebe K, et al. (2003) Identification of Lps2 as a key transducer of MyD88-independent 18. Crozat K, et al. (2006) Analysis of the MCMV resistome by ENU mutagenesis. Mamm TIR signalling. Nature 424:743–748. Genome 17:398–406. 39. Hoebe K, Du X, Goode J, Mann N, Beutler B (2003) Lps2: A new locus required for 19. Woolley GW (1945) Misty dilution in the mouse. J Hered 36:269–270. responses to lipopolysaccharide, revealed by germline mutagenesis and phenotypic 20. Fiedorek FT, Jr, Kay ES (1994) Mapping of PCR-based markers for mouse chromosome screening. J Endotoxin Res 9:250–255. 4 on a backcross penetrant for the misty (m) mutation. Mamm Genome 5:479–485. ϩ ϩ 21. De ME, Dandoy F (1987) Linkage analysis of the murine interferon alpha locus (Ifa) on 40. Janssen EM et al (2005) CD4 T-cell help controls CD8 T-cell memory via TRAIL- chromosome 4. J Hered 78:143–146. mediated activation-induced cell death. Nature 434:88–93. 22. Beutler B, Du X, Xia Y (2007) Precis on forward genetics in mice. Nat Immunol 41. Tabeta K, et al. (2004) Toll-like receptors 9 and 3 as essential components of innate 8:659–664. immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci USA 23. Pinheiro EM, Gertler FB (2006) Nervous Rac: DOCK7 regulation of axon formation. 101:3516–3521. Neuron 51:674–676. 42. Orange JS, Wang B, Terhorst C, Biron CA (1995) Requirement for - 24. Cote J-F, Vuori K (2002) Identification of an evolutionarily conserved superfamily of produced interferon gamma in defense against murine cytomegalovirus infection and DOCK180-related proteins with guanine nucleotide exchange activity. J Cell Sci enhancement of this defense pathway by interleukin 12 administration. J Exp Med 115:4901–4913. 182:1045–1056.

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