Figure 1. (A) Upper panel displays the white-tipped paws typical of this mutant (arrow). (B) Lower panel displays the white belly spot seen occasionally (white arrow). Figure reproduced from reference (1).

Moonlight mice have a slightly lightened coat, white-tipped paws, and hypopigmented genitalia.  Moonlight animals display an occasional belly spot similar to splotch2 mutants, and sometimes a white-tipped tail (Figure 1).  Moonlight females are poor mothers, and their pups require fostering.  Behavior tests displayed no significant differences between wild type and moonlight animals, although there was a trend for moonlight females to show decreased social interaction (1).  Moonlight mice display normal macrophage responses to Toll-like receptor ligands, double-stranded DNA, and the adjuvant alum (TLR Signaling Screen, Double-stranded DNA Macrophage Screen, NALP3 Inflammasome Screen, ), normal proportions of immunological cell types, normal responses to viral infections (MCMV Susceptibility and Resistance Screen, Ex Vivo Macrophage Screen for Control of Viral Infections), and normal natural killer (NK) and T cell cytotoxicity (In Vivo NK Cell and CD8+ Cell Cytotoxicity Screen).  These animals do not develop colitis after being exposed to low doses of dextran sodium sulfate (DSS-induced Colitis Screen), and display a normal humoral immune response to model antigens encoded by a recombinant suicide vector based on the Semliki Forest Virus (rSFV).  From the stock, a single individual with "zebra-like" striping was identified.  This is considered a possible superimposed spontaneous mutation, and has been designated Tigroid, but the Tigroid stock was ultimately lost. 

 

Moonlight mice have a phenotype highly similar to the classical white-spotting mutant, misty, which spontaneously arose in the DBA/J strain, but has been crossed into various backgrounds including C57BL/6J (2).  Misty mapped to the same critical region as moonlight, and complementation testing demonstrated that moonlight is allelic to misty.  Unlike what has been reported for misty animals, moonlight animals do not display a bleeding phenotype (1) (see Background).

Figure 2. (A) Schematic diagram of the deletion located in the Dock7 gene in moonlight mice. The deletion spans exons 22-28. Black signifies amino acid coding regions. (B) Schematic diagram of the retrotransposon insertion located in the Dock7 gene in misty mice. A portion of the inserted nucleotide sequence is shown along with the 10 aberrant amino acids. Figure reproduced from reference (1).

The moonlight mutation was mapped to Chromosome 4.  Sequencing of the critical region found a deletion spanning exons 22-28 (of 48 total exons) of the Dock7 gene  (positions 118824 to 137848 occurring in the middle of introns 21 and 28 in the Genbank genomic region NC_000070 for linear genomic DNA sequence of Dock7; see Figure 2).  If the donor and acceptor splice sites of introns 21 and 28 are still used, amino acids 866-1173 of the DOCK7 protein would be deleted, and a frameshift and insertion of 30 aberrant amino acids before a premature stop codon would occur.

 

A retrotransposon insertion was found in Dock7 in misty mice at nucleotide 2198 of the Dock7 cDNA in exon 18 of 48 total exons. This insertion resulted in the addition of 10 aberrant amino acids followed by a stop codon after amino acid 682 of the DOCK7 protein.

Figure 3. Domain structure of DOCK7. Two domains are shared amongst all DOCK proteins, the DHR-1 domain (DOCK homology region 1) and the catalytic DHR-2 .The moonlight mutation results in the addition of 10 aberrant amino acids followed by a stop codon after amino acid 682 of the DOCK7 protein.

The full-length mouse DOCK7 (dedicator of cytokinesis 7) protein is 2098 amino acids long, and is 97% identical to its human homologue, which contains 2109 amino acids and has a predicted molecular mass of 241 kDa (3).  Several splice variants of DOCK7 exists in humans (3-5), and splice variants are predicted for mouse DOCK7. 

