Development 127, 447-456 (2000) 447 Printed in Great Britain © The Company of Biologists Limited 2000 DEV3100

A critical role for Xdazl, a germ plasm-localized RNA, in the differentiation of primordial germ cells in Xenopus

Douglas W. Houston and Mary Lou King* University of Miami School of Medicine, Department of Cell Biology and Anatomy (R-124), 1011 NW 15th Street, Room 528, Miami, FL 33101, USA *Author for correspondence (e-mail: [email protected])

Accepted 9 November 1999; published on WWW 12 January 2000

SUMMARY

Xdazl is an RNA component of Xenopus germ plasm (PGCs). In the absence of Xdazl, PGCs do not successfully and encodes an RNA-binding protein that can act as a migrate from the ventral to the dorsal and do functional homologue of Drosophila boule. boule is not reach the dorsal mesentery. Germ plasm aggregation required for entry into meiotic cell division during fly and intracellular movements are normal indicating that the . Both Xdazl and boule are related to the defect occurs after PGC formation. We propose that Xdazl human DAZ and DAZL, and murine Dazl genes, which are is required for early PGC differentiation and is indirectly also involved in differentiation. As suggested from necessary for the migration of PGCs through the its germ plasm localization, we show here that Xdazl is endoderm. As an RNA-binding protein, Xdazl may critically involved in PGC development in Xenopus. Xdazl regulate translation or expression of factors that mediate protein is expressed in the cytoplasm, specifically in the migration of PGCs. germ plasm, from blastula to early tailbud stages. Specific depletion of maternal Xdazl RNA results in tadpoles Key words: Xenopus, Germ plasm, Localized RNA, Primordial germ lacking, or severely deficient in, primordial germ cells cell, RNA-binding protein

