DEVELOPMENTAL DYNAMICS 231:871 880, 2004

PATTERNS & PHENOTYPES

Gene Expression in the Axolotl Germ Line: Axdazl, Axvh, Axoct-4, and Axkit
Rosemary F. Bachvarova,1 Thomas Masi,2, Matthew Drum,2 Nathan Parker,3 Ken Mason,4 Roger Patient,5 and Andrew D. Johnson2,5*

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs. Developmental Dynamics 231:871 880, 2004. 2004 Wiley-Liss, Inc. Key words: axolotl; germ cells; dazl; vasa; oct-4; kit Received 14 May 2004; Revised 25 June 2004; Accepted 16 July 2004

INTRODUCTION
Among metazoans, there exist two distinct mechanisms governing the specication of primordial germ cells (PGCs) during embryonic development (Nieuwkoop and Sutasurya, 1979, 1981; Extavour and Akam, 2003; Johnson et al., 2003b). In the embryos of several common model organisms, such as those of Drosophila, Caenorhabditis elegans, Xenopus, and zebrash, germ plasm localized in the egg is segregated into primordial germ cells, and germ

plasm molecules specify PGCs by an as yet unknown mechanism (for reviews see Houston and King, 2000b; Raz, 2003). In contrast, germ plasm is not present in the eggs or embryos of mouse and urodeles, and PGC specication occurs through intercellular signaling (for a review see McLaren, 2003). Indeed, PGCs can be induced ectopically from animal caps or epiblast derived from urodele or mouse embryos, respectively, by exposure to appropriate signals (Sutasurya and Nieuwkoop,

1974; Tam and Zhou, 1996; Johnson et al., 2003a). Bone morphogenetic proteins (BMPs) are known to specify ventral cell fates in vertebrates, and an essential role for BMPs in the development of mouse PGCs has been demonstrated in BMP-4 / mice (Lawson et al., 1999; McLaren, 2003). Similar to mouse embryos, urodele PGCs develop within the presumptive posterior ventrolateral mesoderm (Nieuwkoop, 1947), a region of high BMP signaling. Based on these observations, we have proposed

Department of Cell Biology and Development, Weill Medical College of Cornell University, New York, New York Department of Biological Science, Florida State University, Tallahassee, Florida 3 Division of Integrative Biology, University of Texas, Austin, Texas 4 Department of Biology, Purdue University, Lafayette, Indiana 5 Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom Grant sponsor: The Wellcome Trust; Grant number: 064809. Dr. Masis present address is Department of Pathology, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN 37996-4542. *Correspondence to: Andrew Johnson, Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK. E-mail: andrewdjohnson@nottingham.ac.uk
2

DOI 10.1002/dvdy.20195 Published online 29 October 2004 in Wiley InterScience (www.interscience.wiley.com).

2004 Wiley-Liss, Inc.

872 BACHVAROVA ET AL.

that a common mechanism of germ cell determination has been evolutionarily conserved between urodeles and mammals and is, in fact, the basal mode among vertebrates (Johnson et al., 2003a,b). Relatively little is known about determination of PGCs in urodeles. PGCs have been identied cytologically at mid-late tail bud stages located at the dorsal edge of the lateral plate (DLP) in the posterior trunk (Humphrey, 1925; Maufroid, 1972; Ikenishi and Nieuwkoop, 1978). Fate mapping experiments indicate they are derived from the ventrolateral marginal zone of the early gastrula, adjacent to the precursors of blood (Nieuwkoop, 1947; Smith, 1964). Nevertheless, it remains unknown when embryonic cells are committed to a germ cell fate in urodeles. In earlier work, we examined expression of the dazl-like gene (Axdazl) in the axolotl (Ambystoma mexicanum) (Johnson et al., 2001). Xdazl RNA is localized to the germ plasm in Xenopus (Houston et al., 1998), but we found that, in axolotl Axdazl transcripts are not localized in oocytes or early embryos, consistent with the absence of germ plasm in urodele oocytes. By using Axdazl as a molecular marker, we denitively identied PGCs in the DLP of early larvae (stage 40). To further characterize the development of the germ line in axolotl embryos, here we report the cloning and expression throughout the germ cell cycle of three additional genes known to be involved in development of mouse germ cells as well as further studies on the expression of Axdazl. Two of the genes, dazl and vasa, encode RNA binding proteins. Vh (Vasa homolog) and Dazl-related genes are expressed specically in gonadal germ cells of essentially all animals studied (see Raz, 2003) and are required during or close to the time of meiosis in mice (Ruggiu et al., 1997; Tanaka et al., 2000; Saunders et al., 2003) as well as in invertebrates (see Raz, 2003). Of interest, in Xenopus their products are also found in germ plasm and are required during early development of PGCs (Ikenishi and Tanaka, 1997;

