Research

Non-immune Phenotypes

The functions of many genes are illuminated by the study of mice with visible phenotypes (e.g. changes in pigmentation, metabolism, behavior, or development morphology) induced by ENU.  Mutations in these mice are positionally cloned with interest.

Skin and coat phenotypes

Our lab has identified several mutations in genes involved in melanin production due to visible skin, eye, and/or coat pigment changes (Figure 1). Each strain is mapped and within a few years, putative null or damaging allelic variations will be available for most, if not all, of the proteins involved in pigment production.

Figure 1: Melanasome biogenesis. Premelanosomes arise from the late secretory or endosomal pathway. Stage 1 premelanosomes (depicted here as "Early endosome/Stage I" for simplicity) lacking pigment are thought to correspond to the coated endosome, an intermediate between early and late endosomes densely coated on one face with clathrin. PMEL17, a structural component of the melanosome on which melanins are deposited (not depicted) accumulates in stage I and II; PMEL17 is masked by melanin in later stages. Melanin synthesis begins in Stage II premelanosomes that contain regular arrays of parallel fibers that give these organelles a striated appearance by electron microscopy. During Stage III, these fibers gradually darken and thicken (red arrows, inset) as eumelanin is deposited along them, such that by Stage IV no striations are visible and the melansome is filled with melanin; this action has been illustrated in the inset. All cargoes required for melanin synthesis, processing, and transport (OCA2, SLC45A2, Rab32, Rab38, DCT (alternatively, Tyrp2), Tyr, and Tyrp1) derive from the Golgi and traverse vacuolar and/or tubular elements (not shown) of early endosomes en route to the stage III melanosome. Adaptor protein-3 and -1 as well as biogeneisis of lysosome-related organelle complex-1 (BLOC-1) and BLOC-2 regulate the intracellular trafficking of Tyrp1. SLC45A2 and OCA2 function in the trafficking of Tyrp1 and DCT to melanosomes. OCA2 maintains the proper pH in the melanosomes and transports glutathione, a protein necessary for Tyr and Tyrp1 trafficking to melanosomes. Black arrows represent transport of vesicles; red arrows represent protein-mediated regulation at a specific transport step as indicated in the key. Tyrp1, Tyr, Tyrp2, and Slc45a2 expression are regulated by the microphthalmia transcription factor (MITF). Several mouse models with mutations in components of the pigment-producing pathway are shown below; the mutation name (black) and mutated gene (red) are indicated. The melanosome pathway has been adapted from several sources including: Raposo, G. and Marks, M.S. (2007), Nat. Rev. Mol. Cell Biol., 8:786 and Lakkaraju, A. et al. (2009), J. Cell Biol., 187:161.

Several pigment mutants are models of human diseases (Figure 2). Heterozygote Dalmation mice exhibit white-spotting of the coat. The Dalmation mutation was mapped to Sox10, which encodes the SOX-10 transcription factor that is critical in the formation of tissues and organs during embryonic development. In humans, mutations in SOX10 are linked to Waardenburg-Shah syndrome (1;2). This disease is a combination of Waardenburg syndrome (WS), characterized by deafness, hypopigmentation of the skin, hair, and irises, as well as Hirschsprung’s disease, characterized by lack of nerves and function in part or all of the large intestine (1;3;4). The tigrou strain was identified by the appearance of brown stripes on the back, belly, and face in heterozygous females (5). The mutation in tigrou was mapped to Atp7a, which encodes a copper-transporting ATPase, ATP7A (see New Genes for information about brown, another Atp7a model). Mutations in ATP7A result in copper deficiency in humans and can cause Menkes disease, an inherited neurological disorder characterized by focal cerebral and cerebellar degeneration, seizures, and dementia (6-9)

Figure 2. Coat phenotypes of the Dalmatian and Tigrou strains. Mutations in the Sox10 transcription factor and the copper-transporting ATPase, Atp7a were identified in Dalmatian and Tigrou, respectively.

Visible mutants have also been identified by changes in coat thickness, length, or texture. For example, the Velvet mutant has open eyelids at birth and curly whiskers and as an adult, the coat of the Velvet heterozygotes appears disoriented [Figure 3; (10)]. The mutation in Velvet was mapped to Egfr, which encodes the epidermal growth factor receptor (EGFR), an essential receptor involved in intracellular signaling that promotes cell growth, cell survival, and proliferation as well as lipid biosynthesis and cytoskeleton reorganization. 

Figure 3. The Velvet phenotype. (A) The eyelids of wild-type mice remain closed until 12 days after birth, while (B) the Velvet heterozygote mice are born with eyelids open. Curly vibrissae are observed on Velvet heterozygotes (arrow). (C) The fur of the adult Velvetheterozygotes appears disoriented.

