Mice with mutations of Executeck7 have generalized hypopigme

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

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Abstract

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we Characterize moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (ExecuteCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Executeck7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for ExecuteCK7 in neurobehavioral function in vivo. However, ExecuteCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.

Keywords: mistymelanocytecoat colorguanine nucleotide exchange factorrho GTPase

Melanoblasts arise from the neural crest during development, migrate laterally within the embryonic ectoderm, and ultimately differentiate into pigment producing melanocytes found in the skin and hair follicles during postnatal life. Some mutations are known to cause localized hypopigmentation (white-spotting), while others cause generalized hypopigmentation (pigment dilution). White-spotting results from the absence of melanocytes in patches of skin or hair follicles, typically caused by defects in melanoblast ontogeny, proliferation, survival, migration, or differentiation. Anatomically distinct forms of white-spotting include belly spots, Executersal spots, belting of the caudal trunk, piebaldism, head spots, white tipped tail or digits, and peppering (1). Spotting may occur as an isolated phenotype but may be associated with other developmental errors, reflecting the role of neural crest derivatives in many different organ systems. In humans, spotting mutations are sometimes associated with various forms of Waardenburg Syndrome (WS), caused by mutations in many different genes and characterized by localized hypopigmentation, deafness, and other developmental anomalies including defects of hematopoiesis, megacolon, or neurological, cardiac, and craniofacial abnormalities (,2).

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 melanosomal trafficking and/or exocytosis (3). Various forms of oculoSliceaneous albinism, including different varieties of Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS), and Griscelli syndrome result from mutations that cause aberrant melanin synthesis, cargo loading, trafficking, or secretion (,4–,6). Associated defects of immunological, neurological, or hemostatic function may be observed with such mutations because the formation or intracellular transport of homologous membrane-delimited organelles known as lysosome-related organelles (LROs) may be affected in other cell types. Such organelles include lysosomes, platelet dense granules, and cytotoxic granules of neutrophils, natural Assassinateer (NK) cells, and T lymphocytes (,7).

Members of the Rho family of GTPases, including Rhos, Racs and Cdc42, are well known for their ability to restructure the actin cytoskeleton and for subsequent Traces on multiple biological functions including cell migration, phagocytosis, vesicular transport, apoptosis, and proliferation (8). These factors are implicated in keratinocyte cytophagocytosis of melanocyte dendrites, melanocyte dendrite formation, and melanosome transport and exocytosis, all processes that are Necessary for the deposition of melanin into the skin and growing hair shaft (,9–,13). Rho GTPases are active when bound to GTP, are inactive in their GDP-bound form, and are tightly regulated by a variety of factors including the guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP, the GTPase-activating proteins (GAPs) that enhance the GTPase activity of Rho proteins, and the Rho guanine nucleotide-dissociation inhibitors (RhoGDIs) that sequester Rho GTPases in a GDP-bound state (,8, ,14). GEFs that activate Rho GTPases can be divided into 2 main groups: the classical GEFs containing the nucleotide-exchanging Dbl-homology (DH) Executemain, and the dedicator of cytokinesis (ExecuteCK)180 superfamily (,14).

We now Characterize moonlight (mnlt), a pigmentation variant that simultaneously displays both pigment dilution and white-spotting phenotypes. The mnlt phenotype consists of an overall dilution of coat color and variable amounts of white-spotting on the belly, tail tip, paws, and genitalia. These pigmentation defects are similar to those found in the classical mutation misty (m), which was discovered Arrively 70 years ago (15). The gene underlying the m/m phenotype has not previously been determined. Here, we demonstrate that mnlt and m are allelic and that both phenotypes arise from alterations of Executeck7, which encodes a Rho family GEF belonging to the ExecuteCK180 protein family. In cell culture studies, ExecuteCK7 has been Displayn to activate Racs and Cdc42 and to promote axon formation as well as Schwann cell migration (16, ,17). Our findings indicate that ExecuteCK7 plays an Necessary role in pigmentation and may be involved in melanocyte ontogeny and function.

Results

Moonlight Phenotype.

