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The Chromosomal Mapping of Four Genes Encoding Winged Helix Proteins Expressed Early in Mouse Development
SHORT COMMUNICATION The Chromosomal Mapping of Four Genes Encoding Winged Helix Proteins Expressed Early in Mouse Development PATRICIA A. LABOSKY,* GLENN E. WINNIER,† HIROSHI SASAKI,*,1 MANFRED BLESSING,†,2 AND BRIGID L. M. HOGAN*,3 *Howard Hughes Medical Institute and †Department of Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232-2175 Received September 25, 1995; accepted March 21, 1996
Members of the winged helix family of transcription factors are required for the normal embryonic development of the mouse. Using the interspecific backcross panel from The Jackson Laboratory, we have determined the chromosomal locations of four genes that encode winged helix containing proteins. Mf1 was assigned to mouse Chromosome 8, Mf2 to Chromosome 4, Mf3 to Chromosome 9, and Mf4 to Chromosome 13. Since Mf3 is located in a region of Chromosome 9 containing many well-characterized mouse mutations such as short ear (se), ashen (ash), and dilute (d), we have analyzed deletion mutants to determine the location of Mf3 more precisely. q 1996 Academic Press, Inc.
The temporal and spatial pattern of expression of a gene and the nature of its protein product are important criteria for evaluating its role during embryonic development. Additionally, determining the chromosomal location of a gene can provide clues about its in vivo function. The four genes discussed here (Mf1, Mf2, Mf3, and Mf4) encode members of a family of over 40 transcription factors containing a winged helix motif in their DNA binding domain (see 5 for review). Mf1, Mf2, and Mf3 were cloned on the basis of their sequence similarity to the Xenopus winged helix containing gene XFKH1 (12). The sequence of the winged helix domain of mouse fkh1 (7) is almost identical to that of Mf1 but sequences outside this domain are not yet available for comparison. Mf3 was originally identified as a cDNA known as c43 (12) and was independently cloned and characterized as Hfh-e5.1 (1). The locus here called Mf4 has not yet been characterized The International Committee on Standardized Genetic Nomenclature for Mice at The Jackson Laboratory has approved the locus symbols Mf1, Mf2, Mf3, and Mf4 (mouse/mesoderm/mesenchyme fork-head 1, 2, 3, and 4). 1 Present address: Medical Department, Johannes Gutenberg University, Mainz, Germany. 2 Present address: Laboratory of Developmental Biology, Institute for Molecular and Cellular Biology, Osaka University, Japan. 3 To whom correspondence should be addressed. Telephone: (615) 343-6413. Fax: (615) 343-2033.
in detail, nor have cDNAs been isolated. Its position is determined solely on the basis of hybridization of a winged helix domain probe to specific genomic DNA fragments. The expression patterns of Mf1, Mf2, Mf3, and Mf4 during embryogenesis are distinct (1, 12, and G.E.W. and P.A.L., unpublished results). Mf1 transcripts are found in the presomitic mesoderm and somites, with higher expression in the dorsal region of the somites early in development. Mf2 is expressed in the paraxial mesoderm of the head, as well as the sclerotome of the somites. Mf3 is expressed in the presomitic mesoderm, but the strongest expression is seen in the neural plate and later in the neural tube and restricted regions of the brain. Preliminary data suggest that transcripts hybridizing with the winged helix domain of Mf4 are expressed in the embryo. This expression pattern is similar to that of Mf1 in the somites; however, transcripts for Mf4 are also seen in the branchial arches of the embryo (G.E.W., unpublished results). Targeted null mutations in genes belonging to this family of transcription factors show defects in embryogenesis. For example, embryos homozygous for a null mutation in hepatocyte nuclear factor 3b (Hnf3b) die between 10 and 11 days of embryogenesis with defects in the node, notochord, and neural tube, all sites where transcripts for Hnf3b are detected (2, 13). Embryos homozygous for a null mutation in brain factor 1 (Bf1) have defects in the telencephalon (14). It has also recently been demonstrated that mutations in the winged helix gene winged helix nude (whn) are responsible for the phenotype of the nude mouse (8). Other investigators have determined the chromosomal locations of additional genes encoding winged helix proteins and have shown that they are dispersed throughout the genome (3). We wished to establish the chromosomal locations of Mf1, Mf2, and Mf3 as one step toward determining if mutations in these genes are responsible for known mouse mutations. A mouse 129/Sv genomic library was screened at high stringency with cDNA probes for Mf1, Mf2, and Mf3 (10) using standard methods (11). The resulting phage DNAs were analyzed to generate the restriction maps shown in Fig. 1. The genomic DNA probes indiGENOMICS
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34, 241–245 (1996) ARTICLE NO. 0275
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Partial restriction enzyme maps of the Mf1, Mf2, and Mf3 genomic loci. The probes used to detect polymorphisms are indicated by open boxes, and portions of the loci known to encode a cDNA are indicated by filled boxes. Abbreviations used: A, Asp718; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; N, NotI; Nc, NcoI; P, PstI; Sp, SpeI; and X, XhoI.
