MOLECULAR REPRODUCTION AND DEVELOPMENT 67:15–18 (2004) Germ Cell-Less Expression in Medaka (Oryzias latipes) STEFAN SCHOLZ, 1 * H. DOMASCHKE, 1 A. KANAMORI, 2 K. OSTERMANN, 3 G. RO ¨ DEL, 3 AND H.O. GUTZEIT 1 1 Institut fu ¨r Zoologie, Technische Universita¨t Dresden, Dresden, Germany 2 Division of Biological Sciences, Nagoya University, Nagoya, Japan 3 Institut fu ¨rGenetik, Technische Universita¨t Dresden, Dresden, Germany ABSTRACT The gene germ cell-less (gcl) plays an important role in the early differentiation of germ cells in Drosophila. We isolated the gcl homolog of the model teleost medaka (Oryzias latipes) using degenerated primers and an ovary cDNA bank. The predicted amino acid sequence of medaka gcl showed 92, 68 and 31% overall identity to mouse, human and Drosophila gcl respectively. RT-PCR revealed stronger expression in the ovary and weaker expression in testis, brain, heart, liver and muscle tissue. Expression in early embryos indicates the presence of maternal mRNA. By in situ hybridisation (ISH), gcl could not be detected in embryos. In contrast to vasa, ISH re- vealed expression of gcl in the ovary but not in the testis. Mol. Reprod. Dev. 67: 15–18, 2004. ß 2004 Wiley-Liss, Inc. Key Words: gcl; germ cell differentiation; vasa; teleost; ovary; testis INTRODUCTION The segregation of germ cells from the soma occurs early during embryonic development. In many inverte- brates and vertebrates a specialised cytoplasm contain- ing specific RNAs and proteins, known as germ plasm, or pole plasm (in Drosophila), is asymmetrically localised within the egg. During the early embryonic divisions this specialised cytoplasm becomes incorporated into those cells that will later become the germ cells (pole cells; Eddy, 1975; Wylie, 1999). In Drosophila, vasa protein has been identified as a component of the germ plasm (Hay et al., 1988). Gene expression of vasa is restricted to germ cells and its detection has been used to monitor segregation and migration of germ cells during early development of various organism (Hay et al., 1988; Braat et al., 1999; Tsunekawa et al., 2000; Raz, 2002). Localisation of vasa mRNA along the cleavage plans of 2- and 4-cell stage zebrafish embryos suggests that similar to Drosophila,a specialised germ plasm specifies the germ line in both organisms (Yoon et al., 1997; Wylie, 2000). Further- more, vasa mRNA of zebrafish is embedded in a nuage resembling structure (electron-dense fibrous particles in oocytes, associated with germ plasm organelle; Knaut et al., 2000). However, this early asymmetrically localised mRNA of olvas has not been found in the medaka (Oryzias latipes; Shinomiya et al., 2000; olvas is the vasa homolog of medaka) and further information on the expression of germ cell-specific genes is necessary to unravel the mechanism of the germ line specification in teleosts. In Drosophila, another gene involved in the formation of the germ line has been identified: germ cell less (gcl) mRNA is—similar to vasa—maternally transferred and incorporated in the pole cells in the early cleavage- stage embryo. Transgenic flies with a reduced level of maternal gcl mRNA fail to form germ cells (Jongens et al., 1992). In the mouse, which lacks a germ plasm (Wylie, 2000), a homolog of gcl has been detected as well (Kimura et al., 1999; Leatherman et al., 2000). Its ubiquitous expression during embryonic development does not indicate a role in germ cell formation. However, the predominant expression of gcl in spermatocytes at the pachytene stage points to a potential role in later germ cell development. Mutant mouse lines lacking gcl expression show abnormal nuclear morphologies in different tissues and a significantly reduced fertility in male mice (Kimura et al., 2003). MATERIALS AND METHODS Fish Strain and Culture The medaka d-rR strain was kindly provided by Dr. Y. Wakamatsu (Laboratory of fresh water fish stocks, Bioscience Center, Nagoya University, Japan). Fish were kept under a constant light cycle of 16:8 hr (light:dark) at 268C(Æ18C). Fish were fed ad libitum three times daily, once with artemia and twice with commercial flake food (TetraMin; Tetra GmbH, Melle, Germany). ß 2004 WILEY-LISS, INC. Institution at which the work was performed: Institut fu ¨ r Zoologie, Technische Universita ¨ t Dresden, D-01062 Dresden, Germany. Grant sponsor: DFG (Deutsche Forschungsgemeinschaft). H. Domaschke’s present address is Kinderklinik, Medizinische Fakulta ¨t, Technische Universita ¨t Dresden, D-01307 Dresden, Germany. *Correspondence to: Stefan Scholz, Junior Research Group of Molecular Animal Cell Toxicology, UFZ Centre for Environmental Research, D-04318 Leipzig, Germany. E-mail: stefan.scholz@uoe.ufz.de Received 8 May 2003; Accepted 26 June 2003 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20012