Metabolism

Several of our models display defects in metabolism. For example, a mutation in Tmprss6 causes the mask phenotype of iron deficiency due to impaired iron uptake [Figure 4A; (11)]. The TMPRSS6 protein functions in a pathway that detects iron deficiency and can alter the transcription of genes involved in dietary iron absorption. The mask phenotype was first discovered in unchallenged mice because of hair loss in Tmprss6mask/mask mice. Sublytic is a model of severe anemia due to defects in iron reabsorption [Figure 4B; (12)]. The sublytic mutation was mapped to Atp4a, the gene that encodes ATP4A, an H+, K+-ATPase that functions to regulate the transport of ions across membranes. The zeitgeist mutation in Med30, a gene that encodes a member of the Mediator complex that functions in RNA polymerase II transcriptional machinery, exhibits lethality soon after weaning due to progressive cardiomyopathy [Figure 4C; (13)]. The zeitgeist model established a connection between the Mediator complex and the induction of oxidative phosphorylation and fatty acid oxidation in metabolism. Furthermore, the zeitgeist model revealed a link between these metabolic functions and cardiac function.

 

Behavioral phenotypes

Several mutants have been identified based on changes to observable behavioral patterns. For example, the Possum mutant [see “New Genes” for more details] showed a complex behavioral phenotype with autonomic features emanating from a change in a peripheral voltage-gated sodium channel:  something that would not otherwise have been suspected (14). And a circling mutant, add, led to the identification of a new cause of non-syndromic deafness in humans (15).  

Figure 4. Metabolism phenotypes. (A)The mask strain was identified by hair loss, but subsequently determined to be iron deficient due to a mutation in Tmprss6. (B) The sublytic strain has a mutation in Atp4a and is severely anemic as shown by reduced iron staining (blue) in the spleen. (C) The zeitgeist strain exhibits cardiac defects due to a mutation in Med30. This mutation causes myocardial necrosis and fibrosis. H&E staining of the myocardium from wild-type and zeitgeist mice.

Development

Our lab has characterized mutants with visible defects in development.  The cartoon mouse showed craniofacial anomalies including a shortened head and snout as well as large eyes (Figure 5A). These mice also exhibited arrested physical development, were significantly smaller than wild-type mice, and had a shortened life span. The cartoon mutation was mapped to Mmp14, a gene that encodes the matrix metalloproteinase, MT1-MMP. MT1-MMP forms a complex with fibroblast growth factor receptor 2 (FGFR2) and ADAM9, an essential process that is required for FGFR2-mediated signaling and proper skull development (16). The bat mouse exhibited cryptophthalmos (skin covering the globe of the eye), occasional hindlimb syndactyly (fusion of fingers or toes), and renal anomalies (unilateral agenesis of the kidney) (Figure 5B) (17).  The bat mutation was mapped to the Frem1 gene that encodes FREM1, an extracellular matrix protein that mediates intracellular interactions that control tissue development. The bat strain mimics Fraser syndrome, a human condition characterized by cryptophthalmos, middle and outer ear malformations, and syndactyly.

Figure 5. Visible phenotypes of the cartoon and bat mice. (A) Homozygous cartoon mutant with shortened head and snout and large-appearing eyes. (B) Adult bat mice display cryptophthalmos (left). Bat embryos develop subepidermal blisters around the eyes and sides of the head (right).

Mutagenetix: an ever-expanding collection of mutations

To date, 606 transmissible mutations that cause discernable phenotypes have been set aside for positional cloning in the Beutler laboratory; 269 mutations have been mapped to chromosomes and in 258 instances, molecular identification of the causative mutation has been made. These mutations fall within 171 genes. 402 of the mutations studied affect immunity, and about half of the mutations affecting immunity that are cloned prove to be novel in the sense that no such phenotype had been predicted by knockout mutations, or knockouts had not been generated.

Please visit our Mutagenetix website to view the expanding list of mutations that we have produced and solved. All mutant stocks are deposited with MMRRCJAX, or other repositories when published and are available to the scientific community through these sources.

References

1. Kuhlbrodt, K., Schmidt, C., Sock, E., Pingault, V., Bondurand, N., Goossens, M., and Wegner, M. (1998) Functional Analysis of Sox10 Mutations found in Human Waardenburg-Hirschsprung Patients. J Biol Chem. 273, 23033-23038.

2. Pingault, V., Bondurand, N., Kuhlbrodt, K., Goerich, D. E., Prehu, M. O., Puliti, A., Herbarth, B., Hermans-Borgmeyer, I., Legius, E., Matthijs, G., Amiel, J., Lyonnet, S., Ceccherini, I., Romeo, G., Smith, J. C., Read, A. P., Wegner, M., and Goossens, M. (1998) SOX10 Mutations in Patients with Waardenburg-Hirschsprung Disease. Nat Genet. 18, 171-173.

