Gene content of the 750-kb critical region for mouse embryonic ectoderm lethal tcl-w5 Kuniya Abe, 1,2 Misako Yuzuriha, 1,2 Michihiko Sugimoto, 1 Minoru S.H. Ko, 3 Mgavi Brathwaite, 3 Paul Waeltz, 3 Ramaiah Nagaraja 3 1 Technology and Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan 2 Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan 3 Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6820, USA Received: 4 September 2003 / Accepted: 10 November 2003 Abstract Mice homozygous for the t w5 allele arrest at gastru- lation from defects associated with embryonic ecto- derm development. The mutated gene has been genetically closely linked to the H-2K locus in the mouse MHC region, flanked by markers H-2Pb and D17Mit147. Aiming at the positional cloning of the mutated gene, we constructed a BAC contig spanning about 1 Mb of the genomic region. On the basis of our mapping and sequencing analysis of the BACs com- bined with public genome data, EST database search- es, and gene prediction programs, we delimit the 1.06 cM of the t w5 critical region to 750 kb, and infer 36 genes (1/20 kb) encoded in the interval. All of the 33 genes tested were confirmed as expressed in embry- onic tissues by RT-PCR analyses, and in many cases by EST expression profiles as well. Thus, this highly gene-rich region is essentially totally transcribed during early development and provides priority can- didates to be screened for the t w5 embryonic lesion. Because a number of embryonic lethal mutations map to the ‘‘t-complex’’ on Chromosome (Chr) 17, that genetic region has long intrigued developmental geneticists (Bennett 1975). Interest has been height- ened by the presence in the t-complex of the H-2 major histocompatibility complex (Artzt et al. 1988), critical for immune function. At least three of the recessive ‘‘t-haplotype’’ mutants that disrupt em- bryonic development map in or near H-2 (Shin et al. 1984). One of the embryonic lethal lesions, tcl-w5 (t-complex lethal w5, called t w5 below) is located between H-2Pb and D17Mit147 by detailed genetic mapping with microsatellite markers (Vernet et al. 1998). Embryos homozygous for t w5 arrest at gastr- ulation and exhibit defects in proliferation/differen- tiation of embryonic ectoderm (Bennett and Dunn 1958; Hogan et al. 1980). 1 The t w5 gene is thus in- ferred to be indispensable for the production of all three germ layers from pluripotent embryonic ecto- dermal stem cells. In-depth understanding of the biological role of the t-complex, and positional cloning of the t w5 gene in particular, require detailed analysis of the genomic region. Especially because there are many major histocompatibility (MHC)-associated diseases (Oz- awa et al. 1998; Ota et al. 1999), the region has been preliminarily searched for expressed genes, and a number of transcribed units have been revealed, though of largely unknown functions (Abe et al. 1988; Hanson et al. 1991; Walter and Gunther 2000). Here we extend earlier studies to a systematic anal- ysis of the candidate genomic region for t w5 . From BAC mapping and sequencing, we define the critical region in 750 kb; detail its gene content; and dem- onstrate that 33 of 36 genes, including some priority candidates for t w5 , are expressed in the early embryo. Materials and methods Construction of a BAC contig of the t w5 minimal region. Parts of the cosmid clones corresponding to the ‘extended’ H-2K region (Lai et al. 1994) were subcloned and sequenced. From the sequence data, PCR primers were designed and used for screening of a mouse BAC library (Research Genetics, Huntsville, als.). Primers designed from D17Mit16, 62, and 147 DOI: 10.1007/s00335-003-2329-1 • Volume 15, 265–276 (2004) •Ó Springer-Verlag New York, Inc. 2004 265 Correspondence to: K. Abe; E-mail: abe@rtc.riken.go.jp