Houston DW et al. (1998), A Xenopus DAZ-like gene encodes an RNA componen...

XB-ART-15376

Development 1998 Jan 01;1252:171-80. doi: 10.1242/dev.125.2.171.

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A Xenopus DAZ-like gene encodes an RNA component of germ plasm and is a functional homologue of Drosophila boule.

Houston DW , Zhang J , Maines JZ , Wasserman SA , King ML .

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We have identified a localized RNA component of Xenopus germ plasm. This RNA, Xdazl (Xenopus DAZ-like), encodes a protein homologous to human DAZ (Deleted in Azoospermia), vertebrate DAZL and Drosophila Boule proteins. Human males deficient in DAZ have few or no sperm and boule mutant flies exhibit complete azoospermia and male sterility. Xdazl RNA was detected in the mitochondrial cloud and vegetal cortex of oocytes. In early embryos, the RNA was localized exclusively in the germ plasm. Consistent with other organisms, Xdazl RNA was also expressed in the spermatogonia and spermatocytes of frog testis. Proteins in the DAZ-family contain a conserved RNP domain implying an RNA-binding function. We have shown that Xdazl can function in vitro as an RNA-binding protein. To determine if the function of Xdazl in spermatogenesis was conserved, we introduced the Xdazl cDNA into boule flies. This resulted in rescue of the boule meiotic entry phenotype, including formation of spindles, phosphorylation of histone H3 and completion of meiotic cell division. Overall, these results suggest that Xdazl may be important for primordial germ cell specification in the early embryo and may play a role analogous to Boule in promoting meiotic cell division.

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Species referenced: Xenopus
Genes referenced: azin2 dazl vim