3. Inoue, K., Shilo, K., Boerkoel, C. F., Crowe, C., Sawady, J., Lupski, J. R., and Agamanolis, D. P. (2002) Congenital Hypomyelinating Neuropathy, Central Dysmyelination, and Waardenburg-Hirschsprung Disease: Phenotypes Linked by SOX10 Mutation. Ann Neurol. 52, 836-842.

4. Inoue, K., Khajavi, M., Ohyama, T., Hirabayashi, S., Wilson, J., Reggin, J. D., Mancias, P., Butler, I. J., Wilkinson, M. F., Wegner, M., and Lupski, J. R. (2004) Molecular Mechanism for Distinct Neurological Phenotypes Conveyed by Allelic Truncating Mutations. Nat Genet. 36, 361-369.

5. Siggs, O. M., Cruite, J. T., Du, X., Rutschmann, S., Masliah, E., Beutler, B., and Oldstone, M. B. (2012) Disruption of Copper Homeostasis due to a Mutation of Atp7a Delays the Onset of Prion Disease. Proc Natl Acad Sci U S A. .

6. Danks, D. M., Campbell, P. E., Stevens, B. J., Mayne, V., and Cartwright, E. (1972) Menkes's Kinky Hair Syndrome. an Inherited Defect in Copper Absorption with Widespread Effects. Pediatrics. 50, 188-201.

7. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Isolation of a Candidate Gene for Menkes Disease and Evidence that it Encodes a Copper-Transporting ATPase. Nat Genet. 3, 7-13.

8. Mercer, J. F. (1998) Menkes Syndrome and Animal Models. Am J Clin Nutr. 67, 1022S-1028S.

9. Mercer, J. F., Livingston, J., Hall, B., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., and Siemieniak, D. (1993) Isolation of a Partial Candidate Gene for Menkes Disease by Positional Cloning. Nat Genet. 3, 20-25.

10. Du, X., Tabeta, K., Hoebe, K., Liu, H., Mann, N., Mudd, S., Crozat, K., Sovath, S., Gong, X., and Beutler, B. (2004) Velvet, a Dominant Egfr Mutation that Causes Wavy Hair and Defective Eyelid Development in Mice. Genetics. 166, 331-340.

11. Du, X., She, E., Gelbart, T., Truksa, J., Lee, P., Xia, Y., Khovananth, K., Mudd, S., Mann, N., Moresco, E. M., Beutler, E., and Beutler, B. (2008) The Serine Protease TMPRSS6 is Required to Sense Iron Deficiency. Science. 320, 1088-1092.

12. Krieg, L., Milstein, O., Krebs, P., Xia, Y., Beutler, B., and Du, X. (2011) Mutation of the Gastric Hydrogen-Potassium ATPase Alpha Subunit Causes Iron-Deficiency Anemia in Mice. Blood. 118, 6418-6425.

13. Krebs, P., Fan, W., Chen, Y. H., Tobita, K., Downes, M. R., Wood, M. R., Sun, L., Li, X., Xia, Y., Ding, N., Spaeth, J. M., Moresco, E. M., Boyer, T. G., Lo, C. W., Yen, J., Evans, R. M., and Beutler, B. (2011) Lethal Mitochondrial Cardiomyopathy in a Hypomorphic Med30 Mouse Mutant is Ameliorated by Ketogenic Diet. Proc Natl Acad Sci U S A. 108, 19678-19682.

14. Blasius, A. L., Dubin, A. E., Petrus, M. J., Lim, B. K., Narezkina, A., Criado, J. R., Wills, D. N., Xia, Y., Moresco, E. M., Ehlers, C., Knowlton, K. U., Patapoutian, A., and Beutler, B. (2011) Hypermorphic Mutation of the Voltage-Gated Sodium Channel Encoding Gene Scn10a Causes a Dramatic Stimulus-Dependent Neurobehavioral Phenotype. Proc Natl Acad Sci U S A. 108, 19413-19418.

15. Du, X., Schwander, M., Moresco, E. M., Viviani, P., Haller, C., Hildebrand, M. S., Pak, K., Tarantino, L., Roberts, A., Richardson, H., Koob, G., Najmabadi, H., Ryan, A. F., Smith, R. J., Muller, U., and Beutler, B. (2008) A Catechol-O-Methyltransferase that is Essential for Auditory Function in Mice and Humans. Proc Natl Acad Sci U S A. 105, 14609-14614.

16. Chan, K. M., Wong, H. L., Jin, G., Liu, B., Cao, R., Cao, Y., Lehti, K., Tryggvason, K., and Zhou, Z. (2012) MT1-MMP Inactivates ADAM9 to Regulate FGFR2 Signaling and Calvarial Osteogenesis. Dev Cell. .

17. Smyth, I., Du, X., Taylor, M. S., Justice, M. J., Beutler, B., and Jackson, I. J. (2004) The Extracellular Matrix Gene Frem1 is Essential for the Normal Adhesion of the Embryonic Epidermis. Proc Natl Acad Sci U S A. 101, 13560-13565.