INTRODUCTION equally among daughter cells (Whitington and Dixon, 1975). During subsequent embryogenesis, PGCs remain in the In most sexually reproducing organisms, the are endoderm and are thought to undergo 2-3 cell divisions. derived from a precursor stem cell population, the primordial Around stage 32/33 (late tailbud stage), the PGCs begin to germ cells (PGCs). These cells originate extragonadally early migrate dorsally through the lateral endoderm. By early in development and migrate through somatic tissues to reach tadpole (stage 40), the PGCs accumulate in the dorsal crest of the developing gonads. Once ensheathed by somatic gonad the posterior endoderm and are subsequently incorporated into cells, PGCs divide and differentiate into definitive gametes. the lateral plate that forms the dorsal mesentery. In Discovering how the PGCs are initially specified and develop later stages, PGC migration continues to the dorsal body wall separate from the somatic lineage is a fundamental problem and then laterally to the forming genital ridges (Wylie and in developmental biology. In many organisms, germ plasm Heasman, 1976). material, present in the egg, can be found in PGCs and germ Exactly how the germ plasm functions in PGC specification cells throughout the life of the organism and is thought to act remains largely unknown. Removal of vegetal pole cytoplasm as a determinant of fate. The asymmetric inheritance (Buehr and Blackler, 1970) or low dose ultraviolet (u.v.) of maternal factors is a common strategy used by diverse irradiation of the vegetal pole (Züst and Dixon, 1975; Holwill organisms such as nematodes, insects, crustaceans and Anuran et al., 1987) cause sterility or delayed migration of PGCs. amphibians to specify PGCs (Nieuwkoop and Sutasurya, 1979, Importantly, introduction of vegetal pole cytoplasm into 1981). Germ plasm is morphologically similar in all organisms irradiated eggs can restore fertility (Smith, 1966; Wakahara, where it is found and is typically composed of a fibrillar 1977). This observation led Smith (1966) to conclude that ‘germinal cytoplasm,’ electron-dense germinal granules, indeed germ plasm contained germ cell determinants. Other mitochondria and ribosomes (Czolowska, 1972). evidence, however, suggests that Xenopus germ plasm does not In Xenopus, the germ plasm is present in eggs as numerous act as a ‘true determinant’ as is thought for Drosophila pole discrete islands at the vegetal pole. These islands aggregate plasm. Mis-localization of pole plasm, either through after fertilization, a process that requires a kinesin-like protein, cytoplasmic transfer (Illmensee and Maholwald, 1974) or mis- Xklp-1 (Robb et al., 1996). Germ plasm is segregated expression of oskar RNA, a key pole plasm component, at the unequally during stages until , at which anterior pole (Ephrussi and Lehmann, 1992) can cause the time the germ plasm becomes perinuclear and is divided formation of ectopic, functional pole cells. Similar experiments 448 D. W. Houston and M. L. King have not been successful in Xenopus, although transferred is not required for spermatogenesis (Karashima et al., 2000). vegetal pole cytoplasm can cause the formation of The Drosophila boule gene, on the contrary, is required for supernumerary PGCs when injected into the vegetal pole entry into during spermatogenesis and male fertility, (Wakahara, 1978), the location of endogenous germ plasm. but is not expressed in females (Eberhart et al., 1996). Male Furthermore, ectopic Drosophila pole cells do not differentiate and female mice deficient in Dazl are sterile and exhibit into other cell types (Underwood et al., 1980) while, in defects in germ cell development and survival (Ruggiu et al., Xenopus, transplanted germ plasm-containing and 1997). The phenotypes of human males with deletions of the migrating PGCs can contribute to tissues derived from all three DAZ gene cluster on the Y chromosome range from an germ layers (Wylie et al., 1985). Thus, Drosophila pole plasm absence of germ cells ( only syndrome) to severe appears to function as an instructive determinant while Anuran oligospermia (Reijo et al., 1995). Humans also have an germ plasm may act only in a permissive role or require the autosomal homologue, DAZL (Saxena et al., 1996; Shan et correct endodermal environment. One explanation for the al., 1996; Yen et al., 1996), which may account for the apparent differences in function of germ plasm, despite the variability in DAZ phenotypes. A role for DAZL in high degree of ultrastructural similarity, may be differences in gametogenesis has not been demonstrated. Evidence suggests the specific molecular components of germ plasm, such as that the DAZ proteins are functionally homologous even RNA or proteins, between the two organisms. though the exact roles may differ. Expression of Xdazl in Germ plasm components have been studied primarily in C. boule flies results in rescue of the meiotic entry phenotype elegans, Drosophila and Xenopus. Proteins localized to the (Houston et al., 1998). Additionally, the mouse Dazl germ plasm of C. elegans, in the P-granules, appear to repress phenotype is partially rescued by expression of a human DAZ transcription and prevent somatic differentiation (reviewed in transgene (Slee et al., 1999). These observations suggest that Seydoux and Strome, 1999). Transcriptional repression also the DAZ genes play highly adaptable roles in many contexts occurs in Drosophila pole cells (Seydoux and Dunn, 1997; of germ cell development. Van Doren et al., 1998), although the factors responsible for In this work, we asked whether maternal Xdazl RNA is this have not yet been identified. Drosophila mutants have necessary for PGC formation in Xenopus. We show that Xdazl proved useful in identifying genes involved in the assembly of protein is expressed in the germ plasm from the blastula stage pole plasm components and genes required in somatic cells to early tailbud stages. In embryos derived from for pole cell migration. Of components localized to the pole depleted of Xdazl RNA, we find a severe reduction or a plasm, only a few genes are known to be required for pole cell complete loss of PGCs by stage 42. Additionally, we found that formation or migration. nanos, a zinc-finger gene (Kobayashi Xdazl is required for migration from the ventral endoderm. et al., 1996; Forbes and Lehmann, 1998), and polar granule These findings are the first to provide evidence that a specific component (pgc), an untranslated RNA (Nakamura et al., molecular component of germ plasm, Xdazl, is required for 1996), are required in the pole cells for proper migration out PGC differentiation in Xenopus. of the developing gut and into the somatic gonad. The genes germ cell-less (gcl), which encodes a nuclear pore complex protein (Jongens et al., 1994), and mitochondrial large MATERIALS AND METHODS ribosomal RNA (mtlRNA, Kobayashi and Okada, 1989) are involved in the initial formation of pole cells by still unknown Recombinant Xdazl protein and antibodies mechanisms. The Xdazl coding region (EcoRI insert of pXDZs, Houston et al., In Xenopus, several RNAs have been found localized to the 1998) was subcloned into the NdeI site of pET-16b (Novagen), or the germ plasm (reviewed in King et al., 1999) but none of these BamHI site of pGEX-5X-1 (Pharmacia) by blunt end ligation. This have previously been tested for roles in PGC specification. produced decahistidine or GST tagged Xdazl protein, respectively. Several of these RNAs are similar to important Drosophila pole The plasmid pET-16b-XdzHT was transformed into E. coli strain plasm components; Xcat2 encodes a nanos homologue BL21(DE3); pGEX-5X-1-XdazGST was transformed into BL21. (Mosquera et al., 1993) and Xlsirts encodes an untranslated Protein expression was induced in large-scale cultures by addition of RNA (Kloc et al., 1993), although not related to pgc. In this IPTG to 1 mM for 3 hours at 37¡C. Decahistidine and GST-tagged study, we focus on Xdazl, an RNA that we previously identified Xdazl (XdzHT and XdzGST) were both recovered in the insoluble inclusion body pellet. To purify XdzHT, inclusion bodies were as a germ plasm component in Xenopus (Houston et al., 1998). solubilized in guanidine HCl and then dialyzed against buffer Xdazl is expressed in the mitochondrial cloud of stage I containing 1 M NDSB-201 (non-detergent sulfobetaine, Calbiochem) oocytes, the source of germ plasm material (Heasman et al., to maintain proteins in a soluble state. Buffer was exchanged for 1984), and remains expressed in the germ plasm until the phosphate buffer (20 mM phosphate, pH 7.4, 0.5 M NaCl, 10% neurula stage. Xdazl RNA is also abundantly expressed in germ glycerol) and XdzHT was purified by chromatography on a HisTrap cells of the testis, but not in any of the somatic tissues. Xdazl column (Pharmacia). The isolated protein was approximately 90% encodes an RNA-binding protein and is highly related to genes pure and was used to generate polyclonal antisera to Xdazl in rabbits of the Deleted in Azoospermia (DAZ, Reijo et al., 1995) family, (Zymed). which are involved in germ cell development in a variety of To purify antibodies to Xdazl, XdzGST was prepared as above organisms ranging form C. elegans to humans. except that the final purification was done in PBS over glutathione- Sepharose beads (Pharmacia). Antibodies were purified over an Xdazl Loss-of-function studies have identified roles for DAZ antigen column according to the method of Koff et al. (1992). genes in germ cell development in worms, flies, mice and Antibodies to Xdazl were eluted with 100 mM glycine, pH 2.5. The men. It is interesting to note, however, that the specific unbound antibodies were then incubated with XdzHT protein bound phenotypes differ among the organisms. In C. elegans, the to a nitrocellulose membrane to further deplete the antiserum of gene daz-1 is required for in hermaphrodites, but antibodies to Xdazl. Xdazl is required for PGC formation 449

