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The Human VASA Gene Is Specifically Expressed in the Germ Cell Lineage

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The Human VASA Gene Is Specifically Expressed in the Germ Cell Lineage The human VASA gene is specifically expressed in the germ cell lineage Diego H. Castrillon*†, Bradley J. Quade*, T. Y. Wang‡, Catherine Quigley*, and Christopher P. Crum* *Women’s and Perinatal Pathology Division, Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; and ‡Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan Communicated by Jean D. Wilson, University of Texas Southwestern Medical Center, Dallas, TX, June 14, 2000 (received for review May 16, 2000) To understand the origins and function of the human germ cell posterior of the oocyte (where germ cells will form) and becomes lineage and to identify germ cell-specific markers we have isolated concentrated within early germ cells in the embryo. By gastru- a human ortholog of the Drosophila gene vasa. The gene was lation, vasa protein is detectable only in germ cells, and strong mapped to human chromosome 5q (near the centromere) by cytoplasmic staining in germ cells is maintained during embry- radiation hybrid mapping. We show by Northern analysis of fetal ogenesis in both male and females (8). and adult tissues that expression of the human VASA gene is The vasa gene is conserved in invertebrate and vertebrate restricted to the ovary and testis and is undetectable in somatic species, including C. elegans, Xenopus, zebrafish, mouse, and rat tissues. We generated polyclonal antibodies that bind to the VASA (9–14). Three vasa-like genes have been identified in C. elegans protein in formalin-fixed, paraffin-embedded tissue and charac- (9) whereas in Drosophila and all vertebrate species to date only terized VASA protein expression in human germ cells at various one vasa ortholog has been identified. In all species, members of stages of development. The VASA protein is cytoplasmic and the vasa subfamily are specifically expressed in germ cells expressed in migratory primordial germ cells in the region of the throughout development, and in C. elegans and Xenopus they are gonadal ridge. VASA protein is present in fetal and adult gonadal required for germ cell specification. In zebrafish, studies of vasa germ cells in both males and females and is most abundant in have allowed tracing of the germ cell lineage earlier in devel- spermatocytes and mature oocytes. The gene we have isolated is opment than formerly possible. The RNA exhibits a striking and thus a highly specific marker of germ cells and should be useful for unexpected pattern of localization to the cleavage planes in two- studies of human germ cell determination and function. and four-cell embryos and is localized to exactly four putative PGCs at the 32-cell stage (11, 12). Thus, in zebrafish as in gametogenesis ͉ DEAD box RNA helicase Drosophila, vasa appears to be a specific marker of germ cells at their earliest stages of development, and its pattern of expression erm cells in most animal species undergo two complex and is consistent with a determinative role in germ cell formation. Gremarkably different developmental phases. In the first Mouse and rat vasa genes specifically expressed in the germ cell phase, which occurs during early embryogenesis, primordial lineage also have been identified (13, 14), and mice with a germ cells (PGCs) are formed and then actively migrate to targeted mutation of the vasa locus Mvh recently have been gonadal anlage believed to contain all of the somatic compo- described (15). nents of the mature gonad. In the second phase, the germ cells Here we describe the cloning and characterization of the sense appropriate cues from their environment and initiate one human VASA gene. The predicted protein is similar throughout of two distinct programs of controlled cell division, meiosis and its length to previously identified members of the VASA sub- differentiation-oogenesis or spermatogenesis, to form gametes. family. We show by Northern analysis and immunohistochem- The molecular basis of both gametogenesis and early germ cell istry that the gene is specifically expressed in germ cells in both development is best understood in Drosophila and Caenorhab- sexes, consistent with an evolutionarily conserved role in germ ditis elegans, where systematic genetic screens have identified cell development. VASA gene products (both mRNA and pro- many of the required genes. Studies in these organisms have tein) should serve as valuable reagents for studies of the human shown that the early determination and formation of germ cells germ cell lineage. is controlled by genes expressed by the mother during oogenesis, whereas later steps depend on genes expressed in the embryo Materials and Methods (reviewed in refs. 1 and 2). Cloning of a Human VASA cDNA. Ј The Drosophila gene vasa has a central role in