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	Fig. 1. Analysis of the predicted Xdazl amino acid sequence. (A) Amino acid sequence of Xdazl derived from the full-length cDNA. (B) Diagram showing domains and features of the Xdazl protein. N and C indicate the amino and carboxyl termini respectively, numbers show positions of amino acids beginning with the first in frame methionine. The gray box delineates the RNP domain, while the black stripes show the positions of the RNP-2 hexamer and RNP-1 octamer amino acid motifs respectively. The striped region represents the position of the putative DAZ repeat. Expanded above is the Xdazl sequence in that region compared to a consensus DAZ repeat. Periods indicate gaps in the sequence alignment. The table below the diagram shows the homology of Xdazl to other DAZ-family proteins in percent amino acid identity and similarity. (C) Sequence alignment of the Xdazl RNP domain. Identical residues are shaded black; similar residues are shaded gray. Periods indicate gaps in the alignment. The RNP-1 and RNP-2 motifs are indicated below and above the alignment, respectively. The GenBank accession number for the sequence reported in this paper is AF017778.
	Fig. 2. Molecular analysis of Xdazl mRNA expression. (A) Northern blot of Xdazl RNA expression in adult tissues. Xdazl is shown in the top panel, below is the same membrane stripped and reprobed with EF1-a as a loading control. In the top panel, the size of the Xdazl band is shown at the left. The tissues tested are labeled above each lane. (B) RNase protection assay of Xdazl expression during embryogenesis. RNA from four embryo equivalents of the indicated stages was extracted and analyzed. Stages tested are indicated at the top of each lane. Torula (yeast) total RNA was included as a negative control. A probe for ODC2 was included as a loading control. The ratio of Xdazl:ODC2 relative band intensities as determined by Phosphorimager quantitation is shown below each lane. (C) RNase protection assay of Xdazl mRNA expression in juvenile testis (J. Testis) and ovary (J. Ovary). Positions of the Xdazl and ODC2 protected fragments are indicated by arrows. Included here are examples of undigested probes (Probes) and two equivalents of positive control stage VI oocytes. Arrows indicate the positions of the protected fragments.
	Fig. 3. Localization of Xdazl mRNA during oogenesis. Xdazl DIG-labeled antisense RNA probes were hybridized to previously sectioned material of postmetamorphic froglets (A), juvenile ovary (B), adult ovary (D,E), or defolliculated stage VI oocytes (F). (A) Postmetamorphic froglet ovary. Arrows indicate oocytes stained for Xdazl mRNA, arrowheads show adjacent oocytes lacking in Xdazl signal. (B) Stage I oocyte from a juvenile ovary. Arrow shows Xdazl localization in the mitochondrial cloud. (C) Stage I oocytes from the same tissue sample as (B) hybridized with mtLrRNA to show presence of mitochondrial clouds. (D) Stage I oocyte from adult probed for Xdazl. (E) Xdazl probed stage II oocyte from adult showing Xdazl localization in the fragmenting mitochondrial cloud (arrow) (F) Xdazl probed adult stage VI oocyte. Arrows show islands of Xdazl staining at the vegetal pole (vg) of the oocyte. Scale bars, 200 mm (A-E), 62.5 mm (F). mc, mitochondrial cloud; n, nucleus.
	Fig. 4. Expression of Xdazl mRNA during early embryogenesis analyzed by whole-mount in situ hybridization. (A) Vegetal view of albino embryos at the 2-cell stage hybridized with Xdazl probe, showing staining in discrete patches. (B) Vegetal view of 4-cell embryos. (C) Vegetal view of stage 6 albino embryos. Xdazl is localized in a few cells at the extreme vegetal pole.
	Fig. 5. Expression of Xdazl mRNA during embryogenesis analyzed by in situ hybridization to sectioned material. (A) 8-cell embryo hybridized with Xdazl sense probes. The region of the germ plasm (gp) is shown by the arrow. The vegetal pole (vg) of the embryo is to the lower left. (B) Another section from the same embryo as (A) probed with antisense Xdazl probes. The yolk-free region of the germ plasm is stained. The vegetal pole (vg) is to the lower left. (C) Section from a stage 7 embryo hybridized with Xdazl antisense probes. Staining is seen in streaks near the vegetal pole. (D) Higher magnification of (C). (E) Control stage 7 section immunostained with anti-vimentin antibody Z10. The arrow indicates the vimentin-stained germ plasm. (F) Low power view of a section from a stage 10 embryo hybridized with Xdazl antisense probes. Staining reveals a number of pPGCs near the archenteron (a), indicated individually by arrowheads. (G) The upper panel shows a higher power view of one of the pPGCs in (F), showing perinuclear Xdazl staining (arrow). The lower panel is a section from a different embryo showing a similar staining pattern (arrow). (H) Control stage 10 section immunostained with Z10. The germ plasm is indicated (arrow) and surrounds a nucleus along the floor of the archenteron. Scale bars, 250 mm (C, F), 160 mm (D), 125 mm (E), 62.5 mm (A,B,G,H). n, nucleus.
	Fig. 6. Xdazl expression in the testis. (A) Section of an adult testis stained with antisense Xdazl probes. Spermatogonia (sg) and spermatocytes (sc) are shown by the arrows. (B) Section from the same testis stained with hematoxylin and eosin. Locations of the sperm (sp), spermatids (st) and spermatocytes (sc) are shown (arrows). This staining also revealed meiotic cell division in one of the spermatocyte cysts (arrowhead). This was confirmed at higher magnification (not shown). Scale bar, 160 mm.
	Fig. 7. Xdazl protein can bind RNA. In vitro translated hnRNPC1, a previously characterized RNA-binding protein, (top panel) or Xdazl (lower panel) were incubated with various homopolymeric RNA or control beads (ssDNA, Sepharose 4B). After washing, eluted proteins were separated by SDS-PAGE and visualized by Phosphorimager exposure. Mr is shown at the left. (T), aliquot of the initial translation corresponding to 20% of the input per binding reaction. A lower Mr Xdazl translation product was consistently generated when uncapped RNAs were used. This product was most likely the result of translation initiation at an alternate methionine and bound homopolymeric RNAs with the same specificity as the fulllength protein.
	Fig. 8. Xdazl expression rescues meiotic entry in boule mutant testes. Photographs of squashed, fixed testis contents from newly eclosed boule homozygotes carrying the Xdazl transgene (A-C) or boule alone (D,E). (A) Metaphase of meiosis I. Appearance of the meiotic spindle (green) and phospho histone H3-positive chromosomes (orange) indicates that meiotic entry and the metaphase transition have occurred. (B) Prophase of meiosis I. Assembly of a bipolar spindle (green) and the appearance of condensed DNA labeled with Hoechst (blue) indicates that meiotic entry is beginning. (C) Cytokinesis of meiosis I. Meiotic spindle constriction is evident with daughter nuclei (DNA, blue) segregating to two new cells. (D) Late primary spermatocyte in boule homozygotes. Cytoplasmic array of microtubules (green) is evident while no bipolar spindles are detected. (E) Late primary spermatocyte in boule homozygote. Phospho histone staining coincident with the chromosomes is not evident. Rare phosphohistone-positive chromosomes have been observed in boule mutant spermatocytes, but bipolar spindle formation and cytokinesis has not been observed in boule mutants lacking the Xdazl transgene.