Whole-mount immunostaining platelets are stained yellow or orange (Whitington and Dixon, 1975; Embryos were fixed in MEMFA and stored in methanol. Samples Smith and Neff, 1985). were rehydrated to PBS and some were bisected along the midline with a scalpel. The bisected and intact embryos were then incubated RT-PCR in PBT (PBS, 2 mg/ml BSA, 0.1% Triton X-100) for 15 minutes with RT-PCR analysis of embryos was carried out as previously described rocking. Non-specific binding sites were blocked in PBT containing (Zhang et al., 1998). The primers used were Xpat (Hudson and 10% goat serum for 1 hour at room temperature. Primary antibody Woodland, 1998) and EF1-α (Wilson and Melton, 1994). Xpat incubation (affinity-purified antibodies to Xdazl, 1:100 dilution) was primers were used at an annealing temperature of 58¡C for 22-25 done in blocking solution for 4 hours at room temperature followed cycles while 22 cycles were used for EF1-α. Amplified products were by an overnight wash in PBT at 4¡C. The following day, the embryos analyzed on 6% polyacrylamide/1 × TBE gels and exposed to were washed an additional 4×1 hour each with rocking. This was PhosphorImager screens (Molecular Dynamics). Quantitation was followed by a 4 hour incubation in a 1:2000 dilution of goat anti-rabbit performed with ImageQuant software (Molecular Dynamics). HRP conjugate secondary antibodies (Pierce). Embryos were washed as above. Detection was done with a DAB HRP substrate (Roche Molecular Biochemicals). RESULTS

Oligo injection and host-transfer Expression of Xdazl protein in early embryos The oligos used, XDZ2 and XDZ4, were 18mers with the Our previous work showed that the mRNA for Xdazl was following sequences; XDZ2: A*T*G*AAGCCATACCCC*T*T*G, expressed in the germ plasm from early oocytes until the XDZ4:T*A*A*AACCACACAACC*C*G*A (Genosys, * indicates a phosphorothioate bond, the remainder were phosphodiester linkages). neurula stage (Houston et al., 1998). To determine when the Manually defolliculated stage VI oocytes were injected in the vegetal protein product of this RNA was expressed, we immunostained pole with 3-5 ng of oligo and maintained at 18¡C in culture embryos with affinity-purified antibodies to recombinant Xdazl medium [OCM; 60% L-15, 0.04% BSA, 1 mM l-glutamine, 1 µg/ml protein. Cleavage stage (4-cell) embryos failed to give specific gentamicin]. After 24 hours, capped Xdazl RNA was injected into Xdazl immunoreactivity (Fig. 1A) and the first detectable some of the oocytes to rescue the depletion of endogenous Xdazl. staining was seen in blastulae (stage 7, Fig. 1F). The stain Oocytes were then stimulated to mature and subsequently fertilized was faint and restricted to the germ plasm. Stronger by the host-transfer technique as described (Heasman et al., 1991). immunoreactivity was detected in the germ plasm of gastrula × Resultant embryos were sorted and maintained in 0.1 MMR at 18¡C. (stage 10.5, Fig. 1B,G) and neurula (stage 13, Fig. 1D,J) In rescue experiments, translation of injected Xdazl was verified by embryos. Note that, in the latter two stages, the germ plasm immunoblotting of blastula and gastrula stage embryos with Xdazl antisera. has become juxtanuclear, as expected. Xdazl staining was also seen in PGCs in the endoderm of stage 18 (neural groove stage, Northern blot analysis Fig. 1E) and stage 22 (early tailbud, not shown) embryos and Prior to host-transfer, eggs were frozen in dry ice and stored at −80¡C. was undetectable in later stages. We stained both intact RNA was isolated and 4 egg equivalents were analyzed by northern embryos and embryos bisected along the midline and results blotting for Xdazl as described (Houston et al., 1998). The blot was were similar with both methods. The staining was more readily then stripped and reprobed for Xcat2 (NotI-SalI insert), another germ visible in bisected embryos and clearing was not necessary to plasm localized RNA (Mosquera et al., 1993), as a control for specific see the stain, especially in the neurula and later stages. depletion of Xdazl. To demonstrate the specificity of this staining, we Whole-mount in situ hybridization immunostained mid-gastrula stage embryos with affinity- Whole-mount in situ hybridization was performed essentially as purified antibodies to Xdazl or antiserum depleted of described in Harland (1991). Embryos were fixed in MEMFA and antibodies to Xdazl. While strong signal was seen in the germ stored in methanol. The probe for Xpat was prepared as described plasm of Xdazl-stained embryos (Fig. 1G), the germ plasm was (Hudson and Woodland, 1998). Staining was done overnight at room unstained or very faintly stained in embryos incubated with the temperature in BM Purple (Roche Molecular Biochemicals). Embryos Xdazl-depleted antiserum (Fig. 1H). Subsequent Heidenhain’s were postfixed 1 hour in MEMFA, then bleached overnight in 10% Azan staining of the sections verified that this yolk-free region H2O2/70% methanol, dehydrated in 100% methanol and cleared was indeed germ plasm (Fig. 1I). These results show that in Murray’s (2:1 benzylbenzoate:benzylalcohol). For subsequent endogenous Xdazl protein is expressed in Xenopus embryos sectioning, samples were transferred from Murray’s clear to toluene from the blastula to the early tailbud stage. This is the period and embedded in paraffin (Paraplast X-tra). of time when the population of PGCs is initially established Histology and suggests that Xdazl might function in the early For histological analysis of PGCs, tadpoles were anesthetized in 0.2 development of PGCs. mg/ml MS222 and fixed overnight in Bouin’s. Following dehydration to 100% ethanol, the heads and tails were cut away Depletion of Xdazl RNA with antisense using a scalpel. The torsos were then transferred to toluene and oligodeoxynucleotides embedded in paraffin. Sections (10 µm) were cut on a rotary To determine if Xdazl was important for PGC differentiation, microtome, adhered to SilanePrep slides (Sigma) and stained with we used injection of antisense oligodeoxynucleotides (oligos) Haematoxylin and Eosin. PGCs were counted as in Holwill et al. to deplete Xdazl mRNA from stage VI Xenopus oocytes. Two (1987). To identify germ plasm in early embryos, samples were embedded as above and sections stained with a modified oligos were found that substantially depleted Xdazl RNA Heindenhain’s Azan stain (Smith and Neff, 1985), [0.25% Aniline (XDZ2 and XDZ4). A sense oligo complementary to XDZ2 Blue, 0.5% Orange G, 2% glacial acetic acid] for 40 minutes at room did not affect Xdazl RNA levels and gave results similar to temperature. After staining with Azan, a general histological stain, uninjected controls (not shown). The XDZ2 oligo provided germ plasm and other basophilic areas stain dark blue, while yolk more consistent results and was used in all experiments unless 450 D. W. Houston and M. L. King