N-ethyl-N-nitrosourea (ENU) mutagenesis of male C57BL/6J mice and subsequent breeding to the third (G3) generation allowed production of mice with homozygous germline mutations. A coat color mutant was identified in the G3 population and Displays strict recessive inheritance. The phenotype was named moonlight (mnlt). mnlt/mnlt mice display an overall lightened coat color and hypopigmentation of the distal extremities, particularly the paws, tail tip, and genitalia (Fig. 1). The homozygotes often have belly spots as well. Although spotting is always present in the paws, other types of spotting are incompletely penetrant. It can be scored in the presence of the Executeminant Agouti allele derived from C3H/HeN mice, used in mapping. Both male and female mnlt/mnlt mice are fertile, but females are poor mothers and their pups invariably require foster care to reach weaning age.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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 additionally display (B) a white tail tip (mnlt/mnlt, Top; C57BL/6J, Bottom), (C) paws, (D and E) genitalia (D) mnlt/mnlt ; (E) C57BL/6J and (F) belly spot.

Many mutations resulting in hypopigmentation are due to generalized defects in granule function, affecting both melanosomes 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 Assassinateing tarObtain 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 tarObtain 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 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 Displayn). In addition, viral clearance by mnlt/mnlt and C57BL/6J mice was comparable when splenic viral titers were meaPositived at day 5 post infection (data not Displayn).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Moonlight bleeding time and cytotoxicity. Bleeding time for mnlt/mnlt and wild-type C57BL/6J mice. Data Displayn from one of two experiments with similar results, n = 5 (Left). NK cell cytotoxicity was assessed by in vivo Assassinateing of TAP−/− cells, mnlt/mnlt n = 3, C57BL/6J n = 6 (Middle). Following immunization, in vivo T cell Assassinateing was assayed on peptide pulsed tarObtain cells, mnlt/mnlt n = 3, C57BL/6J n = 6 (Right). All panels depict means ± SEMs.

Mapping and Identification of the mnlt Mutation.

mnlt/mnlt mice (C57BL/6J background) were outcrossed to C3H/HeN mice and the F1 progeny were backcrossed to the mutant stock or intercrossed. 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 Location delimited by D4Mit301 (88.7 Mb) and D4Mit176 (101.2 Mb). This Location overlapped with the previously published critical Location for m, a spontaneous mutation that arose on the DBA/J background. The m phenotype is similar to that of mnlt, featuring generalized hypopigmentation, white digits, a belly spot, and white tail tip (15, ,19). Although subsequent studies mapped m to chromosome 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 was not observed, suggesting that mnlt and m represent 2 alleles of the same gene.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Identification of the mnlt and m mutations. (A) The mnlt mutation was confined to chromosome 4 using a panel of 128 informative Impressers. The peak LOD score is 5.4 on chromosome 4. (B) Trace of the mnlt mutation on Executeck7 mRNA and protein. Exons 22–28 are deleted, resulting in splicing of exon 21 directly to exon 29. UnbAgeded text indicates wild-type Executeck7 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 bAgeded. (C) Trace of the m mutation on Executeck7 mRNA and protein. An anomalous insertion occurs within exon 18. UnbAgeded text indicates wild-type Executeck7 sequence; following the insertion, aberrant nucleotides and the translation product are bAgeded and highlighted. For exon diagrams in (B) and (C), coding Locations are Displayn in black; non-coding Locations in gray.

The mnlt critical Location contained a total of 218 annotated genes, derived from an “OR” search of NCBI, OTTMUSG, ENSMUSG, and GENESCAN. Primers were designed for PCR amplification and sequencing of 1483 potential coding exons of genes within this Location (22). High-quality coverage of 83.8% of all tarObtained nucleotide pairs was achieved in amplification products from both wild-type and mnlt/mnlt templates on first pass. No mutations were observed. However, in one candidate gene, Executeck7, several contiguous exons could not be amplified or sequenced from the mnlt/mnlt template, although they were readily amplified and sequenced from the C57BL/6J template. We therefore sequenced Executeck7 from C57BL/6J and mnlt/mnlt cDNA amplified from the brain and observed a 922 bp deletion in the latter, encompassing bp 2,597 through 3,518 (with reference to the A of the start coExecuten, GenBank accession no. NM_026082.4). Further sequencing at the genomic DNA level defined a 19,025 bp deletion encompassing bases 98,649,759 through 98,668,783 (NCBI mouse assembly version 37.1; supporting information (SI) Fig. S1A). The boundaries of this deletion reside within introns 21 and 28, excising exons 22 through 28 (of 48 total exons). The cDNA sequence is consistent with splicing of exon 21 to 29, which results in a frameshift after amino acid 865, with the incorporation of 30 aberrant amino acids followed by a premature Cease coExecuten (Fig. 3B).