 

DOCK7 belongs to the DOCK180 superfamily of guanine nucleotide exchange factors (GEFs) that have been shown to activate members of the Rho family of small GTPases (5-8).  In mammals, 11 of these proteins have been identified, and can be classified into four subfamilies DOCK-A (which includes DOCK2; see the record for frazz), DOCK-B, DOCK-C (which includes DOCK7) and DOCK-D based on theirdiffering specificities for binding to the Rho GTPases Rac and Cdc42, regulatory domains,and associated subunits (5;7;8).  DOCK A and B subfamilies activateRac, the DOCK D subfamily is specific for Cdc42, whereas theDOCK C subfamily has dual specificity for Rac and Cdc42 (5;6;9;10).  Two domains are shared amongst all DOCK proteins, the catalytic DHR-2 (DOCK homology region 2) or CZH-2 (CDM-zizimin homology 2) domain and the DHR-1 or CZH-1 domain.  The DHR-1 domain is located N-terminal to the DHR-2 domain (7;8).  The sequences between the recognized domains are predicted to be mostly helical, and may fold as armadillo (ARM) repeat domains.  The armadillo repeat is a roughly 40 amino acid long tandemly repeated sequence motif and forms suprahelical structures used to bind to other proteins, especially those containing the same motifs (11).

The DHR-2 domains of several DOCK family members interact with the nucleotide-free form of Rac and/or Cdc42 (7;8), and deletion of the DHR-2 domain in many of these proteins abolishes their ability to activate these GTPases (3;6;9;12).  The DHR-2 domain is large domain containing roughly 450-550 amino acids and is located at residues 1570-2093 in DOCK7 (Uniprot Q8R1A4). The zizimin 1/DOCK9 protein is able to dimerize through its DHR-2 domain (13), while structural analysis of DOCK9-Cdc42 complexes suggests that the presence of a nucleotide sensor contributes to release of guanine diphosphate (GDP) and subsequently, that of activated GTP-bound Cdc42 (PDB 2WMO; 2WMN; 2WM9).  Magnesium (Mg²⁺) exclusion, which promotes GDP release, is mediated by a conserved valine residue (Val 1538 in DOCK2) within the nucleotide sensor.  Binding of GTP-Mg²⁺ to the nucleotide-free DOCK9/Cdc42 complex results in displacement of the sensor to allow discharge of GTP-bound Cdc42.  The structure of the DHR-2 domain differs from that of other GEF catalytic domains and consists of three lobes A, B and C.  The Cdc42 binding site and catalytic center are located in lobes B and C.  Lobe A consists of an antiparallel array of five α helices (α1-α5) and stabilizes the DHR-2 domain through contacts with lobe B.  DOCK9 dimerization occurs through helices 4 and 5.  This interface contains residues that are conserved in other DOCK proteins (Lys 1301, Glu 1308, Leu 1317, and Tyr 1328 for DOCK2).  Lobe B consists of two antiparallel β sheets arranged orthogonally, and lobe C comprises a four-helix bundle (α7-α10).  The α10 helix is the most conserved region of the DHR-2 domain, and is interrupted by the seven-residue loop nucleotide sensor (14).

The roughly 250 amino acid DHR-1 domain (amino acids 561-779 in DOCK2) is not as well defined in these proteins.  For DOCK proteins belonging to the DOCK A and B groups, the DHR-1 domain shares weak homology to the C2 domain, a well characterized Ca²⁺-dependent lipid-binding module (15).  These proteins appear to be localized to the plasma membrane via interaction of its DHR-1 domain with phosphatidylinositol (3,5)-biphosphate [PtdIns(3,5)P₂] and phosphatidylinositol (3,4,5) P₃ (PIP₃) signaling lipids (16-18).  Other studies have shown that the DHR-1 domain for zizimin 1/DOCK9 is able to bind to the DHR-2 domain and inhibit its function (19).  The function of the DOCK7 DHR-1 domain has not been directly tested, although DOCK7 may also interact with PIP₃(3).  In DOCK180 (also known as DOCK1), the DHR-1 domain folds into a type II C2 domain fold consisting of an antiparallel β sandwich consisting of two four-stranded β sheets (β1- β8) (PDB 3L4C). The DHR-1 domain also contains three loops between β1-β2 (L1), β3-β4 (L2), and β5-β6 (L3) on the upper surface of the structure as well as two large insertions between β2 and β3 and β7 and β8.  The loop region creates a positively charged pocket at the top of the molecule that allows recognition of the PIP₃ head group.  The pocket has many of the characteristics of lipid-binding pleckstrin homology (PH) domains.  Residues predicted to contact phospholipid are Lys 437, Lys 440, Arg 444, Tyr 482, Glu 483, His 513, Ser 515, Glu 517 and Lys 522, and mutagenesis of Lys 437, Lys 440, Arg 444 (a lysine in DOCK180) and Lys 522 reduces or abolishes phospholipid binding.  Unlike many C2 domains, the DHR-1 domain binds phospholipids independently of calcium.  The DHR-1 domain also contains another highly basic pocket on its concave surface often called the β-groove in many C2 domains, but this does not participate in phospholipid binding (20).