Fig. 1. Xdazl protein is expressed in the germ plasm of Xenopus embryos. (A) Vegetal view of albino 4 cell embryos lacking Xdazl immunoreactivity. (B) Bisected (upper) and intact (lower) albino stage 10.5 embryos stained with purified antibodies to Xdazl. Arrows indicate Xdazl-stained PGCs below the (bl, upper embryo) and in the yolk plug (lower embryo) (C) Bisected (upper) and intact (lower) albino stage 10.5 embryos stained with antiserum depleted of Xdazl antibodies. (D). Bisected (left) and intact (right) stage 13 embryos stained for Xdazl and cleared in Murray’s. Arrows indicate stained PGCs in the endoderm below the (ar). Anterior is toward the left. (E) Bisected stage 18 cleared embryo showing Xdazl-stained PGCs (arrows). Anterior is to the left. (F) Stage 7 section immunostained for Xdazl showing reactivity in the germ plasm (gp). The vegetal pole (vp) is toward the bottom. (G) Section through a stage 10.5 embryo showing perinuclear Xdazl staining (arrow), nucleus, n. (H) Control section from a stage 10.5 embryo incubated with antiserum depleted of Xdazl antibodies showing unlabelled germ plasm. (I) The same section in H stained with Heindenhain’s Azan confirming the presence of germ plasm (arrow, dark blue) around a nucleus (n, pale blue). (J) Stage 13 section with Xdazl-stained germ plasm. Scale bar, 50 µm.

Fig. 3. Gonads of Fig. 2. Depletion of Xdazl RNA Xdazl-depleted in oocytes. Northern blot of RNA tadpoles (stage 58) from two sets of oocytes probed lack definitive germ for Xdazl or Xcat2 RNA. U, cells. (A) Section of uninjected control oocytes. 5 ng an uninjected control and 3 ng, dose of antisense oligo ovary stained with XDZ2 injected. haematoxylin and eosin. Note the characteristic ovarian cavity (oc) and oocyte in meiosis (arrow). (B) Section through the otherwise stated. Northern blot analysis of uninjected and undifferentiated gonad of an Xdazl-depleted tadpole. Germ cells are oligo-injected oocytes showed that Xdazl RNA was specifically not detected and the tissue lacks cavity or tubule formation, µ eliminated while Xcat2 RNA, which co-localizes with Xdazl in indicative of either ovary or testis differentiation. Scale bar, 35 m. the germ plasm (Mosquera et al., 1993; Forristall et al., 1995), was unaffected (Fig. 2). mesentery and total numbers per embryo were counted. The number of PGCs in control embryos was variable (means from Tadpoles depleted of maternal Xdazl RNA are 12-16.6, n=35; Table 1), but similar to previously reported deficient in PGCs numbers (Holwill et al., 1987). In contrast, PGC numbers in In Xenopus, PGCs are readily identifiable in the dorsal Xdazl-depleted embryos were greatly reduced (means from mesentery of swimming tadpole stage embryos (stage 43/44). 2.7-5.7, n=52; Table 1). These observations were consistent in Our initial studies focused on whether depletion of maternal at least four independent experiments using different donor and Xdazl could cause a deficiency in histologically identifiable host females and two different oligos. Note that, in many cases, PGCs in tadpoles. To this end, we fertilized uninjected control PGCs were completely eliminated (3/26 for XDZ4 and 10/26 and Xdazl-depleted oocytes by the host-transfer technique for XDZ2). (Heasman et al., 1991) and raised the embryos to the tadpole It was reported by Züst and Dixon (1977) that PGCs could stage (stage 43/44). In a typical experiment, 20-40% of populate the gonads of u.v.-irradiated embryos at stage 48, transferred eggs developed to the desired stage. Overall growth even though PGCs were absent from the dorsal mesentery of and were similar in control and Xdazl-depleted earlier stages. To determine if this was also the case for Xdazl- embryos, suggesting that oligo injection did not affect these depleted embryos, we examined the gonads of control and processes. Importantly, we saw normal gut coiling in over 80% Xdazl-depleted tadpoles at stage 58 (n=3 for each). In the of depleted embryos. Because abnormal gut coiling could controls, well-differentiated gonads had formed (ovary in all affect PGC migration, we excluded these from analysis. To three cases) with clearly developed early stage oocytes (Fig. analyze the numbers of PGCs in control and Xdazl-depleted 3A). In 2/3 Xdazl-depleted tadpoles, the gonads were devoid tadpoles, we fixed the embryos and stained serial sections with of germ cells and contained only masses of somatic gonadal haematoxylin and eosin. PGCs were clearly visible within the cells (Fig. 3B). The third Xdazl-depleted tadpole had a Xdazl is required for PGC formation 451

Fig. 4. PGCs are reduced in number or absent in Xdazl-depleted early tadpoles (stage 39/40). (A) RT-PCR analysis of Xpat RNA levels in individual uninjected (Un), oligo injected (Oligo) or rescued (Oligo+Xdazl) stage 39 embryos. EF1-alpha was assayed as a loading control. (B-F) Whole-mount in situ hybridization for Xpat RNA in uninjected (B, D) and Xdazl-depleted (C,E,F) stage 39/40 embryos. (B) Uninjected control embryos showing numerous Xpat- labelled PGCs in the posterior dorsal endoderm (arrowheads). (C) Xdazl-depleted embryos showing examples of reduced (arrowhead) or absent Xpat-labelled PGCs. (D) Close up view of PGCs in an uninjected tadpole. (E) Close up of an oligo-injected tadpole showing only four PGCs. (F) Example of an oligo-injected tadpole with no PGCs.

