Molecular Cloning and Genomic Organization of Mouse Homologue of Drosophila germ cell-less and Its Expression in Germ Lineage Cells 1 Tohru Kimura,* ,2 Kentaro Yomogida,† Naomi Iwai,* Yukiko Kato,* and Toru Nakano* *Department of Molecular Cell Biology, †Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita-shi, Osaka 565-0871, Japan Received July 12, 1999 Primordial germ cells (PGCs) are founder cells of all gametes. A number of genes which control PGCs de- velopment have been identified in invertebrates, whereas such genes are by and large unelucidated in mammals. Here we describe cloning, genomic struc- ture and expression of mouse homologue of germ cell- less (gcl) gene which is required for PGCs formation in Drosophila. The mouse gcl shows 34% identity com- pared with Drosophila gcl protein and contains BTB/ POZ domain. The gcl gene consists of 14 exons and spans more than 50 kb. The CpG islands are found around exon 1 of the gene. Putative promoter region contains potential binding sites for various transcrip- tion factors. Northern blot analysis showed that its mRNA is highly expressed in adult testis with lower expression in ovary, ES (embryonic stem) cells, and various other organs. In situ hybridization analysis revealed strong expression of the gcl gene in the pachytene stage spermatocytes. The expression was also observed in post-migratory PGCs, but was not apparent in migratory and pre-migratory PGCs. Fur- ther studies including gene disruption analysis would provide an important insight into mammalian germ lineage development. © 1999 Academic Press Key Words: germ lineage; development; primordial germ cell; spermatogenesis. All gametes are known to arise from primordial germ cells (PGCs) (1, 2). In mouse, PGCs are set aside from other cell lineages during gastrulation and appear as a small population of cells near the base of allantois at embryonic day 7.5 (7.5E). They become incorporated into the hindgut endoderm and migrate to the genital ridges through dorsal mesentery by 10.5E. PGCs inter- act with each other and somatic cells in genital ridges to form sex cords which are the pre-structures of sem- iniferous tubules and ovarian follicles in the male and in the female, respectively. By 12.5E, sex differences become apparent: PGCs are aggregated in a striped pattern along the sex cords in the male gonads whereas they are in a dotted pattern in the female gonads (3). The genes involved in these processes are poorly elucidated. In contrast, a large number of genes have been iden- tified as regulators of PGCs development in Drosophila melanogaster and Caenorhabditis elegans (1). In Dro- sophila, PGCs, also called as pole cells, appear at the posterior pole of blastula. During cellularization, nu- clei arriving at the posterior pole are directed to enter the germ line by the molecules stored in the posterior egg cytoplasm (the pole plasm). A large number of maternal effect genes are required for the pole cell formation. germ cell-less (gcl) is one of such genes and has several characteristics of germ cell determinants (4, 5). At first, gcl is specifically incorporated into pole cells. Second, its posterior localization requires the function of all the genes necessary for pole cell forma- tion. Most importantly, the reduced gcl expression re- sults in failure of pole cell formation and the overex- pression of gcl results in transient increase of pole cells. Thus, gcl is believed to be one of the essential components during germ cell specification pathway. In this study, we have cloned mouse homologue of gcl and determined its gene structure in order to gain more insights into the molecular basis of PGCs devel- opment in mammals. The mouse gcl was highly ex- pressed in pachytene stage spermatocytes and PGCs in genital ridges of the male and the female, which im- 1 Sequence data from this paper have been submitted in GenBank under Accession Nos. AF163665 and AF163666. 2 To whom correspondence should be addressed. Fax: +81-6-6879- 8362. E-mail: tkimura@biken.osaka-u.ac.jp. Biochemical and Biophysical Research Communications 262, 223–230 (1999) Article ID bbrc.1999.1160, available online at http://www.idealibrary.com on 223 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.