cated in Fig. 1 were then used to analyze 94 progeny from The Jackson Laboratory interspecific backcross panel BSS (10). In all three cases, probes were derived from closely flanking DNA instead of cDNA. This maximized the chances of detecting restriction fragment length variants (RFLVs) and also ensured that the probes would correspond only to the gene of interest as opposed to pseudogenes or any related sequences. RFLVs between C57BL/6J and Mus spretus from The Jackson Laboratory colony were identified for each gene with the following genomic DNA probes: for Mf1, a 2.4-kb BglII fragment showed a RFLV with the restriction enzymes MspI and TaqI; a 3.5-kb BglII fragment of the Mf2 locus detected RFLVs with SacI and TaqI; and a 4.5-kb BamHI–Asp718 fragment of the Mf3 locus detected variants with SacI, MspI, and TaqI. In all cases, 94 of the (C57BL/6JEi 1 SPRET/Ei)F1 1 SPRET/Ei progeny were typed for inheritance of the Mus domesticus or Mus spretus alleles, and the distribution pattern of each allele was used to place the loci onto the interspecific backcross map. For Mf1, RFLVs were detected using the restriction enzymes MspI and TaqI. In the case of MspI, the indicated probe 2 revealed 1.2- and 1.0-kb hybridizing fragments for the M. spretus allele and 1.4-kb and 900-bp hybridizing fragments for the M. domesticus allele. For TaqI, the same probe hybridized to a 900-bp fragment for the M. spretus allele and 1.6-kb and 700-bp fragments for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot hybridizations with both MspI and TaqI, Mf1 was assigned to Chromosome 8, 2.1 cM distal to Cox4 (Fig.
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2A). The position of Mf1 is close to the chromosomal location of another winged helix containing gene, Hfh8 (3). However, DNA sequence analysis establishes Mf1 and Hfh8 as two distinct genes (data not shown). When a fragment containing only the Mf1 winged helix domain (probe 1, shown in Fig. 1) was used as a probe, two distinct sets of RFLVs were detected between the M. spretus and M. domesticus alleles using the enzyme DraI. This probe revealed a 1.3-kb fragment for the M. domesticus allele and a 1.2-kb fragment for the M. spretus allele that confirmed our initial mapping of Mf1 to Chromosome 8. Additionally, this probe detected a cross-hybridizing 5-kb fragment for the M. domesticus allele and a 6-kb fragment for the M. spretus allele, which assigned a second locus to Chromosome 13 (Fig. 2D) near Hfh1 (3). The gene corresponding to this locus is presumed to have a winged helix domain identical or closely related to that of Mf1. This gene is designated Mf4. For Mf2, RFLVs were detected using the restriction enzymes SacI and TaqI. In the case of SacI, the indicated probe revealed 7.5-kb and 800-bp hybridizing fragments for the M. spretus allele and 4.2- and 5.5-kb hybridizing fragments for the M. domesticus allele. For TaqI, the same probe hybridized to a 5.5-kb fragment for the M. spretus allele and a 6.8-kb fragment for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot hybridizations with both SacI and TaqI, Mf2 was assigned to Chromosome 4 near Mpl (myeloproliferative leukemia virus oncogene; see Fig. 2B). For Mf3, RFLVs were detected using the restriction
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FIG. 2. The chromosomal location of Mf1 (A), Mf2 (B), Mf3 (C), and Mf4 (D). The four genes encoding winged helix proteins were mapped using The Jackson Laboratory interspecific backcross panel BSS. Shown are partial chromosomal linkage maps with surrounding loci. In all cases, 94 of the (C57BL/6JEi 1 SPRET/Ei)F1 1 SPRET/Ei) progeny were typed for inheritance of the M. domesticus or M. spretus alleles. Haplotype maps are also shown for each gene. All data generated were analyzed using the Map Manager program. The black boxes indicate inheritance of a M. domesticus allele while the white boxes indicate inheritance of a M. spretus allele. Loci indicated in parentheses are from The Jackson Laboratory Encyclopedia of the Mouse Genome, and their exact positions relative to Mf1, Mf2, Mf3, and Mf4 are unknown. Genotypes for some untyped animals for surrounding markers were inferred, and these data and references can be accessed at the internet address http://www.jax.org/resources/documents/cmdata. Additional references for some of the loci can be found in The Jackson Laboratory Encyclopedia of the Mouse Genome. R, recombination distances in centimorgans; SE, standard error.
