Figure 1.
Nucleotide sequence of the medaka boule cDNA.
Obol and its deduced protein OBol: shown in bold are the translation start codon, stop codon and putative poly-adenylation signal. Highlighted are RRM motif (Turquoise) and DAZ motif (light grey). Underlined are primer sequences for 5′ RACE (dash) and RT-PCR (solid, fragment for RNA probe) with arrows depicting their extension directions.
Figure 2.
Identification of fish boule and its expression in medaka.
(A) Multiple sequence alignment of the RRM. Boule and Dazl proteins share 27 invariant residues (asterisks) and seven conserved positions (%). There are 20 invariant or conserved residues characteristic of Boule (&) or 21 of Dazl (#) proteins each. Ol, Oryzias latipes (medaka); Ga, Gasterosteus aculeatus (stickleback); Gg, Gallus gallus (chicken); Oa, Ornithorhynchus anatinus (platypus); Bt, Bos taurus (cattle); Mm, Mus musculus (mouse); Xl, Xenopus laevis (African clawed frog); Cp, Cynops pyrrhogaster (newt); Hs, Homo sapiens (human). (B) Phylogenetic tree of DAZ family proteins. Notably, the branching between Boule and Dazl clades coincides with the branching between the fish and tetrapod lineages, and molecular trees on the basis of either Boule or Dazl sequences are well in accordance with organism relationships, indicating that generation of boule and Dazl took place in early vertebrate evolution. Followed species are gene accession numbers. (C) The phenogenic relationship of major bony fish with other vertebrates and their divergence time: Bony fish groups in blue; Mammal groups in green and other transit groups in dark; drawings are based on references [27], [42] and data from http://en.wikipedia.org/wiki/Timeline_of_human_evolution#Primates.
Figure 3.
Comparisons of genomic organizations between fish and mammal.
A. boule. B.dazl. Exons are shown in scale. The sizes of primary transcripts are indicated. The 5′ and 3′ untranslated regions (UTR) are not known for the stickleback boule and dazl. The fish genes are generally smaller than the human genes except the medaka dazl that is the largest. Notably, both medaka boule and dazl have introns in the region coding for 3′-UTRs. There is a gap (*) in the intron between the medaka boule exons.
Figure 4.
Chromosome synteny of fish boule and dazl genes.
(A) Chromosomal synteny of boule gene. (B) Chromosomal synteny of dazl gene. Chr, chromosome; grp, linkage group; UC, ultracontig. Numericals in parentheses are chromosomal positions.
Figure 5.
Embryonic, adult and spermatogenic RNA expression.
(A-B) Adult (A) and embryonic expression (B) by RT-PCR. (C-I) Spermatogenic expression by in situ hybridization. Adult testicular cryosections were hybridized to antisense RNA probes and the signals were visualized by chemical (C-E) and fluorescent staining (F-I). Nuclei were stained with DAPI (blue). (C-E) Chemical SISH. (C) Obol probe. (D) Odazl probe. (E) Olvas probe. (F-I) Dual color FISH. (F and G) Obol and Odazl FISH. (H and I) Obol and Olvas FISH. Obol, Odazl and Olvas show distinct stage-preferential expression patterns. The Obol signal peaks in primary spermatocytes and reduces in secondary spermatocytes and spermatids. Notably, spermatogonia exhibit a low and detectable Obol signal. In contrast, the Odazl signal peaks in spermatogonia and sharply reduces in spermatocytes, whereas the Olvas signal peaks in spermatogonia and persists at reduced levels from spermatocytes to spermatids. sg, spermatogonium; sc 1 and sc2, primary and secondary spermatocyte; st, spermatid; sm, sperm.
Figure 6.
Adult ovarian cryosections were hybridized to antisense RNA probes and the signals were visualized by chemical (A-C) and fluorescent staining (D-I). Nuclei were stained with DAPI (blue). (A-C) Chemical SISH. (A) Obol probe. (B) Odazl probe. (C) Olvas probe. (D-F) Dual color FISH. (D) Obol signal (E) Odazl signal. (F) Merge of Obol and Odazl signals. The transcripts of Obol, Odazl and Olvas all are not detectable in somatic cells, and their germline expression persists throughout oogenenesis that proceeds in 10 stages (I – X). However, there are distinct differences. Notably, Obol and Odazl exhibit detectable expression in oogonia, albeit at a lower level than Olvas. (G-I) Colocalization of the Odazl RNA with mitochondrial cloud in the Balbiani's body of oocytes. (G) Odazl probe. (H) MitoTracker Red580 staining for mitochondrial cloud, the characteristic component of BB (arrows). (I) Merge of Odazl signal and MitoTracker staining. Intriguingly, Odazl concentrates in the Balbinani's body (asterisks), whereas Obol is diffuse in the ooplasm and essentially absent in Balbinani's body. Both Obol and Odazl are absent in the nuclei (nu). It is noteworthy that fluorescent dyes DAPI and propidium iodide do stain nuclei of oogonia but do not stain nuclei of oocytes in medaka, a similar situation has been described also in zebrafish [43].
Figure 7.
Obol is maternally supplied and expressed in PGCs.
(A-I) Chemical WISH, showing maternal inheritance (A and C) and PGC expression (D-I) of Obol, Odazl and Olvas. PGCs are seen in two clusters bilateral to the body axis. (J-M) Dual color fluorescent SISH of Obol and Odazl. (N-P) Dual color fluorescent SISH of Obol and Olvas. At stage 27, Obol, Odazl and Olvas RNAs colocalize in gondal PGCs in two clusters. so, somites; no, notochord. (A-C) Top view. (D-P) Lateral view. The anterior is to the left.
Figure 8.
Phylogeny and ontogeny of DAZ family genes.
Left, Phylogeny and sex-specificity. The ancient member boule exists in all metazoans, whereas dazl is in vertebrates and daz is restricted to human and certain primates. Evolutionary branching and two gene duplication events (R1 and R2) are indicated. Sex specificity of expression is indicated in different colors. Right, Ontogenic expression. Expression pattern of each member is indicated by extent of horizontal lines. Drawings are originals by author or redrawn based the references [7], [10], [12], [15], [19], [21], [23], [40], [44], [45], [46]. Genes (in italics) refer to RNA expression while Proteins represent the protein expression profiles. Major stages of germline development are diagramed as a timeline for DAZ family gene expression. Expression of dazl is detected in many or all stages of germline development in both sexes. Daz has premeiotic male expression. Meiotic expression of Boule occurs in male fly, mouse and human (most abundantly in primary spermatocytes) and mitotic and meiotic in female worm. Data obtained in this study from medaka clearly demonstrate that boule is also expressed in embryos at the earliest stage, in primordial germ cells (PGCs) and adult premeiotic and meiotic germ cells of both sexes. The expression of medaka boule is similar to that of dazl in medaka and other organisms, despite of clear differences as detailed in the text.