Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development Eiri´kur Steingri´msson*†, Lino Tessarollo‡, Bhavani Pathak§¶, Ling Houʈ**, Heinz Arnheiterʈ, Neal G. Copeland§, and Nancy A. Jenkins§ *Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland; ‡Neural Development Group and §Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD 21702-1201; and ʈLaboratory of Developmental Neurogenetics, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Communicated by Liane B. Russell, Oak Ridge National Laboratory, Oak Ridge, TN, February 6, 2002 (received for review December 1, 2001) The Mitf-Tfe family of basic helix–loop–helix-leucine zipper (bHLH- neuroepithelial-derived retinal pigment epithelium of the eye, Zip) transcription factors encodes four family members: Mitf, Tfe3, ultimately resulting in microphthalmia, and some Mitf mutations Tfeb, and Tfec. In vitro, each protein in the family can bind DNA as result in a reduction in mast cell numbers. Thus, the Mitf a homo- or heterodimer with other family members. Mutational mutations are pleiotropic, affect many different cell types, and studies in mice have shown that Mitf is essential for melanocyte can be arranged in an allelic series (4). and eye development, whereas Tfeb is required for placental A mutation has been made in the Tfeb gene (TfebFcr) and vascularization. Here, we uncover a role for Tfe3 in osteoclast results in embryonic lethality because of defects in placental development, a role that is functionally redundant with Mitf. vascularization; the labyrinthine cells fail to express vascular Although osteoclasts seem normal in Mitf or Tfe3 null mice, the endothelial growth factor, the embryonic vasculature is unable combined loss of the two genes results in severe osteopetrosis. We to invade the placenta, and the embryos degenerate as a result also show that Tfec mutant mice are phenotypically normal, and of hypoxia (5). Unfortunately, the embryos die before melano- that the Tfec mutation does not alter the phenotype of Mitf, Tfeb, cytes or mast cells are formed thus it is unclear whether Tfeb is or Tfe3 mutant mice. Surprisingly, our studies failed to identify any important for the development of these cell types as well. phenotypic overlap between the different Mitf–Tfe mutations. Molecular analysis of the semidominant Mitf mutations shows These results suggest that heterodimeric interactions are not that they affect the basic or transcriptional activation domains of essential for Mitf-Tfe function in contrast to other bHLH-Zip fam- ͞ ͞ the protein (6–8). These mutant proteins cannot bind DNA; ilies like Myc Max Mad, where heterodimeric interactions seem to however, they can still dimerize with proteins such as Tfe3 and be essential. thereby interfere with DNA binding of the wild-type partner (9). The dominant negative behavior of these mutant proteins likely asic helix–loop–helix-leucine zipper (bHLH-Zip) proteins accounts for the phenotype seen in heterozygous mice. Consis- Bregulate gene expression by binding to the E-box tent with this hypothesis, the recessive Mitf mutations affect (CANNTG) as hetero- or homodimers; some dimers activate the dimerization domain of the protein or lack Mift expression gene expression whereas others repress it. The prototypic (6, 7, 10). ͞ ͞ bHLH-Zip family is Myc Max Mad. Work from many labora- Interestingly, a few of the strong semidominant Mitf mutations GENETICS tories suggests that these proteins function through a complex also produce osteopetrosis because of osteoclast defects, which network of interacting proteins (1). The ubiquitous Max protein is not seen in Mitf mutant mice carrying loss of function is at the heart of this network. When Myc concentration is high, mutations at the locus. This phenotype is limited to homozygous it complexes with Max, resulting in increased gene expression animals indicating that the mutations are recessive with respect with concomitant effects on the cell. In contrast, when the to the osteoclasts. Bone marrow transplantation experiments concentration of a Mad family member is high, Max complexes performed on the original Mitf microphthalmia (Mitf mi) mutation with Mad, resulting in reduced gene expression. Mad-mediated (one of the semidominant Mitf mutations that produces osteo- repression results from the interaction of Mad proteins with petrosis) showed that osteopetrosis can be rescued by trans- mSin3 proteins, which in turn interact with the histone deacety- planting cell suspensions from wild-type spleen and bone mar- lases to moderate gene expression (2). The emerging complexity row to the mutant animals (11). Similarly, transplantation