Houston et al., 1998; Houston and King, 2000a). A third gene, Oct-4, is of particular interest, because homologs of the mammalian genes have been difcult to identify in lower animals. Oct-4 encodes a class V POUdomain transcription factor (Phillips and Luisi, 2000) expressed in mouse embryos in the inner cell mass (ICM), epiblast and in female germ cells throughout most of their life cycle (Yeom et al., 1996; Pesce et al., 1998). It is required in the ICM of the blastocyst (Nichols et al., 1998) and in embryonic stem (ES) cells to maintain the pluripotent state (Niwa et al., 2000; Pesce and Scholer, 2001). Mammalian c-kit is expressed in a variety of tissues and is required for development of several cell types, including germ cells, hematopoietic stem cells, and melanocytes (see Morrison-Graham and Takahashi, 1993).

RESULTS Axolotl vasa and dazl Are First Expressed Specically in Germ Cells of Late Tail Bud Stage Embryos
A full-length axolotl vasa (Axvh) cDNA (accession no. AY542375) was obtained by screening a stage 18 embryo cDNA library (Busse and Seguin, 1993) using a polymerase chain reaction (PCR) fragment amplied from testes RNA by degenerate reverse transcriptase (RT) -PCR (see Experimental Procedures section). The amino acid sequence obtained shares greater similarity with the mammalian vasa homolog than with any other sequence in the database. It has an overall amino acid identity of 61% to human, 59% to mouse, 56% to Xenopus, 55% to zebrash, and 52% to chicken vasa homologs. A survey of adult tissues by RT-PCR found expression of Axvh only in ovary and testis (Fig. 1A) similar to Axdazl (Johnson et al., 2001). In developing embryos, Axvh RNA is present from cleavage to gastrula and declines gradually after stage 12, with very little present by stage 40 (Fig. 1B). This presumably reects maternal RNA, because it is wide-

spread in the dissected parts of embryos (Fig. 1EG), and no specic expression could be detected from stage 8 35 by in situ hybridization (ISH; not shown). Maternal Axdazl RNA also showed a wide distribution at these stages (Johnson et al., 2001). By stage 40, Axvh RNA is predominantly in the posterior and dorsal region (data not shown) of the embryo, and by stage 45, is localized in the posterior trunk, which includes the germ cells (Fig. 1H). By ISH, specic expression of Axvh was observed starting at a low level in PGCs in the DLP at approximately stage 38 and then at a higher level in germ cells in the forming gonads at stage 45 (Fig. 2G,H). Expression of Axdazl RNA in embryos and ovary has been described (Johnson et al., 2001). In more recent experiments, Axdazl RNA was rst detected in PGC at late tail bud (stage 33, earlier than previously reported). Labeled cells are located in the DLP in the posterior half of the trunk (Fig. 2A,B), where previous workers have placed PGCs (see above). Axscl (accession no. AF313414), a marker of hemangioblast precursors (for a review see Begley and Green, 1999) is expressed in the ventral blood island and DLP in a pattern similar to that of Xscl in Xenopus (Ciau-Uitz et al., 2000). At stage 35, Axscl RNA is declining in the DLP as Axdazl expression is increasing, and the two genes are apparently coexpressed in some cells of the posterior DLP (Fig. 2CF).