aggregation to PGC migration or survival. To identify PGCs in developing embryos, we stained control, Xdazl-depleted and rescued embryos for Xpat RNA, a PGC-specific molecular marker (Hudson and Woodland, 1998), by whole-mount in situ hybridization. Xpat, a maternal RNA, is localized in the germ plasm of oocytes and embryos and persists until stage 40. We first examined stage 39/40 embryos to discover if PGCs were lost before or after incorporation into the dorsal mesentery in Xdazl-depleted embryos. Control embryos had numerous Xpat-stained PGCs located along the dorsal endoderm (Fig. 4B,D). Consistent with the results seen at stage 43, Xdazl-depleted stage 40 embryos showed a severe reduction in the number of PGCs. Most embryos had no detectable Xpat expression while some had a normally formed ovary (not shown). These results suggest that few PGCs that completed migration to the dorsal endoderm PGCs in Xdazl-depleted embryos either do not form or cannot (Fig. 4C,E,F). Overall, PGCs were reduced in 6/20 embryos migrate properly to the dorsal mesentery. (30%) and absent in 13/20 cases (65%, Table 2). RT-PCR analysis showed that Xpat RNA levels in Xdazl-depleted PGCs in Xdazl-depleted embryos are lost prior to embryos were reduced to about 40% that of controls (Fig. 4A). entering the dorsal mesentery To ensure that the reduction in Xpat abundance and PGC We next wanted to establish when PGC development was numbers were specific to depletion of Xdazl RNA, we disrupted in Xdazl-depleted embryos. Xdazl could be required performed rescue experiments by injecting in vitro synthesized for any of the steps in PGC formation from germ plasm Xdazl RNA (300 pg) into oocytes previously depleted of

Fig. 5. PGC migration from the ventral endoderm is impaired in Xdazl-depleted, late tailbud (stage 35) embryos. (A-I) Whole-mount in situ hybridization for Xpat RNA. (A) uninjected control embryo. (B) Xdazl- depleted embryo. (C) Close up view of the embryo in A showing Xpat-labelled PGCs in the process of migration. Note that many are equal or dorsal to level of the gut tube (dashes). (D) Section of the embryo in C showing position of PGCs in the lateral endoderm (arrowhead). (E) Close up of the Xdazl-depleted embryo in B. Xpat-labelled cells are located exclusively below the level of the gut tube (dashes). (F) Section through the embryo in E demonstrating the ventral location of PGCs (arrowhead). (G) Uninjected embryo from another experiment showing distribution of Xpat- labelled PGCs (arrowhead). (H) Xdazl-depleted embryo in which PGCs are absent. (I) Xdazl-depleted embryo rescued with injected Xdazl RNA. Note the restoration of migration and Xpat expression (arrowhead). (J) RT- PCR analysis of Xpat expression in stage 35 embryos (3 embryos pooled for each lane). Scale bar, 150 µm. 452 D. W. Houston and M. L. King

Table 1. Injection of Xdazl oligos inhibits PGC formation Series Treatment n Number of PGCs per embryo (stage 43/44) Mean±s.d. 1 Uninjected 12 6,7,9,10,12,12,13,13,14,15,19,21 12.6±4.4 XDZ4, 3.5 ng 15 0,0,0,2,3,3,3,4,4,4,5,7,7,8,10 *4±3 2 Uninjected 14 8,8,9,10,12,12,13,13,14,14,14,15,17,19 12.7±3.2 XDZ4, 5 ng 11 2,3,3,3,4,6,8,8,8,9,9 *5.7±2.8 3 Uninjected 19 10,13,13,13,14,14,14,15,15,16,16,17,19,19,20,20,22,22,24 16.6±3.7 XDZ2, 3 ng 27 0,0,0,0,0,0,0,0,0,0,0,1,1,1,1,1,2,3,4,6,6,6,7,8,8,8,11 *2.7±3.4

PGCs located in the dorsal mesentery of control uninjected or oligo (XDZ2 or XDZ4)-injected embryos were counted in Haematoxylin and Eosin-stained sections. Numbers of PGCs in individual embryos from three different experiments are shown, as well as the means and standard deviations (s.d.). Asterisks (*) indicate significant differences (P<0.00001) between oligo-injected and control groups as determined by the Student’s t-test.

Table 2. Summary of Xpat expression in Xdazl-depleted embryos Stage 12 Stage 26 Stage 35 Stage 39/40 Treatment Xpat expression %%%% Uninjected Normal 5/5 (100) 3/3 (100) 11/12 (92) 7/7 (100) Reduced 0/5 (0) 0/3 (0) 0/12 (0) 0/7 (0) Absent 0/5 (0) 0/3 (0) 1/12 (8) 0/7 (0)

Oligo (3 ng XDZ2) Normal 8/8 (100) 4/4 (100) 1/14 (7) 1/20 (5) Reduced 0/8 (0) 0/4 (0) 5/14* (36)* 6/20 (30) Absent 0/8 (0) 0/4 (0) 8/14 (57) 13/20 (65)

Rescue (oligo +300 pg Xdazl RNA) Normal N.D. N. D. 6/11 (55) 3/12 (25) Reduced 4/11 (36) 6/12 (50) Absent 1/11 (9) 3/12 (25)