enzymes SacI, MspI, and TaqI. SacI was used to map the gene, and the indicated probe revealed a 6.5-kb hybridizing fragment for the M. spretus allele and a 4.5kb hybridizing fragment for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot, Mf3 was assigned to Chromosome 9, 1.1 cM proximal to the microsatellite marker D9Mit8 (Fig.
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2C). Because this placed Mf3 within the region of ashen (ash), dilute (d), and short ear (se), we analyzed two deletion mutants specific for this region of Chromosome 9 (Fig. 3). The two deletions we examined, 18FATWb and 37FBFo, were induced by X-ray mutagenesis (9) and are two independent deletions spanning d and se. In each case, the deletion chromosomes were made in
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FIG. 3. Possible location of Mf3 with respect to the deletion chromosomes 37FBFo and 18FATw. Southern analysis of DNA from animals containing deletion chromosomes 37FBFo and 18FATw. These tissues were kindly provided by Drs. Dabney Johnson and Gene Rinchik (Oak Ridge National Laboratories, TN). Lane 1, M. spretus from ORNL; Lane 2, (C3H 1 101)F1 hybrid; Lane 3, Animal 50799 (M. spretus/ 37FBFo); Lane 4, Animal 50987 (M. spretus/nondeletion balancer chromosome); Lane 5, Animal 50990 (M. spretus/18FATW); Lane 6, Animal 50797 (M. spretus/37FBFo). Mf3 was shown to be outside the region of Chromosome 9 deleted in 37FBFo and 18FATw.
a (C3H 1 101)F1 hybrid and maintained by crossing the mice with M. spretus from the outbred colony at Oak Ridge National Laboratories (ORNL) (9). Genomic DNAs from animals carrying the deletion chromosomes were restricted with MspI and the same probe used to map Mf3 on The Jackson Laboratory backcross now revealed 2.8- and 2.5-kb hybridizing fragments with M. spretus from the ORNL outbred colony and 6.2- and 3.2-kb hybridizing fragments with the (C3H 1 101)F1 hybrid (Fig. 3). When DNA from an animal with the genotype M. spretus/nondeletion balancer chromosome (Animal 50987, lane 4) was analyzed, hybridization indicated the presence of a M. spretus allele as well as an additional hybridizing band indicating either the C3H or the 101 Chromosome. When DNAs from animals carrying the deletion chromosome were analyzed in the same manner, hybridizing fragments for both the M. spretus allele and the C3H or 101 allele for Mf3 were present in these animals (Fig. 3). This indicates that Mf3 is not included in either deletion, despite its location proximal to D9Mit8 and distal to D9Mit21, leading us to conclude that Mf3 must be further proximal than prenatal lethal factor 2 (pnlf2). The only nearby mutations are Snell’s waltzer (sv), staggerer (sg), and small thymus (sty). Both sv and sg have been cloned recently and encode a novel myosin heavy chain (4) and a member of the nuclear hormone-receptor family (6), respectively. The only other candidate mutation within the region predicted to contain Mf3 is pnlf1, a prenatal lethal factor. In conclusion, Mf1 and Mf2 do not map closely to any known mutations consistent with the expression pattern of these genes. However, the location of Mf3 does suggest one possible mutant locus, pnlf1. Gene targeting experiments are underway to determine the null mutant phenotypes of Mf1, Mf2, and Mf3.