The moonlight mutation results in truncation of the DOCK7 protein, but further studies need to be completed to determine the exact limits of the deletion, and also whether an aberrant DOCK7 protein is expressed in moonlight mice.  In misty mice, the retrotransposon is inserted into the DHR-1 domain of the protein and also results in protein truncation.

In postnatal day (P) 5 rats, DOCK7 protein is highly expressed in the developing brain and the heart, and is expressed at lower levels in the lung, kidney, skeletal muscle, liver, and spleen.  In the brain, DOCK7 protein levels were higher at late embryonic and early postnatal stages and decreased gradually with age.  Early during hippocampal neuronal development, DOCK7 is expressed throughout the cell body, but not in actin-rich areas.  Strong DOCK7 staining is observed around the cell periphery, and accumulates at the basal side of the cell where it attaches to the substrate.  Later in development, DOCK7 is detected in the cell body and in neurites, but stronger staining is observed in the neurite that later differentiates into the axon.  DOCK7 is highly expressed in the axon where it co-localizes with microtubules (3).  DOCK7 is also abundantly expressed in Schwann cells (4).  

A microarray comparing branching and non-branching regions of mouse embryonic lung (E11.5) found dock7 mRNA to be enriched in branching regions of the developing lung (21).  In situ hybridization confirmed this result, and found Dock7 mRNA present in both the distal mesenchyme and epithelium, which will eventually form the alveoli. 

Figure 4. Rho GTPase activation cycle. Rho GTPases cycle between the inactive GDP-bound state and active GTP-bound state which interacts with effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP. GTPase-activating proteins (GAPs) inactivate Rho GTPases by stimulating GTP hydrolysis. Rho guanine nucleotide-dissociation inhibitors (RhoGDIs) recruit inactive GDP-bound Rho GTPases from the membrane.

The Rho GTPases are known regulators of the actin cytoskeleton and affect multiple cellular activities including cell morphology, polarity, migration, proliferation and apoptosis, phagocytosis, cytokinesis, adhesion, vesicular transport, transcription and neurite extension and retraction.  The Rho GTPases are active when bound to GTP and are inactive in their GDP-bound form [reviewed in (22)].  Regulation of Rho GTPase activity is complex and involves 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.  GEFs that activate Rho GTPases can be divided into two main groups; the classical GEFs containing the nucleotide-exchanging Dbl-homology (DH) domain, and the DOCK180 superfamily, including DOCK7 (11).

Recently, DOCK7 was identified as interacting with and activating the Rho GTPase, Rac1 (3).  Rac1 controls the actin cytoskeleton and regulates microtubule dynamics, processes critical for cell migration and lamellipodia formation (23-26).  In developing neurons, DOCK7 is located preferentially in the neuron that later becomes the axon.  Knockdown of DOCK7 expression in neuronal cultures prevents axon formation, and overexpression induces formation of multiple axons.  Rac1 activation by DOCK7 in the developing axon leads to phosphorylation and inactivation of the microtubule destabilizing protein stathmin/Op18 (3;27).  Since microtubule formation is crucial for axon development, inactivation of this protein by DOCK7/Rac1 promotes axon formation.  It is possible DOCK7 localization in these neurons is regulated via interaction with PIP₃.  Laminin induces the production of PIP₃via PI 3-kinase in the developing axon, and inhibitors of PI 3-kinase prevented the ability of DOCK7 to induce multiple axons in overexpression experiments (3). 

In addition to being necessary for axonal formation, DOCK7 is also critical for Schwann cell migration and subsequent myelination of axons (4).  The axonal signal, neuregulin-1 (NRG1), binds to the tyrosine kinase ErbB receptors present on Schwann cells (28;29), which then phosphorylates DOCK7 on Tyr-1118.  Phosphorylation of DOCK7 by the ErbB complex activates DOCK7 and promotes association with the Rho GTPases Rac1 and Cdc42.  Rac1 and Cdc42 then acts through the c-Jun N-terminal (JNK) kinase cascade to regulate Schwann cell migration (4).  Some targets of JNK implicated in cell migration is the focal adhesion adaptor protein paxillin (30) and microtubule-associated proteins (MAPs) that are important for microtubule stability (31;32).  The closely-related protein DOCK6 has also been found to interact with both Rac1 and Cdc42 and is necessary for neurite growth (10).