Embryos were analyzed for Xpat expression at the indicated stages by whole-mount in situ hybridization. The data are shown as the number of embryos with the indicated expression out of the total number tested. Values are represented as percentages in parentheses. Normal Xpat expression: ≥4 expressing cells/embryo at stage 12 and ≥10 expressing cells at later stages. Reduced expression in stage 26, 35 and 40 embryos: 1-9 expressing cells/embryo. Absent represents no detectable Xpat expression. Asterisks (*) indicate Xpat-expressing embryos with abnormal migration. N.D., not determined. endogenous Xdazl. Rescued stage 40 embryos showed a migration phenotype is striking in sectioned embryos (compare restoration of both PGCs (Table 2) and Xpat RNA levels (Fig. Fig. 5D with F). RT-PCR analysis confirmed that Xpat RNA 4A). was reduced in abundance (~60% control levels, Fig. 5J). In Although our results with stage 43/44 tadpoles suggested rescued stage 35 embryos, normal Xpat expression and PGC otherwise, we asked whether there were PGCs present in the migration were seen in 6/11 cases (55%) compared to 1/14 stage 40 embryos that lacked Xpat RNA. We analyzed serial (7%) in depleted embryos (Fig. 5I, Table 2). Also, the number sections of three Xdazl-depleted stage 40 embryos that lacked of embryos lacking Xpat expression declined from 8/14 (57%) Xpat RNA and failed to find cells with the morphological in oligo-injected embryos to 1/11 (9%) in rescued Xdazl- features of PGCs (not shown). This observation suggests that depleted embryos. The restoration of Xpat expression in PGC migration did not occur in the absence of Xpat RNA. rescued embryos was also confirmed by RT-PCR (Fig. 5J). Additionally, the lack of Xpat-containing cells remaining in the These findings show that, in Xdazl-depleted embryos, PGCs gut indicates that PGC migration was not merely delayed and are lost at the late tailbud stage, at or near the time when PGCs that residual PGCs do not persist in the endoderm of Xdazl- normally begin their dorsal migration. Migration is the first depleted embryos. overt indication of PGC differentiation. Xdazl-depleted PGCs fail to migrate from the ventral Germ plasm aggregation and early PGC formation endoderm of late tailbud embryos are normal in Xdazl-depleted embryos We next analyzed progressively earlier stages by staining for Embryos at stage 12 (gastrula), and 26 (tailbud) were next Xpat to determine when the defect in PGC formation in Xdazl- examined to determine if initial events in PGC specification depleted embryos became apparent. PGCs in uninjected stage had occurred properly in Xdazl-depleted embryos (Figs 6, 7; 35 embryos were typically found in the process of migration Table 2). Analysis of Xpat expression by RT-PCR in both from the ventral and lateral endoderm with some cells reaching stages confirmed that the abundance of Xpat RNA was similar the dorsal endoderm (Fig. 5A,C,G). In stage 35 Xdazl-depleted in control and depleted embryos (Fig. 6G). Azan staining (Fig. embryos, Xpat-containing cells were either lost (8/14 cases, 6A-D) or Xpat in situ hybridization (Fig. 6E,F; Table 2) of Table 2), or reduced in number compared to controls (5/14 control and Xdazl-depleted gastrulas showed a similar number embryos). Additionally, the PGCs that were detected in and distribution of PGCs located in the presumptive endoderm. depleted embryos remained located in the ventral endoderm Germ plasm aggregation occurred in both controls and Xdazl- (Fig. 5B,E). In only one Xdazl-depleted embryo (out of 14) did depleted embryos, as judged by the presence of large pools of we see PGCs reaching the dorsal endoderm. This impaired germ plasm positively stained with Azan. Importantly, the Xdazl is required for PGC formation 453 germ plasm was perinuclear at this stage, indicating that re- plasm-localized RNA, Xcat2, is under similar repression localization was not affected by depletion of Xdazl (Fig. 6C,D). (MacArthur et al., 1999). The factors that mediate this Sectioning confirmed that Xpat expression was restricted to the repression must also be present early in oogenesis and may be germ plasm (Fig. 6E,F). part of the RNA localization apparatus. Derepression of At stage 26, Xdazl-depleted embryos and uninjected controls translation of maternal germ plasm RNAs might be one of the had similar numbers of PGCs, as judged by Xpat staining initial steps in PGC specification. It will be important to (Fig. 7A,B). Additionally, these numbers were increased from establish whether this occurs cell autonomously or as a result gastrula embryos, suggesting that the germ plasm in Xdazl- of inductive interactions. depleted embryos was segregated equally into dividing In addition to demonstrating expression of Xdazl protein in daughter cells. Although the PGCs were situated in similar early PGCs, these results indicate an essential role for Xdazl in positions within the endoderm, PGCs in oligo-injected PGC development. This was shown in several ways. First, embryos seemed to cluster together more than in the control tadpoles grown from oocytes that were depleted of Xdazl embryos (Fig. 7B, arrows). Sectioning also confirmed that RNA have few PGCs or none at all. Second, gonads from PGCs were located in similar positions within the endoderm. metamorphosing