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ACKNOWLEDGMENTS P.A.L. and H.S. are Associates and B.L.M.H. is an Investigator of the Howard Hughes Medical Institute. We thank Dr. Dabney Johnson, Oak Ridge National Laboratories, and Mary Barter at The Jackson Laboratory for their assistance. Dr. Lucy Rowe, The Jackson Laboratory, provided invaluable advice and encouragement. We also thank Drs. Linda Siracusa and Leslie Lock for helpful comments on the manuscript. Note added in proof. We have now obtained the complete sequence of the cDNA for the locus we call Mf1 and have mapped to Chromosome 8 in this study. This is identical to approximately 3 kb of genomic sequence from the same locus and is also identical to the gene Mfh-1 (N. Miura, A. Wanaka, M. Tohyama, and K. Tanaka (1993) MHF-1, a new member of the forkhead domain family, is expressed in developing mesenchyme. FEBS Lett. 326: 171–176). The Mfh-1 and Mf1 cDNA sequences differ by only 23 of the 330 nucleotides in the winged helix encoding domain. We therefore conclude that the locus on Chromosome 8 corresponds to Mfh-1, and the locus on Chromosome 13 probably corresponds to Mf1 of Ref. 12.
REFERENCES 1. Ang, S., Wierda, A., Wong, D., Stevens, K. A., Cascio, S., Rossant, J., and Zaret, K. S. (1993). The formation and maintenance of the definitive endoderm lineage in the mouse: Involvement of HNF3/forkhead proteins. Development 119: 1301–1315. 2. Ang, S., and Rossant, J. (1994). HNF-3b is essential for node and notochord formation in mouse development. Cell 78: 561– 574. 3. Avraham, K. B., Fletcher, C., Overdier, D. G., Clevidence, D. E., Lai, E., Costa, R. H., Jenkins, N. A., and Copeland, N. G. (1995). Murine chromosomal location of eight members of the hepatocyte nuclear factor 3/fork head winged helix family of transcription factors. Genomics 25: 388–393. 4. Avraham, K. B., Hasson, T., Steel, K. P., Kingsley, D. M., Russell, L. B., Mooseker, M. S., Copeland, N. G., and Jenkins, N. A. (1995). The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner ear hair cells. Nature Genet. 11: 369–375.
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11.
12.
13.
14.
V. A., Eppig, J. T., Ko, M. S. H., Thurston, S. J., and Birkenmeier, E. H. (1994). Maps from two interspecific backcross DNA panels available as a community genetic mapping resource. Mamm. Genome 5: 253–274. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ Second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Sasaki, H., and Hogan, B. L. M. (1993). Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118: 47–59. Weinstein, D. C., Ruiz i Altaba, A., Chen, W. S., Hoodless, P., Preziosos, V. R., Jessell, T. M., and Darnell, J. E. (1994). Transcription factor HNF-3b is required for notochord development in the mouse embryo. Cell 78: 575–588. Xuan, S., Baptista, C. A., Balas, G., Tao, W., Soares, V. C., and Lai, E. (1995). Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 14: 1141–1152.
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Members of the winged helix family of transcription factors are required for the normal embryonic development of the mouse. Using the interspecific backcross panel from The Jackson Laboratory, we have determined the chromosomal locations of four genes that encode winged helix containing proteins. Mf1 was assigned to mouse Chromosome 8, Mf2 to Chromosome 4, Mf3 to Chromosome 9, and Mf4 to Chromosome 13. Since Mf3 is located in a region of Chromosome 9 containing many well-characterized mouse mutations such as short ear (se), ashen (ash), and dilute (d), we have analyzed deletion mutants to determine the location of Mf3 more precisely. q 1996 Academic Press, Inc.