The only identified mutations in Dock7 are those found in moonlight (Dock7^mnlt/mnlt) and misty (Dock7^m/m) mice.  The misty mouse mutant is a classical white-spotting mutant that arose spontaneously in the DBA/J strain of mice over 60 years ago (2).  White-spotting mouse mutants exhibit white spots that include belly spots, head spots, belts spanning the caudal trunk region, piebald spotting and peppering. Unlike color variations in mice which are typically due to alterations in melanin production, distribution, or deposition into hair and/or skin (please see the records for ghost, cardigan, quicksilver, bullet gray, toffee, and souris), white-spotting mutants are often due to defects at various stages of melanocyte development including proliferation, survival, migration, invasion of the integument, hair follicle entry and melanocyte stem cell renewal (33;34). During embryonic development, melanoblasts show variable density along the rostral-caudal axis with large numbers seen around the optic cup, in the cervical region, and caudal to the hindlimbs.  Hypopigmentation of the head, trunk, feet, tail and ventral regions common in white-spotting mutants likely reflect initially reduced melanoblast number, and a longer distance of melanoblast migration to reach these areas (34).  A number of other genes cause these phenotypes including ones encoding the transcription factors MITF, PAX3, and SOX10 (mutated in Dalmatian), the KIT receptor tyrosine kinase (mutated in Casperand Pretty2), a G-protein coupled receptor and its ligand (endothelin receptor type B, EDNRB, and endothelin-3, EDN3), a transmembrane protein (Mucolipin 3) as well as ADAMTS-20, a putative ECM associated metalloprotease (mutated in splotch2and whitebelly) (33;34).

Homozygous misty animals typically show paler pigmentation interpreted as a dilution of eumelanin, but also have a white tail-tip, feet and a belly spot, suggestive of improper melanoblast migration or survival (2;33;34).  The pigmentation defects are consistent with poor pigmentation in histological sections of neonatal misty mouse skin.  Primary cultures of misty melanoblasts and melanocytes are also abnormal (2), containing a few, small melanocytic colonies with hyperpigmented melanocytes of defective morphology.  Misty melanocyte lines do not proliferate well, display increased cell death, and respond poorly to cAMP, which normally induces growth and differentiation of melanocytes.  Decreased cell survival may be due to hyperpigmentation as high levels of melanin intermediates are toxic (35).

In addition to the pigmentation defects, misty mice display a number of other defects, including absence of heat-generating brown fat found as interscapular and lateral thoracic pads in rodents  (2).  Another report found that adult misty mice were smaller and leaner than wild type littermates, displaying a reduction in white adipose tissue (36).  Misty homozygote animals also display a significant increase in bleeding time over wild type controls (2).  Prolonged bleeding occurred despite normal platelet counts and serotonin levels, and was likely caused by reduced levels of platelet ADP, which is known to stimulate platelet aggregation (37). 

Although a role for DOCK7 in human disease has not been identified, the closely related DOCK8 is mutated in patients with an autosomal recessive form of hyper-IgE recurrent infection syndrome (HIES; OMIM #243700), a disease characterized by recurrent bacterial and viral infections, increased serum IgE, and eosinophilia (38;39).  Mutations in DOCK3, a less similar protein, are associated with attention-deficit hyperactivity disorder (ADHD) in humans (40) (OMIM #143465).  DOCK7 has also been found to interact with the tuberous sclerosis (TSC) protein, hamartin (41;42).  Tuberous sclerosis (OMIM #191100) is a multisystem disorder characterized by tumor-like growths, and the TSC protein complex has been shown to have GTPase activity (42). 