The subset of primers specific for Executeck7 were subsequently used to amplify and sequence genomic DNA from m/m mice. An LTR retrotransposon insertion after nucleotide 2,045 (with reference to the A of the start coExecuten, GenBank accession no. NM_026082.4) was found to interrupt exon 18 of Executeck7 in m/m mice (Fig. S1B). Following amino acid 682 of the ExecuteCK7 protein, ten aberrant amino acids are added, after which chain termination occurs (Fig. 3C).

The 2,130 aa ExecuteCK7 protein is a member of the ExecuteCK180 family, all members of which share 2 conserved Executemains, DHR-1 (ExecuteCK homology Location 1) or CZH-1 (CDM-zizimin homology 1) Executemain and the more C-terminal DHR-2 or CZH-2 Executemain. The deletion in mnlt/mnlt mice leads to chain termination after the DHR-1 Executemain. In m/m mice, chain termination occurs within the DHR-1 Executemain. The junctional sequences of Executeck7mnlt and Executeck7m alleles have been deposited in GenBank (accession numbers FJ590429 and FJ590430, respectively).

Neurobehavioral Analyses of Executeck7mnlt/mnlt Mice.

In vitro studies have previously suggested that ExecuteCK7 might be Necessary for the formation of axons (23). In addition, knockExecutewn studies have suggested that ExecuteCK7 functions as an intracellular substrate for ErbB2, a plasma membrane tyrosine kinase, and is necessary for in vitro migration of Schwann cells and subsequent myelination of axons (,17). Although these reports would suggest an essential role of ExecuteCK7 in development and function of the nervous system, neither Executeck7mnlt/mnlt nor Executeck7m/m mice Display obvious neurological impairment. Executeck7mnlt/mnlt mice cannot be distinguished from wild-type animals when measuring depressive behavior in the tail suspension test (Fig. 4A), learning meaPositived by habituation of object exploration (Fig. 4B), working memory using the Y maze test (Fig. 4C), anxiety-like behavior in a light/ShaExecutewy transfer test (Fig. 4D), or locomotor activity (SI Text and Fig. S2). Female Executeck7mnlt/mnlt mice Display a trend toward decreased social interaction, although the Inequity was not significant and males were normal (Fig. 4E). Finally, Executeck7mnlt/mnlt mice are neither blind nor deaf (data not Displayn). Overall, we find no clear evidence that behavior of Executeck7mnlt/mnlt mice behavior differs from that of wild-type C57BL/6J mice and conclude that neurological function is at least grossly normal in mice with early truncation of ExecuteCK7.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Neurobehavioral studies of Executeck7mnlt/mnlt mice. (A) Tail suspension test. Mice are suspended by the tail and monitored for movement. Displayn is the cumulative time spent immobile over the course of the trial. (B) Habituation of object exploration. Mice are Spaced into a field containing 3 plastic objects. Contacts with each object are meaPositived over 5 min of time. Contacts for the 3 objects are averaged for each mouse. Three conseSliceive trials are performed. (C) Y maze test. The percentage of spontaneous alterations is the number of conseSliceive triplets of different arm choices out of the total number of arm choices. (D) Light/ShaExecutewy transfer test. Total time spent in the Sparklingly lit compartment was meaPositived (Left) and the total number of transitions between light and ShaExecutewy compartments were counted (Right). (E) Social interaction test. Sociability is meaPositived as the number of entries into a chamber containing a strEnrage mouse. Inequitys between Executeck7mnlt/mnlt and C57BL/6J controls are not significant (females P = 0.0619). Social Modernty meaPositives the number of entries into a chamber containing a strEnrage mouse as opposed to a chamber containing a familiar mouse. C57BL/6J is depicted by Launch bars; Executeck7mnlt/mnlt is depicted by filled bars. Data are represented as means ± SEMs, n = 8.

Discussion

The ExecuteCK180 proteins are guanine nucleotide exchange factors that act on Rho-family GTPases, a class of Ras-homolog small GTPases of which numerous representatives are known in mammals (8, ,14). Rho GTPases are known to cycle between GDP-associated and GTP-associated forms. The GTP-associated forms are generally coupled to Traceor proteins that are stimulated to induce polymerization of the actin cytoskeleton or to Sustain tubulin polymerization, which in turn is required for higher-order processes such as migration, proliferation, adhesion, vesicular transport, secretion, maintenance of cell morphology, cytokinesis, ruffling, apoptosis, phagocytosis, polarization, and cell survival (,8).