Axolotl Oct-4 Is Expressed in Presumptive Ectomesoderm in Blastulae and Gastrulae but Not in PGCs
Axoct-4 cDNA (accession no. AY542376) was obtained by screening an axolotl ovary cDNA library with a PCR fragment obtained by degenerate RT-PCR (see Experimental Procedures section). The 3111base pair sequence contains the conserved POU-specic and POU homeodomains (comprising the DNA binding domain) found in all POU proteins (see Spaniol et al., 1996; Remenyi et al., 2000; Phillips and Luisi, 2000). Based on the sequences available in the database

GENE EXPRESSION IN AXOLOTL GERM LINE 873

Fig. 1. Tissue specicity and temporal and spatial expression of Axvh and Axoct-4 RNAs in axolotls. A: Axvh RNA expression in adult tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). Equal amounts of total RNA from the indicated tissues were reversed transcribed and used as template for PCR with specic primers for each gene; the products were then detected as previously described (Johnson et al., 2001). AxEF-1 serves as a control for input RNA. K, kidney; Sk, skin; L, liver; Lu, lung; Sp, spleen; B, brain; O, ovary; T, testes; Sm, skeletal muscle; H, heart; St, stomach; RT(-), no reverse transcriptase. B: Axvh RNA expression throughout embryogenesis by RT-PCR. Total RNA was extracted from ve embryos per indicated stage, and one-half embryo equivalent was reverse transcribed. PCR reactions were performed and products detected as described (Johnson et al., 2001). AxEF-1 serves as a control and is transcribed from the mid-blastula transition (stage 10). Cl, cleavage stage embryos. C: Axoct-4 RNA expression in adult tissues by Northern blotting. Ten micrograms of total RNA from the indicated tissues was loaded per lane. 18S ribosomal bands show loading of RNA. I, intestine; other abbreviations as above. D: Axoct-4 RNA expression during embryogenesis by Northern blotting. One-half embryo equivalent of total RNA from the indicated stages was loaded per lane. Hybridization to 18S ribosomal bands shows loading of RNA. EH: Axvh and Axoct-4 RNAs in dissected embryos parts. Ten embryos from each stage were dissected into parts indicated by gray lines. Total RNA was extracted from each region, and Axvh or Axoct-4 RNA detected by RT-PCR. Regions indicated in the accompanying diagram. E: Stage 8 embryos. Zones dissected as described in Johnson et al. (2001). F: Stage 10.75 embryos. An, animal portion; DMZ, dorsal marginal zone; VMZ, ventral marginal zone; Lat, lateral; Veg, vegetal region. Arrow indicates position of dorsal lip of the blastopore. G: Stage 34 embryos. Ventral, ventral body wall; Lat, lateral body wall; Endo, endoderm. H: Stage 45 embryos. AV, anterior ventral mesoderm; AD, anterior dorsal mesoderm; Gut, gut tube; Post, posterior ventral mesoderm; SE, surface ectoderm.

(GenBank), this region is 7375% identical and 88% similar to mammalian Oct-4 and is the most closely related ortholog of mammalian Oct-4 s identied to date (Table 1).

In adult tissues Northern blotting revealed Axoct-4 RNA only in ovary (not in testis; Fig. 1C). The time course of embryonic expression was examined in total RNA by Northern blotting. Maternal Axoct-4 RNA is detected in

cleavage stage embryos. A signicant increase in the amount of Axoct-4 RNA at stage 11 indicates that embryonic synthesis has been initiated by this time (Fig. 1D). From stages 1530, it is low or absent. A similar time