of of this network suggests that the specificity of this protein family bone marrow from Mitf mi mice to lethally irradiated wild-type is because of protein–protein interactions as well as different mice induces osteopetrosis (12). Ultrastructural analysis shows target gene specificity (3). It has been difficult, however, to mi confirm these results in vivo, as a result of the complexity of this that osteoclasts from Mitf mice are smaller than normal, lack family and that many of the mutations are embryonic-lethal. ruffled borders (13), and exhibit fusion disability (14). These Another well studied bHLH-Zip protein family is Mitf-Tfe. strong dominant negative Mitf alleles therefore seem to induce Four Mitf-Tfe family members have been identified: microph- thalmia (Mitf), Tfe3, Tfeb, and Tfec. Mutations in Mitf were Abbreviation: bHLH, basic helix–loop–helix. recognized as early as 1942 and since then over 20 spontaneous †To whom reprint requests should be addressed. E-mail: [email protected]. or induced mutations have been identified at the locus (4). ¶Present address: American Association for the Advancement of Science, 1200 New York Interestingly, about half of the mutations are semidominantly Avenue, NW, Washington, DC 20005. inherited (i.e., they show a partial phenotype in the heterozygous **Present address: Genetic Diseases Research Branch, National Human Genome Research condition) and about half are recessively inherited. All Mitf Institute, National Institutes of Health, Bethesda, MD 20892. mutations result in defects in neural crest-derived melanocytes, The publication costs of this article were defrayed in part by page charge payment. This manifested by reduction or lack of pigment in the coat and inner article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. ear. Many of the mutations also affect differentiation of the §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.072071099 PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 ͉ 4477–4482 Downloaded by guest on September 29, 2021 osteopetrosis by affecting osteoclast differentiation; the defect is Mitf mi-vga9͞Mitf mi-vga9 mice were maintained and propagated at cell-autonomous with respect to osteoclasts. the National Institutes of Health. Several observations suggest that Mitf-Tfe proteins mediate their effects as heterodimers, like Myc͞Max͞Mad. For example, Results the Mitf-Tfe family members are coexpressed in several cell Tfe3 and Tfec Mutations. To begin to analyze the functions of Tfe3 types, including cells affected by Mitf mutations such as oste- and Tfec in vivo and determine whether any of these functions oclasts (15) and melanocytes (16). Furthermore, all possible overlap with Mitf and Tfeb, we made germ-line null mutations in combinations of Mitf-Tfe homo- and heterodimers can form in both genes by means of gene targeting in mouse embryonic stem vitro (9), and Mitf-Tfe heterodimers have been shown to exist in cells. Part, or all, of the bHLH-Zip domain of each protein was cell cultures (15). Taken together, these data suggest that the replaced with pGKNeo (Fig. 1 A and D), and the mutations, absence of any one protein would produce a phenotype that is designated Tfe3Fcr and TfecFcr, respectively, were introduced into at least partially overlapping with one or more of the other family the mouse germ line by standard techniques (Fig. 1 B and E). As members. Here, we produce germ-line null mutations in Tfe3 expected, the Tfec message expressed in homozygous TfecFcr and Tfec and show that the osteopetrosis associated with the mice is missing 132 bp of the Tfec bHLH-Zip domain and dominant negative Mitf alleles is most likely a result of dominant contains a frameshift mutation, which deletes the remainder of negative interference with Tfe3. Surprisingly, none of the Mitf- the protein (Fig. 1F). In contrast, the Tfe3Fcr mutation destabi- Tfe phenotypes studied overlap, suggesting that the homodimer, lizes the Tfe3Fcr message, and no stable mRNA is made in not the heterodimer, is the essential functional unit of the homozygous Tfe3Fcr mice (Fig. 1C). Surprisingly, homozygous Mitf-Tfe family. Tfe3Fcr and TfecFcr mice are indistinguishable from their wild- type littermates; they are viable and fertile, normally pigmented, Materials and Methods have normal eyes and mast cells, and show no osteopetrosis (Fig. Targeted Disruption of Tfe3 and Tfec. Tfe3 and Tfec genomic clones 2F, Table 1, and data not shown). Furthermore, detailed analysis were isolated from a 129͞Sv mouse library (Stratagene) by using of homozygous Tfe3Fcr mice (C. Tunyaplin and K. Calame, human Tfe3 and rat Tfec cDNA probes, respectively (17, 18).