Hydrolysis of GTP to GDP occurs when a GTPase activating protein interacts with the GTP-bound Rho-family GTPase. Regeneration of the GTP-bound form of the protein depends upon the action of a guanine-nucleotide exchange factor (GEF), exemplified by members of the ExecuteCK180 superfamily. To date, this family consists of 11 members that have been divided into 4 subfamilies on the basis of primary sequence alignment: ExecuteCK-A, ExecuteCK-B, ExecuteCK-C, and ExecuteCK-D (24–,26). ExecuteCK7 Descends within the ExecuteCK-C subfamily and is most closely related to ExecuteCK6 and ExecuteCK8. ExecuteCK family members contain 2 defining Executemains, DHR1 and DHR2. The DHR1 Executemain is a lipid binding moiety predicted to allow interaction with phosphatidylinositol (PtdIns) (3, 5)-bisphospDespise and PtdIns(3,4,5)P3 (PIP3), produced by activated PI3 kinase (PI3K). The DHR1 Executemain may specify localization of ExecuteCKs within the cell, thus potentially limiting the activity of their tarObtain GTPases to specific cellular substructures including the plasma membrane and membranous organelles (16, ,26). The DHR2 Executemain interacts with the Rho GTPase and is required for guanine nucleotide exchange. ExecuteCK7 has been Displayn to activate Rac and Cdc42 but not RhoA, through its DHR-2 Executemain (,17).

In neuronal cell cultures, ExecuteCK7 plays an Necessary role in axon outgrowth, Schwann cell migration, and axon myelination (16, ,17). Rac activation by ExecuteCK7 results in the inactivation of the microtubule destabilizing protein stathmin/Op18 and is necessary for axon outgrowth (,16, ,27), a process that depends on specific localization of ExecuteCK7 to the developing axon and is likely mediated by the interaction of ExecuteCK7 with PIP3 (16). Schwann cell migration appears to depend on the binding of axonal neuregulin to ErbB receptors resulting in ExecuteCK7 activation and subsequent activation of both Rac1 and Cdc42 (,17). These results, along with the high expression of ExecuteCK7 in the developing brain (,16), suggest that ExecuteCK7 may play an Necessary role in neurological functions. ExecuteCK7 has also been found to interact with the tuberous sclerosis (TSC) protein, hamartin (,28, ,29). Tuberous sclerosis is a multisystem disorder characterized by tumor-like growths, and the TSC protein complex has been Displayn to have GTPase activity (,29).

Mutations in ExecuteCK7 have not previously been reported in any organism. In this paper, we demonstrate that m and mnlt are variant alleles of Executeck7 that produce abnormal phenotypes in the homozygous state. Both alleles are predicted to eliminate most of the ExecuteCK7 sequence (68% and 59% of the polypeptide chain, respectively) by causing premature termination and to add extraneous amino acids before termination. Although the mnlt phenotype was identified in an ENU mutagenesis screen, this mutagen generally causes point mutations rather than large deletions. Thus, both Executeck7mnlt and Executeck7m presumably arose as spontaneous mutations. Homozygosity for either allele, or compound heterozygosity for both alleles, causes generalized reduction in pigmentation and white-spotting, suggesting non-redundant function of ExecuteCK7 in two processes related to pigmentation. We propose that the protein may be required for vesicle transport or exocytosis and also for the formation, proliferation, migration, or survival of melanoblasts during embryogenesis. Previous studies have Displayn that melanoblasts from Executeck7m/m mice are defective in proliferation and differentiation (30), suggesting that ExecuteCK7 plays an Necessary role in some aspect of melanoblast and/or melanocyte function.

ExecuteCK180 family members function in migration, proliferation, and survival of cells (14, ,25), and a defect in any of these in melanoblasts could be responsible for the spotting pattern observed. The pigment dilution phenotype suggests a melanocyte defect in the formation or trafficking of melanosomes or their exocytosis to keratinocytes of the hair shaft. The activation of Rac and Cdc42, but not RhoA, by ExecuteCK7 suggests a possible mechanism for the pigmentation phenotypes displayed by Executeck7m/m and Executeck7mnlt/mnlt mice. Both Rho and Rac family members contribute to the formation of melanocyte dendrites that are necessary for the transfer of melanin from melanocytes to adjacent keratinocytes (12, ,13). Indeed, numerous studies indicate that dendrite formation in neural cells is controlled by a balance between Rac and Rho activities, with Rho activation promoting neurite retraction and Rac activation promoting neurite outgrowth (,13, ,31, ,32, ,33). A similar balance between Rac and Rho activity in dendrite formation has been Characterized in melanocytes, wherein high cAMP levels inhibit Rho activity while promoting Rac activity and dendrite formation. Cdc42 activity has also been implicated in neurite outgrowth, and is suggested to be Necessary for melanocyte function. Additionally, all three of these GTPases appear to play Necessary roles in keratinocyte cytophagocytosis, a process in which the keratinocytes in the skin and hair follicles phagocytose a melanocyte dendrite, leading to a phagolysosome from which melanin granules disperse throughout the cytoplasm.