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Fig. 2. Expression of Axdazl, Axscl, and Axvh in axolotl embryos visualized cross-sections of the posterior region. A: Mid-posterior trunk of a stage 33 embryo, showing Axdazl expression (arrows) in clusters of cells in the dorsal edge of the lateral plate. B: A more posterior section of the same embryo, showing Axdazl expression (arrows). C,D: Adjacent sections of the same stage 35 embryo hybridized to the Axdazl and the Axscl probes, respectively. Both genes are expressed at relatively low levels in the dorsal lateral plate (arrows). Overlays reveal that the Axscl and Axdazl stained cells overlap. Axscl is strongly expressed in the ventral blood island at the ventral tip of the embryo (arrowhead). E,F: A similar pair of sections from a different stage 35 embryo. In D and F, nuclei were stained red with safranin, and the sections were dehydrated and mounted in Permount. G: Axvh expression in a stage 38 embryo (arrows). H: Axvh expression in cells in the forming gonads of a stage 45 embryo. n, notochord. Scale bar in A 200 m in A,B, 150 m in CF, 100 m in G, 75 m in H.

Fig. 2.

course of expression was obtained by RT-PCR (data not shown). Analysis of expression in dissected embryonic parts (Fig. 1F,H) shows Axoct-4 RNA is present in all regions at stage 10.75, and signicantly elevated in the dorsal region at stage 15 (data not shown). By stage 45, only minor amounts remain, primarily in the head. By ISH on sections, localized Axoct-4 expression is rst seen at stage 8 in the perinuclear cytoplasm of cells in the animal half (not shown). At stages 10 11, cells of the surface layer of ectoderm and presumptive mesoderm express Axoct-4 as they extend vegetally toward the blastopore; expression decreases as the cells round the lips of the blastopore to a very low level in the ingressed dorsal mesoderm (Fig. 3A). At stage 12, this pattern continues with strong expression in the ectoderm on the dorsal side from the posterior to the anterior pole (Fig. 3CF). The later ingressing posterior mesoderm expresses Axoct-4 strongly (Fig. 3C), with expression diminishing more anteriorly (Fig. 3D,E). Some endoderm adjacent to the archenteron is also labeled (Fig. 3E). Little or no signicant expression was seen after stage 15. Importantly, no expression was seen in PGCs through early larval stages (see below for gonadal stages).

Fig. 3. Expression of Axoct-4 in gastrula stage embryos, using in situ hybridization to sections. A: Sagittal section of a stage 10.75 embryo, showing the dorsal lip (arrow). B: Diagram of orientation of sections in CF. CF: Cross-sections within the posterior half of a stage 12 embryo. Dorsal is up in all sections. C: A section at the yolk plug. DF: Successively more anterior sections. arch, archenteron. Scale bar 250 m in A (applies to all panels).

Fig. 4. Axkit expression in embryos and larvae, as shown by in situ hybridization to crosssections. A: Stage 35 embryo, showing expression in isolated cells in the skin (arrow shows one of the four). Note also expression in two cells within the notochord (n). B: High-magnication view of a labeled cell in the skin of the stage 35 embryo. At the arrow, it can be seen that the labeled cytoplasm is associated with pigment. C: Stage 44 embryo, showing scattered labeled cells in the skin, strong staining in the cells of the notochord, and scattered stained cells on the surface of the gut, presumed interstitial cells of Cajal cells (one is shown by the arrow). D: Stage 45, showing expression in scattered cells in the liver, presumed hematopoietic stem or progenitor cells. Two of the several labeled cells are indicated by arrows. n, notochord. Scale bar 100 m in A (applies to A,C,D; 20 m in B).

GENE EXPRESSION IN AXOLOTL GERM LINE 875

TABLE 1. Percentage Amino Acid Identity and Similarity of AxOct-4 and Mouse Oct-4 to Other Vertebrate Class V POU Domain Sequencesa Organism Identity Similarity

Axolotl Oct-4 compared to other vertebrates Human 75% 88% Mouse 73% 88% Zpou2 73% 86% Xoct-25 67% 86% Xoct-79 66% 83% Xoct-91 66% 83% XLPOU60 60% 80% Mouse Oct-4 compared to other vertebrates Human 92% 98% Axolotl 73% 88% Zpou2 63% 85% Xoct-91 63% 80% Xoct-79 63% 78% Xoct-25 61% 84% XLPOU60 53% 78%
a

Data were calculated by CLUSTAL analysis over the amino acids corresponding to 152 amino acids of the mouse sequence (amino acids 131282), including the POUS and POUH DNA binding domains (Spaniol et al., 1996; Remenyi et al., 2001).