tadpoles depleted of Xdazl are deficient in These results show that the reduction in PGC numbers in germ cells. Third, analysis of Xpat RNA levels in Xdazl- Xdazl-depleted tadpoles is not caused by defects in the physical depleted embryos showed that PGCs fail to migrate to the integrity or movements of the germ plasm, nor in the initial dorsal endoderm at the correct time and are subsequently lost. expansion of the PGCs. These effects were all specific to depletion of Xdazl as injection of synthetic Xdazl RNA rescued PGC development to a large extent. In our experiments, we injected Xdazl RNA 24 hours DISCUSSION after the oligos, which is sufficient time for the oligos to be degraded (Zhang et al., 1998). Thus, rescue of the phenotype Numerous genes are known to be required for PGC formation occurs by supplying Xdazl protein to embryos that lack it, in Drosophila and C. elegans (reviewed in Wylie, 1999). In rather than by competition with endogenous Xdazl for oligo contrast, until this study, no germ plasm-localized molecules binding. The rescue experiments control for both degradation have been shown to be necessary for PGC formation in of RNAs other than Xdazl as well as non-specific oligo effects. Xenopus. This is mainly because the genetic screens used to Several pieces of evidence indicate that while early PGC identify genes in flies and worms are unavailable for frogs. The formation is normal, PGC migration is severely affected. Azan results presented here show for the first time that a specific staining or Xpat in situ hybridization of gastrula embryos molecule in frog germ plasm, Xdazl, has a role in PGC showed that germ plasm aggregation and perinuclear development in Xenopus. Xdazl is an RNA-binding protein accumulation occur normally. These observations rule out a homologous to the human Deleted in Azoospermia (DAZ) and role for Xdazl in germ plasm movements, even though the related proteins in many species (Houston et al., 1998). Both protein is made prior to the perinuclear accumulation of the DNA and protein sequences are highly similar in DAZ-family germ plasm. Evidence also argues against the involvement in genes and functional conservation has been demonstrated in PGC proliferation because PGC numbers and Xpat RNA levels some cases (Houston et al., 1998; Slee et al., 1999). Despite appear normal in stage 26 embryos. Also, although Xdazl this conservation, roles of DAZ genes in germ cells appear to protein is expressed during the first wave of PGC mitoses differ significantly between organisms. This work shows that between stages 10 and 22 (Dziadek and Dixon, 1977), Xdazl Xdazl is involved in still another aspect of germ cell is not detectable during the time of the second wave of PGC development, PGC migration in Xenopus. cell division (stages 26-31). Previous work demonstrated that Xdazl RNA is expressed in The first visible effect on PGC development in Xdazl- the germ plasm until the neurula stage. The immunostaining depleted embryos is a clustering of PGCs during the early results presented here demonstrate that Xdazl protein is also tailbud stages. The significance of this aggregation is unknown. localized to the germ plasm of embryos. The protein becomes Increased self-adhesion of PGCs could be due to acquisition of detectable in blastula stages and persists until early tailbud endodermal characteristics. Alternatively, this could be the first stages (stage 22). The increase in protein accumulation occurs indication of inhibited migration. Abnormal PGC clustering is during the time when the RNA levels decrease. This is also seen in Drosophila pole cells lacking nanos activity consistent with degradation of the maternal transcript during (Forbes and Lehmann, 1998), suggesting that regulation of translation. Within the cell, Xdazl protein is cytoplasmic and cell adhesion is important for PGC development in multiple localized to the germ plasm. Other DAZ proteins are also species. cytoplasmic (Ruggiu et al., 1997; Cheng et al., 1998), arguing In normal development, PGCs begin to migrate from the against roles in nuclear RNA metabolism such as splicing. The ventral endoderm during late tailbud stages (stages 30-36). At period of time when Xdazl protein is present spans the shift in first the PGCs move laterally and then dorsally until they reach germ plasm position from plasma membrane to perinuclear and the dorsal crest of the endoderm by stage 40. In Xdazl-depleted continues until a stable PGC population is formed. The protein embryos, PGCs appear laterally, but do not migrate dorsally. then becomes undetectable during the migratory phase of PGC This was seen dramatically in sections, with PGCs in Xdazl- development. These results suggest a role for Xdazl during depleted embryos restricted below the level of the gut tube. The early PGC differentiation. lateral movement of PGCs in Xdazl-depleted embryos could be Although Xdazl RNA is present from early oogenesis, the the result of diminished migratory activity or may be the result protein is not detected until the blastula stage. This implies that of passive displacement due to normal morphogenetic Xdazl RNA is under translational repression. Another germ movements of the gut endoderm. In subsequent stages, the 454 D. W. Houston and M. L. King