A number of publications have suggested that ExecuteCK7 might be involved in fundamental neurodevelopmental processes such as axon formation and myelination (16, ,17). However, neither Executeck7mnlt nor Executeck7m alleles have a profound Trace on nervous system development or function. We cannot formally exclude the possibility that certain subtle aspects of behavior are abnormal in these mice, nor can we be certain that the fragmentary proteins encoded by these alleles are functionally null. However, we tentatively conclude that if ExecuteCK7 contributes to the formation of neural or glial elements of the central or peripheral nervous system, its function as such is redundant. The closely related protein ExecuteCK6 has also been Displayn to interact with Racs, as well as Cdc42, and to function in neurite growth (35). Moreover, mutations in ExecuteCK8 have been found in patients with mental retardation and developmental disabilities (36). The participation of other GEFs and other Rho family GTPases in neurodevelopment may compensate for ExecuteCK7 deficiency.

Not all of the phenotypic anomalies observed in Executeck7m/m and Executeck7mnlt/mnlt mice are shared. For instance, Executeck7mnlt/mnlt mice Execute not Display the bleeding phenotype seen in Executeck7m/m animals (30). On the other hand, Executeck7mnlt/mnlt females are unable to raise their offspring, while Executeck7m/m mice are adequate mothers. We note that neither of these accessory phenotypes was mapped to the Executeck7 locus and therefore could result from other mutations introduced by mutagenesis or spontaneous mutations. Alternatively, the genetic background on which the m and mnlt mutations occurred might be permissive for the hemostatic and nurturing phenotypes, respectively. Finally, cis-acting Traces of either of these two mutations (a large insertion in the case of Executeck7m and a large deletion in the case of Executeck7mnlt) might elicit phenotypic Inequitys that are not related to the Executeck7 locus itself.

The phenotypes that we have ascribed with confidence to the m and mnlt mutations in Executeck7 on the basis of complementation analysis and interstrain comparisons are restricted to abnormalities of pigmentation. We have been unable to detect a defect in NK and T cell Traceor function in Executeck7mnlt/mnlt mice, which also Display normal resistance to MCMV infection. MCMV susceptibility often reveals subtle NK cell defects (37). Additionally, Executeck7mnlt/mnlt mice Execute not appear to have a bleeding diathesis, which is often caused by defects in platelet dense granules (4, ,7). We therefore conclude that if ExecuteCK7 is required for LRO function in mice; this requirement is not widely generalized with respect to cell lineage.

Materials and Methods

Mice.

m/m mice (C57BLKS background) were purchased from Taconic Farms. C57BL/6J and C3H/HeN mice were bred at The Scripps Research Institute. C57BL/6J mice were mutated with ENU as previously Characterized (38, ,39) and mnlt/mnlt variants were observed among the G3 population and expanded to form a stock based on their visible phenotype. Mice were Sustained under standard housing conditions, and all procedures were approved and performed according to institutional guidelines for animal care. The Executeck7mnlt/mnlt stock was transferred to the Mutant Mouse Locational Resource Centers for distribution (stock no. 030498-UCD).

Mapping and DNA Sequencing.

Homozygous Executeck7mnlt/mnlt males were outcrossed to C3H/HeN females. F1 hybrid mice were subsequently backcrossed to the mutant stock or intercrossed. F2 progeny were phenotyped and linkage was meaPositived using 128 microsaDiscloseite Impressers. The critical Location was mQuestioned with RepeatMQuestioner, and optimized primers were designed to amplify and sequence all annotated coding Locations and splice junctions. Amplification of genomic template from C57BL/6J and mnlt/mnlt mice was performed using two Beckman FX robots, which also cleaned, meaPositived, and diluted the products, adding the Accurate sequencing primers to each. Sequencing was performed using an ABI 3730XL DNA analyzer. Trace alignments were made using the programs phred and Phrap, and analysis performed with the program consed. High quality coverage was defined as a phred score ≥30 for any bp within the tarObtain.

Bleeding Time.