Embryonic Expression of Axkit Resembles Its Expression in the Mouse but Is Not Found in PGCs
Axolotl kit cDNA (accession no. AY578915) was obtained by screening a larval library with an RT-PCR product obtained using nested degenerate PCR primers. The 950 amino acid sequence obtained showed 39% identity to mouse c-kit, 44% to a Xenopus kit-related kinase (Xkrk), and 32% to zebrash kit. A probe made from the Axkit cDNA was used to examine its expression in embryos by ISH. In axolotl as in mammals (Bernex et al., 1996), c-kit RNA is found in a variety of tissues, and only key results will be presented here. No expression was observed in cleavage, gastrula, and neurula stages. Axkit expression is found in all three of the tissues shown in classic studies to be affected by

Fig. 5. Expression of Axdazl, Axvh, Axoct-4, and Axkit in gonadal germ cells. The rst four rows show gonads of feeding larvae with oocytes at the indicated stages. The label meiosis indicates that the larger oocytes are in the leptotenepachytene stages of meiosis, as indicated by their condensed chromosomes. At diplotene, chromosomes are no longer visible. The last row shows adult ovaries. AE: Axdazl. A: Gonia. B: Larger germ cells are leptotenepachytene oocytes. C: Early diplotene oocytes are not expressing (arrows). D: A small growing oocyte with a distinct nuclear spot (arrow). E: Adult ovary, with a small growing oocyte, and labeled oogonia (arrows). FJ: Axvh. F: Gonia. G: Leptotene pachytene oocytes. Later oocytes have a distinct nuclear spot (arrows). H: Early diplotene oocytes with a large pale nucleus and a thin rim of pale cytoplasm that is distorted upon xation. Three oocytes show a prominent nuclear spot. I: Three intact oocytes with a high expression in the cytoplasm and in a prominent nuclear spot; in two the spots appears partially double (arrows). J: A small growing oocyte from adult ovary with two adjacent spots (arrow) in the nucleus. K: Axoct-4 probe, no expression in gonia. L: Gonad xed in Bouin's solution and stained with hematoxylin and eosin (H&E) with leptotenepachytene oocytes in the cortical region. Two pachytene oocytes are indicated by arrows. MO: Axoct-4. M: Larger oocytes in early diplotene show expression in the cytoplasm. N: Slightly larger oocytes. O: A large vitellogenic oocyte of 800 m from an adult ovary. c, perinuclear cytoplasm, showing a moderate level of transcripts; y, outer yolky layer shows background expression. PT: Axkit. P: Gonia. Q: Little expression in leptotenepachytene oocytes. Gonia are still expressing. R: Little expression in early diplotene oocytes. Strong spots of expression represent gonia. S: Small growing oocytes. T: A large vitellogenic oocyte 880 m in diameter. Scale bars 100 m in A (applies to A,B,F,G,K,P,Q), 100 m in C (applies to CE,HJ,M,N,R,S), 25 m in L, 200 m in O (applies to O,T).

c-kit mutations: melanocytes, hematopoietic cells, and germ cells (see Morrison-Graham and Takahashi, 1993). It is expressed in scattered cells in the skin (Fig. 4A,B); these are identied as melanocytes, because they are associated with pigment and labeling is initiated at stage 35 when melanocytes are rst evident

on the surface of the larvae (Bordzilovskaya et al., 1989). Unlike mice, Axkit is not expressed in unpigmented melanoblasts migrating out of the neural crest. Individual labeled cells in the larval liver (Fig. 4D) are likely to correspond to hematopoietic stem cells and progenitor cells that express and require c-kit at