Although DOCK7 function has only been assessed in in vitro cultures of neurons and Schwann cells, it is likely that DOCK7 has roles in numerous tissues based on its expression pattern and ability to interact with both Rac1 and Cdc42, both of which regulate a wide variety of cellular processes in multiple cell types.  The pattern of Dock7 mRNA expression in the developing lung (21), as well as localization in the kidney (3) suggests that DOCK7 may have a role in controlling cell migration and branching morphogenesis in several tissue types.  The phenotypes of Dock7 mutant mice suggest that DOCK7 also has a role in melanocyte development or survival, and may affect the appropriate migration of melanoblasts by mechanisms similar to those used in axonal outgrowth or Schwann cell migration.  Indeed, previous studies have shown that melanoblasts from Dock7^m/m mice are defective in proliferation and differentiation (2), suggesting that DOCK7 plays an important role in some aspect of melanoblast and/or melanocyte function.  These same mechanisms may also underlie the overall dilution of coat color observed in these animals.  Poor melanocyte growth has been associated with a disturbance in melanogenic pathways (43), and misty melanocytes appear to overproduce melanin, at least in culture.  In vivo toxic melanin intermediates could lead to cell death and overall hypopigmentation.  Additionally, members of the Rho family of GTPases, including Rhos, Racs and Cdc42, are implicated in keratinocyte cytophagocytosis of melanocyte dendrites, melanocyte dendrite formation and melanosome transport and exocytosis, all processes that are important for the deposition of melanin into the skin and growing hair shaft (44-48).  The formation of melanocytic dendrites is analogous to the formation of neurites, and is controlled by a balance between Rac and Rho activities (45;48).

Dock7^m/mmice also display a defect in platelet ADP levels.  Phenotypes exhibited by these animals are reminiscent of Hermansky-Pudlak syndrome (HPS; OMIM #203300), Chideak-Higashi syndrome (CHS; OMIM #214500), and Griscelli syndrome type II (GS2; OMIM #607624).  These diseases can be caused by mutations in multiple genes that are involved in protein trafficking to and secretion of lysosome-related organelles (LROs) including melanosomes, platelet-dense granules, and lytic granules necessary for normal NK and T cell cytotoxicity (please see toffee, dorian gray, bullet gray, pam gray, minnie, stamper-coat, sooty, souris, grey wolf and concrete) (49-51).  There are no reports that mutations in Rho GTPases or GEFs cause these diseases,  but Rho GTPases are involved in many cellular processes including vesicular transport (22).  Mutations in the Rab GTPase, Rab27a, result in GS2 in humans and block the proper docking and fusion of LROs with the plasma membrane.  Rho GTPases, regulated by DOCK7, may be involved in similar mechanisms.  However, our studies show that Dock7^mnlt/mnltanimals do not have an overall LRO defect as they display normal bleeding times as well as NK and T cell cytotoxicity. 

Unlike Dock7^mnl/mnlt females, Dock7^m/m females are able to nurture their young.  The reason for this phenotype in Dock7^mnl/mnlt females is unknown as these females appear to be normal in a wide variety of behavioral tests (1).  Other phenotypes found in Dock7^m/m mice, such as the platelet defect and decreased fat levels, have not been observed in Dock7^mnlt/mnlt mice.  The differences in phenotypes between Dock7^mnlt/mnlt and Dock7^m/m mice may be due to differing genetic backgrounds, additional mutations, or cis-acting effects of the moonlight or misty mutations.  The lack of neurological phenotypes in animals with mutations in Dock7 suggests that the neurological role associated with DOCK7 function in vitro may be redundant in vivo, although production and partial function of truncated proteins from the moonlight and misty alleles cannot be discounted.

Moonlight genotyping is performed by amplifying the region spanning the deletion in moonlight mice, and by amplifying a region within the moonlight deletion in wild type mice using two PCR reactions. 

 

PCR primers spanning the moonlight deletion

moon(F): 5’-TGCTTACCTGTTTGCTGTACAAGCAATC-3’

moon(R): 5’-CGACTGGAGGGAGCGAGGTT-3’

 

PCR primers within the moonlight deletion

moonwt(F): 5’-TGTGAGGGATAGCTGAGTCTCCAC-3’

moonwt(R): 5’-GACAGGGGCATCCAGTTCATTACC-3’

 

PCR program

1) 94°C             2:00

2) 94°C             0:30

3) 56°C             0:30

4) 72°C             1:00

5) repeat steps (2-4) 29X

6) 72°C             7:00

7) 4°C               ∞

 

The first set of primers will amplify a product of 463 nucleotides from moonlight mice.  No product will be seen from wild type mice.  The second set of primers will amplify a product of 767 nucleotides from wild type mice.  No product will be seen from moonlight homozygotes.  Products from both PCR reactions will be seen in heterozygote animals.  Please refer to Genbank genomic region NC_000070 for linear DNA sequence of Dock7.

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