The temporal and spatial pattern of expression of a gene and the nature of its protein product are important criteria for evaluating its role during embryonic development. Additionally, determining the chromosomal location of a gene can provide clues about its in vivo function. The four genes discussed here (Mf1, Mf2, Mf3, and Mf4) encode members of a family of over 40 transcription factors containing a winged helix motif in their DNA binding domain (see 5 for review). Mf1, Mf2, and Mf3 were cloned on the basis of their sequence similarity to the Xenopus winged helix containing gene XFKH1 (12). The sequence of the winged helix domain of mouse fkh1 (7) is almost identical to that of Mf1 but sequences outside this domain are not yet available for comparison. Mf3 was originally identified as a cDNA known as c43 (12) and was independently cloned and characterized as Hfh-e5.1 (1). The locus here called Mf4 has not yet been characterized The International Committee on Standardized Genetic Nomenclature for Mice at The Jackson Laboratory has approved the locus symbols Mf1, Mf2, Mf3, and Mf4 (mouse/mesoderm/mesenchyme fork-head 1, 2, 3, and 4). 1 Present address: Medical Department, Johannes Gutenberg University, Mainz, Germany. 2 Present address: Laboratory of Developmental Biology, Institute for Molecular and Cellular Biology, Osaka University, Japan. 3 To whom correspondence should be addressed. Telephone: (615) 343-6413. Fax: (615) 343-2033.
in detail, nor have cDNAs been isolated. Its position is determined solely on the basis of hybridization of a winged helix domain probe to specific genomic DNA fragments. The expression patterns of Mf1, Mf2, Mf3, and Mf4 during embryogenesis are distinct (1, 12, and G.E.W. and P.A.L., unpublished results). Mf1 transcripts are found in the presomitic mesoderm and somites, with higher expression in the dorsal region of the somites early in development. Mf2 is expressed in the paraxial mesoderm of the head, as well as the sclerotome of the somites. Mf3 is expressed in the presomitic mesoderm, but the strongest expression is seen in the neural plate and later in the neural tube and restricted regions of the brain. Preliminary data suggest that transcripts hybridizing with the winged helix domain of Mf4 are expressed in the embryo. This expression pattern is similar to that of Mf1 in the somites; however, transcripts for Mf4 are also seen in the branchial arches of the embryo (G.E.W., unpublished results). Targeted null mutations in genes belonging to this family of transcription factors show defects in embryogenesis. For example, embryos homozygous for a null mutation in hepatocyte nuclear factor 3b (Hnf3b) die between 10 and 11 days of embryogenesis with defects in the node, notochord, and neural tube, all sites where transcripts for Hnf3b are detected (2, 13). Embryos homozygous for a null mutation in brain factor 1 (Bf1) have defects in the telencephalon (14). It has also recently been demonstrated that mutations in the winged helix gene winged helix nude (whn) are responsible for the phenotype of the nude mouse (8). Other investigators have determined the chromosomal locations of additional genes encoding winged helix proteins and have shown that they are dispersed throughout the genome (3). We wished to establish the chromosomal locations of Mf1, Mf2, and Mf3 as one step toward determining if mutations in these genes are responsible for known mouse mutations. A mouse 129/Sv genomic library was screened at high stringency with cDNA probes for Mf1, Mf2, and Mf3 (10) using standard methods (11). The resulting phage DNAs were analyzed to generate the restriction maps shown in Fig. 1. The genomic DNA probes indiGENOMICS
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34, 241–245 (1996) ARTICLE NO. 0275
0888-7543/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Partial restriction enzyme maps of the Mf1, Mf2, and Mf3 genomic loci. The probes used to detect polymorphisms are indicated by open boxes, and portions of the loci known to encode a cDNA are indicated by filled boxes. Abbreviations used: A, Asp718; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; N, NotI; Nc, NcoI; P, PstI; Sp, SpeI; and X, XhoI.