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a corresponding stage in the mouse (see Ogawa et al., 1993). Moreover, individual labeled cells in the gut wall (Fig. 4C) are likely to correspond to a fourth cell type more recently discovered to be affected by c-kit mutations, the interstitial cells of Cajal (ICC) located in the gut (Maeda et al., 1992). c-kit is expressed in gonadal germ cells as in mice (see below). However, in mouse embryos, c-kit expression appears in germ cells shortly after they are segregated in the late gastrula, continues during germ cell migration to the gonad (Manova and Bachvarova, 1991), and is required for normal survival and proliferation of PGCs (see Sette et al., 2000). Expression was not found in PGCs in tail bud embryos or early larvae; it was also absent in early gonadal germ cells (not shown). In frog, a kit-related gene, Xkrk, is expressed in adult ovary, presumably in growing oocytes, but probably not in PGCs (Baker et al., 1995), nor has it been reported that kit-related genes are expressed in melanocytes, or blood precursors (Baker et al., 1995; Kao and Bernstein, 1995; Martin and Harland, 2001). Axkit is expressed in lateral line (not shown), as is Xkrk, and in notochord (Fig. 4A,C), as is another kit-related gene of Xenopus, Xkl1 (Kao and Bernstein, 1995). The c-kit ortholog of zebrash, encoded at sparse, is expressed and has a role in developing melanocytes but apparently not in germ cells, blood cell precursors, or ICC (Parichy et al., 1999). Chicken kit is expressed in gonadal germ cells, ICC, hematopoietic cells, and melanocytes, and has a role in at least the latter two cell types (Sasaki et al., 1993; Lahav et al., 1994; Lecoin et al., 1995; Siatskas and Boyd, 2000; Reedy et al., 2003), similar to mice. Thus, the kit expression pattern in axolotl resembles that in amniotes more closely than does that of the frog or zebrash.

Expression of Axdazl, Axvh, Axoct-4, and Axkit During Oogenesis

Axdazl expression is found in PGCs before the gonad is formed (John-

son et al., 2001) and in gonia of feeding larvae (Fig. 5A). After sexual differentiation of the gonads occurs, expression continues in females in gonia and in meiotic oocytes (Fig. 5B). Meiotic cells (in leptotene, zygotene, or pachytene stages of meiosis) were identied in hematoxylin and eosin (H&E) -stained sections of the same gonads (Fig. 5L), and their nuclear diameter (20 25 m) agreed with that reported previously (Humphrey and Fankhauser, 1946). Curiously, expression was not seen at the next stage, in early diplotene oocytes (diameter, 30 75 m; Fig. 5C), although it reappeared in larger oocytes (Fig. 5D). From this point, expression was examined in adult ovaries. Cytoplasmic staining continues in growing oocytes and gradually declines during vitellogenesis as described (Johnson et al., 2001), although signicant amounts of maternal RNA remain in the egg as measured by RT-PCR (Johnson et al., 2001). Stained oogonia were observed in adult ovaries (Fig. 5E). Expression was seen in testes of all larvae examined up to 8 cm in length, but was not analyzed in detail. Axvh RNA is expressed continuously in gonadal germ cells. Expression is strong at the gonial stage (Fig. 5F), whereas at meiotic stages, leptotene to pachytene, most of the Axvh RNA is concentrated in a nuclear spot (Fig. 5G; see below). The spot is even more striking in small diplotene oocytes (Fig. 5H). Then Axvh RNA begins to accumulate in the cytoplasm of diplotene growing oocytes up to 200 m in diameter (Fig. 5I,J) and decreases in concentration thereafter (not shown, similar to that described for Axdazl; Johnson et al., 2001). As in Xenopus (Ikenishi and Tanaka, 2000), Axvh is not localized within the oocyte cytoplasm at any stage. In full-grown oocytes, the signal has decreased to background levels (not shown). In males, expression was observed in testes of larvae up to 8 cm in length (not shown). No expression of Axoct-4 was observed in PGCs, or in gonadal germ cells from the gonial stages in feeding female larvae through leptotene-pachytene meiotic oocytes (Fig. 5K and not shown). Axoct-4