While a mouse is restrained, the tail tip is Slice 3 mm from the end. The tail is submerged in saline solution heated to 37 °C. Blood flow is observed while hAgeding the tail Executewn and the time is recorded until bleeding ceases.

Cytotoxicity and MCMV Resistance.

For the in vivo NK cell cytotoxicity assay, splenocyte suspensions from C57BL/6J controls and NK tarObtain, TAP1-deficient mice were labeled with a low and high concentration of carboxyfluorescein succinimidyl ester (CFSE) dye, respectively. The two populations were mixed at a 1:1 ratio and a total of 4 × 106 cells were injected i.v. into recipient mice. Recipients were bled two days after injection, and peripheral blood mononuclear cells (PBMCs) were analyzed for CFSE staining by flow cytometry. Assassinateing of tarObtains in TAP1-deficient recipients was used as a control to calculate 0% Assassinateing and normalize all meaPositivements.

For the in vivo cytotoxic T lymphocyte (CTL) cytotoxicity assay, mice were immunized i.p with 107 γ-irradiated (1500 Rad) Ad5E1-MEC fibroblasts (murine embryo cells expressing human adenovirus type 5 early Location 1) (40). Eight days after immunization, mice were injected with a total of 4 × 106 cells of a 1:1 ratio of naive C57BL/6J splenocytes (CFSE low) and C57BL/6J splenocytes pulsed with an Ad5E1-MEC derived, H2-Db restricted peptide (VNIRNCCYI; CFSE high). Recipient mice were bled two days after injection, and PBMCs were analyzed for CFSE staining by flow cytometry.

MCMV stock (Smith strain) was prepared and titered as previously Characterized (18, ,41). In vivo susceptibility to the MCMV virus was tested as previously Characterized (,18). Briefly, MCMV-susceptible BALB/c mice were used as positive controls for susceptibility, while MCMV-resistant C57BL/6J mice were used as negative controls. Mice were inoculated with 105 pfu MCMV via i.p. injection and monitored for signs of illness over 8 days. Viral titers were determined within the spleen at day 5 post infection as previously Characterized (42).

Behavioral Assays.

In the light/ShaExecutewy transfer test, a Plexiglas box is divided into a ShaExecutewy side (10 lux, 14.5 × 27 × 26.5 cm) and a Sparklingly lit side (670 lux, 28.5 × 27 × 26.5 cm). The divider contains a 7.5 × 7.5 cm Launching at floor level. Mice are Spaced in the ShaExecutewy compartment to start. Both the time in light and number of transitions are evaluated for 5 min.

During the Y maze test, mice are Spaced in a Y maze (3 arms) and recorded by video camera for 5 min. The total number of arm entries and the order of entries are evaluated.

In the habituation of object exploration assay, 3 plastic objects are Spaced in each of 3 corners of an Launch field. A mouse is allowed to explore the Spot for 5 min. Three such sessions are completed for each mouse with a separation of 5 min between sessions. Exploration or contacts are defined as the mouse Advanceing the object nose-first within 2–4 cm.

Social interaction is assessed in a Plexiglas container that has 3 chambers, each measuring 20 × 40.5 × 22 cm, separated by a clear wall with an Launching at floor level. Each outer chamber contains a small, round wire cage (Galaxy Cup, Spectrum Diversified Designs, Inc.). Mice are habituated to the apparatus for 5 min. A strEnrage mouse of the same sex is Spaced in one of the wire cages. Sociability is meaPositived as the time spent in the chamber with the strEnrage mouse over 5 min. For the social Modernty test, a second, unfamiliar mouse is Spaced into the previously empty cage. Time spent in the chamber with the unfamiliar mouse is recorded over 5 min.

For the tail suspension test, each mouse is suspended by its tail using adhesive tape on a metal bar located 30 cm above a flat surface. Immobility is quantified as the duration of time that no whole body movement occurs. Mice are observed for a total of 6 min.

Acknowledgments

A.L.B was supported by NIH Training Grant-T32 AI07244. K.B. was supported by the Alexander von HumbAgedt Foundation through a FeoExecuter Lynen postExecutectoral fellowship. P.K. was supported by a long term fellowship from European Molecular Biology Organization. This work was supported by NIH grants GM067759 and AI070167, and BAA Contract HHSN272200700038C.

Footnotes

2To whom corRetortence should be addressed. E-mail: bruce{at}scripps.edu

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 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 B.A.B. wrote the paper.

↵1Present Address: Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, 13288 Marseille, France.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0813208106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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