cated in Fig. 1 were then used to analyze 94 progeny from The Jackson Laboratory interspecific backcross panel BSS (10). In all three cases, probes were derived from closely flanking DNA instead of cDNA. This maximized the chances of detecting restriction fragment length variants (RFLVs) and also ensured that the probes would correspond only to the gene of interest as opposed to pseudogenes or any related sequences. RFLVs between C57BL/6J and Mus spretus from The Jackson Laboratory colony were identified for each gene with the following genomic DNA probes: for Mf1, a 2.4-kb BglII fragment showed a RFLV with the restriction enzymes MspI and TaqI; a 3.5-kb BglII fragment of the Mf2 locus detected RFLVs with SacI and TaqI; and a 4.5-kb BamHI–Asp718 fragment of the Mf3 locus detected variants with SacI, MspI, and TaqI. In all cases, 94 of the (C57BL/6JEi 1 SPRET/Ei)F1 1 SPRET/Ei progeny were typed for inheritance of the Mus domesticus or Mus spretus alleles, and the distribution pattern of each allele was used to place the loci onto the interspecific backcross map. For Mf1, RFLVs were detected using the restriction enzymes MspI and TaqI. In the case of MspI, the indicated probe 2 revealed 1.2- and 1.0-kb hybridizing fragments for the M. spretus allele and 1.4-kb and 900-bp hybridizing fragments for the M. domesticus allele. For TaqI, the same probe hybridized to a 900-bp fragment for the M. spretus allele and 1.6-kb and 700-bp fragments for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot hybridizations with both MspI and TaqI, Mf1 was assigned to Chromosome 8, 2.1 cM distal to Cox4 (Fig.
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2A). The position of Mf1 is close to the chromosomal location of another winged helix containing gene, Hfh8 (3). However, DNA sequence analysis establishes Mf1 and Hfh8 as two distinct genes (data not shown). When a fragment containing only the Mf1 winged helix domain (probe 1, shown in Fig. 1) was used as a probe, two distinct sets of RFLVs were detected between the M. spretus and M. domesticus alleles using the enzyme DraI. This probe revealed a 1.3-kb fragment for the M. domesticus allele and a 1.2-kb fragment for the M. spretus allele that confirmed our initial mapping of Mf1 to Chromosome 8. Additionally, this probe detected a cross-hybridizing 5-kb fragment for the M. domesticus allele and a 6-kb fragment for the M. spretus allele, which assigned a second locus to Chromosome 13 (Fig. 2D) near Hfh1 (3). The gene corresponding to this locus is presumed to have a winged helix domain identical or closely related to that of Mf1. This gene is designated Mf4. For Mf2, RFLVs were detected using the restriction enzymes SacI and TaqI. In the case of SacI, the indicated probe revealed 7.5-kb and 800-bp hybridizing fragments for the M. spretus allele and 4.2- and 5.5-kb hybridizing fragments for the M. domesticus allele. For TaqI, the same probe hybridized to a 5.5-kb fragment for the M. spretus allele and a 6.8-kb fragment for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot hybridizations with both SacI and TaqI, Mf2 was assigned to Chromosome 4 near Mpl (myeloproliferative leukemia virus oncogene; see Fig. 2B). For Mf3, RFLVs were detected using the restriction
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FIG. 2. The chromosomal location of Mf1 (A), Mf2 (B), Mf3 (C), and Mf4 (D). The four genes encoding winged helix proteins were mapped using The Jackson Laboratory interspecific backcross panel BSS. Shown are partial chromosomal linkage maps with surrounding loci. In all cases, 94 of the (C57BL/6JEi 1 SPRET/Ei)F1 1 SPRET/Ei) progeny were typed for inheritance of the M. domesticus or M. spretus alleles. Haplotype maps are also shown for each gene. All data generated were analyzed using the Map Manager program. The black boxes indicate inheritance of a M. domesticus allele while the white boxes indicate inheritance of a M. spretus allele. Loci indicated in parentheses are from The Jackson Laboratory Encyclopedia of the Mouse Genome, and their exact positions relative to Mf1, Mf2, Mf3, and Mf4 are unknown. Genotypes for some untyped animals for surrounding markers were inferred, and these data and references can be accessed at the internet address http://www.jax.org/resources/documents/cmdata. Additional references for some of the loci can be found in The Jackson Laboratory Encyclopedia of the Mouse Genome. R, recombination distances in centimorgans; SE, standard error.
enzymes SacI, MspI, and TaqI. SacI was used to map the gene, and the indicated probe revealed a 6.5-kb hybridizing fragment for the M. spretus allele and a 4.5kb hybridizing fragment for the M. domesticus allele. When 94 animals from the backcross were analyzed by Southern blot, Mf3 was assigned to Chromosome 9, 1.1 cM proximal to the microsatellite marker D9Mit8 (Fig.