mRNA rst appears in germ cells during early diplotene in growing oocytes approximately 70 m in diameter (Fig. 5M). From this point, it is highly concentrated in the cytoplasm of small growing oocytes (Fig. 5N) and is still present in the perinuclear cytoplasm during yolk deposition (Fig. 5O). It then declines but is still detectable in a large region of inner cytoplasm in full-grown oocytes (not shown). Expression was not observed in larval testes (not shown). Axkit expression rst appears in germ cells during the gonial stage when germ cells are proliferating in the gonad of feeding larvae more than a week after stage 45 (Fig. 5P). Expression then declines to undetectable levels in meiotic oocytes (Fig. 5Q) and reappears in growing diplotene oocytes (Fig. 5RT) at approximately the same point that Axoct-4 mRNA appears, a pattern similar to that in mice (Manova and Bachvarova, 1991). From the earliest diplotene stage, hybridization of the Axvh probe to one or two closely spaced distinct spots 6 12 m in diameter was observed within the nucleus (Fig. 5HJ). Most or all oocytes up to 500 m in diameter contained at least one spot; larger oocytes were not examined carefully because many sections are required to survey one oocyte. For Axdazl, such spots were also observed (Fig. 5D), but they were smaller and not consistently present. For Axkit, they were reduced even more, and few if any were detected for Axoct-4. These spots most likely represent the lampbrush loops on which the gene is transcribed. This interpretation is supported by the nding of two pairs of spots in several oocytes hybridized simultaneously to Axvh and Axdazl probes (data not shown). Thus, the paired spots represent the loops on a pair of homologs. The size of the spot would depend on the rate of transcription and processing. The dramatic Axvh spot could coincide with one of the special features of axolotl lampbrush chromosomes as previously described (Callan, 1966), such as the stiff loops or one of the pairs of uffy loops.

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TABLE 2. Summary of Axdazl, Axvh, Axoct-4, and Axkit Expression in Germ Cells Expression in blastulagastrula Axdazl Axvh Axoct-4 Widespread maternal Widespread maternal Widespread maternal; zygotic expression in ectoderm and posterior mesoderm of gastrula None PGC expressiona Yes, from stage 33 Yes, from stage 38 No Expression during oogenesis All stages except very early diplotene All stages From early diplotene

Axkit
a

No

In gonia and diplotene oocytes

PGC, primordial germ cell.

DISCUSSION
Urodeles represent an important model system, in addition to the mouse, for the study of the inductive mode of germ cell formation in vertebrates. We and others have proposed that this mode is basal among vertebrates (Extavour and Akam, 2003; Johnson et al., 2003b), and urodeles most likely reect the basal mode among tetrapods. Thus, further study of this process in urodeles may provide important insights into the basic biology of germ cell development. The gene expression patterns we have described are summarized in Table 2. The patterns found in gonadal germ cells of axolotl are similar to those in the mouse, but we nd signicant differences in pregonadal germ cells. In mice, germ cells are segregated from somatic lineages at embryonic day (E) 7.2, late gastrulation (Lawson and Hage, 1994), whereas in axolotl, germ cell segregation may occur signicantly later.

Axvh and Axdazl

As in other animals, Axvh is expressed throughout the life cycle of gonadal germ cells and Axdazl throughout almost all of this period. Mutational and expression studies in mice and other animals (Raz, 2000; Tanaka et al., 2000; Saunders et al., 2003) suggest that the principal role for Dazl and Vasa proteins is during the mitotic proliferation of gonadal germ cells and the early phases of meiosis, and that this role may be a conserved feature in all metazoans. In addition, products of these genes may play another role in early germ cells of those animals with germ plasm. For example, maternal gene products of vasa and dazl