Fig. 7. Clustering of PGCs in Xdazl-depleted tailbud embryos (stage 26). Whole-mount in situ hybridization for Xpat in uninjected (A,C) and Xdazl-depleted embryos (B,D). (A) Uninjected embryos showing spaced distribution of Xpat-labelled PGCs (arrowheads) in the posterior endoderm. (B) Xdazl-depleted embryos, arrows indicate groups of tightly clustered PGCs. (C) Section of an uninjected embryo showing position of a PGC in the endoderm. (D) Section of a Xdazl-depleted embryo. nt, neural tube, g, gut tube. Scale bar, 150 µm.

that germ plasm-containing blastomeres and migrating PGCs could both differentiate into types when placed ectopically. This demonstrates that transdifferentiation of PGCs is possible in Xenopus, contrary to the situation in Fig. 6. Germ plasm aggregation and perinuclear positioning occurs Drosophila (Underwood et al., 1980). Also, initial stages in normally in Xdazl-depleted embryos. Uninjected (A,C,E) or Xdazl- PGC formation are normal in Xdazl-depleted embryos and depleted embryos (B,D,F) were fixed at stage 10, sectioned and Xpat RNA persists until the migration phenotype is stained with Heindenhain’s Azan (A-D), or were fixed at stage 12 for pronounced. This also differs from the case in Drosophila. In Xpat whole-mount in situ hybridization and subsequently sectioned pole cells lacking nanos and pgc, ectopic pole cells continue (E,F). (A) Section of an uninjected stage 10 embryo showing PGCs (arrowhead) in the vegetal endoderm. (B) Section of an Xdazl- to express PGC-specific markers such as Vasa (Forbes and depleted stage 10 embryo with a PGC in the vegetal endoderm Lehmann, 1998; Nakamura et al., 1996). Xdazl-depleted (arrowhead). (C) High magnification view of the germ plasm (gp) embryos do not exhibit ectopic PGCs and PGC-specific from one of the PGCs in A. (D) High magnification view of the PGC markers are lost. Although more experiments are needed to in (B) demonstrating perinuclear germ plasm accumulation in Xdazl- determine if Xdazl-depleted PGCs are indeed differentiating as depleted embryos. (E) Xpat staining of germ plasm in an uninjected other cell types, the above evidence taken together suggests stage 12 embryo. (F) Xpat staining in the germ plasm of an Xdazl- that the PGCs lose their pluripotency and differentiate depleted stage 12 embryo showing two adjacent PGCs. (G) RT-PCR according to the surrounding environment. Also, because analysis of Xpat expression in individual stage 12 and 26 embryos pluripotency seems to be linked with Xpat expression, it will showing equal RNA abundance in uninjected and oligo-injected be interesting to determine the role of this novel molecule in samples. Scale bars, 625 µm (A,B), 80 µm (C,D), 50 µm (E,F). PGC development. One plausible model for the effects seen in Xdazl-depleted majority of PGCs are lost and cannot be identified either embryos, is that Xdazl is required for the establishment of the histologically or by Xpat in situ hybridization. The few PGCs migrational competence of PGCs during the formation of the that do make it past this point migrate normally into the dorsal initial PGC population. It is unlikely that Xdazl is directly mesentery. The variation seen in the number of PGCs in Xdazl- involved in migration in later stages (stages 30-40) because the depleted embryos probably results from incomplete destruction protein is only detected earlier. As migration is the first aspect of maternal Xdazl. of differentiation displayed by PGCs, and as Xdazl is unlikely The loss of PGCs could be due either to death or to affect migration directly, it is more correct to state that Xdazl differentiation into other cell types. We did not see any is necessary for PGC differentiation. Xdazl encodes an RNA- evidence of apoptotic nuclei in DAPI-stained sections or in binding protein (Houston et al., 1998) and the protein is whole-mount TUNEL analysis (not shown) of Xdazl-depleted cytoplasmic, thus possible roles for Xdazl include regulation stage 35 embryos. These observations do not prove that the of translation, RNA storage, RNA transport or regulation of PGCs are not dying, however other evidence suggests that RNA stability. In establishing migrational competence, Xdazl differentiation into other cell types is more likely. First, Wylie could regulate the translation or expression of specific cell et al. (1985) demonstrated in PGC transplantation experiments surface or cytoskeletal proteins needed to respond to Xdazl is required for PGC formation 455 migrational cues. Alternatively, Xdazl could be involved in Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by repressing expression of factors that inhibit migration. PGC oskar. Nature 358, 387-392. migration is often regulated by signals from the environment Forbes, A. and Lehmann R. (1998). Nanos and Pumilio have critical roles in the development and function of Drosophila stem cells. (reviewed in Wylie, 1999). In this model, PGCs lacking Xdazl Development 125, 679-690. are not competent either to receive or respond to such signals, Forristall, C., Pondel, M., Chen, L. and King M. L. (1995). Patterns of resulting in the PGCs remaining in the ventral endoderm. In localization and cytoskeletal association of two vegetally localized RNAs, the absence of migration, the same signals or competing ones Vg1 and Xcat-2. 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Peng), pp. 685-695. New the identification of PGC-specific markers unique to different York: Academic Press. stages of development would aid in determining the exact Holwill, S., Heasman, J., Crawley, C. R. and Wylie C. C. (1987). Axis and time that PGC development is perturbed in Xdazl-depleted germline deficiencies caused by u. v. irradiation of Xenopus oocytes cultured in vitro. Development 100, 735-743. embryos. Houston, D. W., Zhang, J., Maines, J. Z., Wasserman, S. A. and King, M. The predominant view in the literature about the function of L. (1998). A Xenopus DAZ-like gene encodes an RNA component of germ germ plasm is that it acts to ‘protect’ the pluripotent state of plasm and is a functional homologue of Drosophila boule. Development PGCs (Blackler, 1958; Dixon, 1994). The data presented here 125, 171-180. Hudson, C. and Woodland, H. R. (1998). Xpat, a gene expressed specifically do not directly support or refute the idea of germ plasm in germ plasm and primordial germ cells of Xenopus laevis. Mech. Dev. 73, protection. However, PGCs in Xdazl-depleted embryos fail to 159-168. migrate while still expressing the maternal PGC-specific Illmensee, K. and Mahowald, A. P. (1974). Transplantation of posterior polar marker Xpat. Thus Xdazl does not appear to be involved in plasm in Drosophila. Induction of germ cells at the anterior pole of the egg. specifying cells as PGCs, rather it is required for a specific Proc. Natl. Acad. Sci. USA 71, 1016-1020. Jongens, T., Ackerman, L., Swedlow, J., Jan, L. and Jan, Y. (1994). Germ aspect of PGC differentiation. It is possible that some germ cell-less encodes a cell type-specific nuclear pore associated protein and plasm components are critical for conferring pluripotency functions early in the germ–cell specification pathway of Drosophila. Genes while others are responsible for differentiation. Alternatively, Dev. 8, 2123-2136. pluripotency could be a consequence of PGC differentiation, Karashima, T., Sugimoto, A. and Yamamoto, M. (2000). Caenorhabditis not its cause. The experiments described here demonstrate the elegans homologue of the human azoospermia factor DAZ is required for oogenesis but not for spermatogenesis. Development, in press. utility of antisense oligo depletion of maternal mRNAs in King, M. L., Zhou, Y. and Bubunenko, M. (1999). Polarizing genetic studying the function of germ plasm components. By studying information in the egg: RNA localization in the frog oocyte. BioEssays 21, other maternal germ plasm components, a clearer picture of 546-557. how PGCs develop in Xenopus should emerge. Kloc, M., Spohr, G. and Etkin, L. D. (1993). Translocation of repetitive RNA sequences with the germ plasm in Xenopus oocytes. Science 262, 1712- 1714. We would like to thank J. Heasman and C. Wylie for hosting D. W. Kobayashi, S. and Okada, M. (1989). Restoration of pole-cell-forming H. to learn the host-transfer technique, and H. Woodland for providing ability to u. v.-irradiated Drosophila embryos by injection of mitochondrial the Xpat cDNA. We also thank Tom Warren (Boehringer Ingelheim lrRNA. Development 107, 733-742. Pharmaceuticals) for the suggestion to use NDSBs for solubilizing Kobayashi, S., Yamada, M., Asaoka, M. and Kitamura, T. (1996). Essential inclusion body proteins. We also acknowledge J. Zhang for the initial role of the posterior morphogen nanos for germline development in isolation of Xdazl. This work was supported by a grant from the NIH Drosophila. Nature 380, 708-711 (GM33932) to M. L. K. Koff, A., Giordano, A., Desai, D., Yamashita, K., Harper, J. W., Elledge, S., Nishimoto, T., Morgan, D. O., Franza, B. R. and Roberts, J. M. (1992). Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 257, 1689-1694. 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