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2C). Because this placed Mf3 within the region of ashen (ash), dilute (d), and short ear (se), we analyzed two deletion mutants specific for this region of Chromosome 9 (Fig. 3). The two deletions we examined, 18FATWb and 37FBFo, were induced by X-ray mutagenesis (9) and are two independent deletions spanning d and se. In each case, the deletion chromosomes were made in
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FIG. 3. Possible location of Mf3 with respect to the deletion chromosomes 37FBFo and 18FATw. Southern analysis of DNA from animals containing deletion chromosomes 37FBFo and 18FATw. These tissues were kindly provided by Drs. Dabney Johnson and Gene Rinchik (Oak Ridge National Laboratories, TN). Lane 1, M. spretus from ORNL; Lane 2, (C3H 1 101)F1 hybrid; Lane 3, Animal 50799 (M. spretus/ 37FBFo); Lane 4, Animal 50987 (M. spretus/nondeletion balancer chromosome); Lane 5, Animal 50990 (M. spretus/18FATW); Lane 6, Animal 50797 (M. spretus/37FBFo). Mf3 was shown to be outside the region of Chromosome 9 deleted in 37FBFo and 18FATw.
a (C3H 1 101)F1 hybrid and maintained by crossing the mice with M. spretus from the outbred colony at Oak Ridge National Laboratories (ORNL) (9). Genomic DNAs from animals carrying the deletion chromosomes were restricted with MspI and the same probe used to map Mf3 on The Jackson Laboratory backcross now revealed 2.8- and 2.5-kb hybridizing fragments with M. spretus from the ORNL outbred colony and 6.2- and 3.2-kb hybridizing fragments with the (C3H 1 101)F1 hybrid (Fig. 3). When DNA from an animal with the genotype M. spretus/nondeletion balancer chromosome (Animal 50987, lane 4) was analyzed, hybridization indicated the presence of a M. spretus allele as well as an additional hybridizing band indicating either the C3H or the 101 Chromosome. When DNAs from animals carrying the deletion chromosome were analyzed in the same manner, hybridizing fragments for both the M. spretus allele and the C3H or 101 allele for Mf3 were present in these animals (Fig. 3). This indicates that Mf3 is not included in either deletion, despite its location proximal to D9Mit8 and distal to D9Mit21, leading us to conclude that Mf3 must be further proximal than prenatal lethal factor 2 (pnlf2). The only nearby mutations are Snell’s waltzer (sv), staggerer (sg), and small thymus (sty). Both sv and sg have been cloned recently and encode a novel myosin heavy chain (4) and a member of the nuclear hormone-receptor family (6), respectively. The only other candidate mutation within the region predicted to contain Mf3 is pnlf1, a prenatal lethal factor. In conclusion, Mf1 and Mf2 do not map closely to any known mutations consistent with the expression pattern of these genes. However, the location of Mf3 does suggest one possible mutant locus, pnlf1. Gene targeting experiments are underway to determine the null mutant phenotypes of Mf1, Mf2, and Mf3.
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ACKNOWLEDGMENTS P.A.L. and H.S. are Associates and B.L.M.H. is an Investigator of the Howard Hughes Medical Institute. We thank Dr. Dabney Johnson, Oak Ridge National Laboratories, and Mary Barter at The Jackson Laboratory for their assistance. Dr. Lucy Rowe, The Jackson Laboratory, provided invaluable advice and encouragement. We also thank Drs. Linda Siracusa and Leslie Lock for helpful comments on the manuscript. Note added in proof. We have now obtained the complete sequence of the cDNA for the locus we call Mf1 and have mapped to Chromosome 8 in this study. This is identical to approximately 3 kb of genomic sequence from the same locus and is also identical to the gene Mfh-1 (N. Miura, A. Wanaka, M. Tohyama, and K. Tanaka (1993) MHF-1, a new member of the forkhead domain family, is expressed in developing mesenchyme. FEBS Lett. 326: 171–176). The Mfh-1 and Mf1 cDNA sequences differ by only 23 of the 330 nucleotides in the winged helix encoding domain. We therefore conclude that the locus on Chromosome 8 corresponds to Mfh-1, and the locus on Chromosome 13 probably corresponds to Mf1 of Ref. 12.
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