several aspects The primer (oVAS2a) for 5 rapid amplification of cDNA ends was 5Ј CTC TGC ATC AAA ACC of germ cell development. It encodes a member of the DEAD Ј (Asp-Glu-Ala-Asp) box family of ATP-dependent RNA heli- ACA GAC TTG AAG G 3 (28-mer). Marathon human testis cases (3, 4), and the protein exhibits ATP-dependent RNA cDNA (CLONTECH) was used as a template per the manufac- helicase activity in vitro (5). The vasa protein appears to regulate turer’s instructions except that the high-fidelity LA Taq DNA the translation of multiple downstream mRNAs and has been polymerase (Takara Shuzo, Kyoto) was used. Parameters for o shown to interact with a Drosophila homologue of the yeast PCR were: 35 cycles of denaturation at 94 for 30 sec, annealing o o translation initiation factor 2 (6). Vasa protein is an essential at 56 for 30 sec, and extension at 72 for 4 min. Ten picomoles Ј component of germ plasm, a poorly understood complex of RNA of oVAS2a and the 5 rapid amplification of cDNA ends AP1 ␮ and proteins that is required for germ cell determination (2). A primer (CLONTECH) were used in a 50- l reaction. The single null mutation that removes the entire vasa coding region results in female sterility with severe defects in oogenesis, including abnormal germ-line differentiation and oocyte determination Abbreviation: PGC, primordial germ cell. (5). Females homozygous for partial loss-of-function alleles Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY004154). produce eggs that can be fertilized, but the resulting embryos †To whom reprint requests should be addressed at: Brigham and Women’s Hospital, lack germ cells (7). Therefore, vasa function is not only required Department of Pathology, 75 Francis Street, Amory Building LB3–117, Boston, MA 02115. during gametogenesis in the adult but is also essential for the E-mail: [email protected]. specification of the germ cell lineage during embryogenesis. The Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.160274797. MEDICAL SCIENCES vasa protein is localized during Drosophila oogenesis to the Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.160274797 PNAS ͉ August 15, 2000 ͉ vol. 97 ͉ no. 17 ͉ 9585–9590 Downloaded by guest on September 29, 2021 discrete 2.2-kb product was gel-purified, subcloned, and se- sequence, an oligonucleotide corresponding to the 3Ј untrans- quenced on both strands by primer walking. lated region was used in a 5Ј rapid amplification of cDNA ends reaction with an adult human testis cDNA source. A single 2.2-kb Radiation Hybrid Mapping of VASA. The GENEBRIDGE 4 human product corresponding to a human VASA cDNA was obtained. genome radiation hybrid mapping panel (Research Genetics, This cDNA encodes the full-length protein, based on sequence Huntsville, AL) was screened by PCR, with DNA from the alignments that show a similar N terminus to other vasa sub- parental hamster cell line 4A23 (Research Genetics) as a family members (Fig. 1). negative control and normal human genomic DNA as a positive control. PCRs (50 ␮l) contained 20 mM Tris⅐HCl (pH 8.4), 50 Comparison of the Predicted Human VASA Protein Sequence with mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 mM of the VASA Other Species. The cDNA encodes a predicted protein of 724 aa primers oVAS3 and oVAS4, and 0.02 unit͞␮l Taq DNA poly- and a molecular mass of 79 kDa. Eight conserved domains merase (Life Technologies, Gaithersburg, MD). The primer (I–VIII) directly involved in RNA helicase activity have been sequences were 5Ј-ACA GGA TGT TCC TGC ATG GT-3Ј identified in all DEAD box proteins to date (16). All eight of (oVAS3, 20-mer) and 5Ј-TGC CCT TTC TGG TAT CAA these domains, including domain V, which contains the DEAD CTG-3Ј (oVAS4, 20-mer). The PCR protocol entailed 40 cycles motif, are present in the human VASA protein (Fig. 1). The of denaturation at 94o for 30 sec, annealing at 52o for 30 sec, and 302-aa sequence encompassing this conserved core region (do- extension at 72o for 1 min. Ten microliters of each PCR was mains I–VIII including intervening sequences) shows greater electrophoresed in 2% agarose gels. A reaction was scored similarity to VASA homologues in other species (9–14) than to positive if a band at 110 bp was visualized by ethidium staining. other known human DEAD box proteins: rat (94% identity), Results were submitted to the radiation hybrid mapping server mouse (90%), zebrafish (76%), Xenopus (72%), and Drosophila at the Whitehead Institute͞Massachusetts Institute of Technol- (56%). The human DEAD box protein exhibiting greatest ogy Center for Genome Research (http:͞͞www-genome.wi. similarity in this core region was the Y-chromosome encoded mit.edu͞cgi-bin͞contig͞rhmapper.pl). DBY, which had lower similarity (70% vs. 75%) and greater overall gap size (4% vs. 2%) than the Drosophila vasa gene. The Collection of Tissue Samples, RNA Preparation, and Northern Analysis.
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