orthologs are associated with the germ plasm in Xenopus embryos, and they play essential roles during early migration of PGCs to the genital ridge (Ikenishi and Tanaka, 1997; Houston and King, 2000a). In mice, Mvh is rst expressed or is greatly up-regulated as the germ cells enter the gonad in response to signals from gonadal cells (Toyooka et al., 2000). In axolotl, Axvh is rst expressed at a level detectable by ISH in the early larva at stage 38, but it is not clear whether this is in response to signals from gonadal cells. Germ cells may be located from an early stage adjacent to precursors of the gonad. Cytologically distinctive gonadal cells rst appear in the larva at approximately stage 43. Perhaps there is a functional maturation of the gonadal precursor cells around stage 40, which induces a response in the germ cells similar to that seen in mouse, i.e., up-regulation of vasa. In axolotl Axdazl expression is clearly detectable at late tail bud, stage 33 35, earlier than Axvh. Thus, PGCs must undergo some developmental maturation during the pregonadal phase of their development. In mice, dazl transcripts are apparently present at a higher level than Mvh transcripts in pregonadal germ cells at E10.5 (Molyneaux et al., 2004), and in fact, dazl is expressed in ES cells and then at a low level in germ cells differentiating from them (Geijsen et al., 2004). It is not clear whether dazl may be up-regulated in mouse germ cells at a point corresponding to this event in axolotl.

Axoct-4
In mammals, the POU domain transcription factor Oct-4 is considered

to be a marker of pluripotentiality. It is expressed in the ICM of blastocysts, throughout the early epiblast, and in embryonic stem cells and germ cells (Yeom et al., 1996; Pesce and Scholer, 2001). To date, it has been difcult to identify close homologs of Oct-4 in lower vertebrates. Thus far, of the several class V POU domain transcription factors identied in nonmammalian species, zebrash Pou2 (Zpou2) has the highest amino acid similarity to mammalian Oct-4 (Table 1). Moreover, it is present in the early embryo as maternal RNA, and injection of high concentrations of morpholino directed against zpou2 mRNA arrests development at the early gastrula stage (Burgess et al., 2002). For these reasons, it has been proposed that Zpou2 is a true ortholog of mammalian Oct-4 (Burgess et al., 2002). Here, we report the sequence and expression prole of the axolotl Oct-4 ortholog, Axoct-4. Axoct-4 is expressed in the animal hemisphere and presumptive ectoderm and mesoderm of the blastula and gastrula in a pattern similar to that of the mouse. Signicantly, Axoct-4 is expressed in tissue known to give rise to PGCs, the posterior mesoderm. AxOct-4 is 7375% identical to mammalian Oct-4 over the 152 amino acid conserved region, including the two POU domains and the linker region. By comparison, Zpou2 has 63% identity (Table 1). We propose that axolotl and mouse Oct-4 proteins represent a subclass of class V POU factors that is characteristic of organisms whose germ cells are formed by induction, as in axolotls and mice. In this regard, we have isolated genes with signicant

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sequence identity to mammalian Oct-4 from primitive sh (sturgeon and lungsh) as well as turtles (Masi and Johnson, unpublished observations), species selected on the basis of a prediction that they use regulative germ cell specication (see Johnson et al., 2003b). Within this context, it is striking that a close homolog of Oct-4 has not been found in chick (Soodeen-Karamath and Gibbins, 2001; conrmed by search of Sanger expressed sequence tag database), an amniote with germ plasm (Tsunekawa et al., 2000). Xenopus contains several members of the class V POU domain transcription factors (Xoct-25, Xoct-79, Xoct-91, XLPOU60), showing an overall identity of between 53% and 63% to the conserved region of murine Oct-4, whose transcripts are present in oocytes or early embryos (Frank and Harland, 1992; Hinkley et al., 1992; Whiteld et al., 1993). We were unable to detect equivalent genes in axolotls. In fact, a search for maternally expressed POU proteins yielded only one sequence, Axbrn-1 (Masi and Johnson, 2001), whose later embryonic expression is restricted primarily to the central nervous system. It is interesting that, when the individual expression patterns of each of the Xenopus class V POU genes are considered together, the additive expression pattern resembles that of Axoct-4. One possibility is that the several class V POU genes in Xenopus have arisen as a result of multiple duplications of a single ancestral Oct-4 related gene. In this regard, the duplication-degeneration-complementation (DDC) model (Prince and Pickett, 2002) predicts that a complex pattern of ancestral gene functions might be subfunctionalized after duplications, followed by loss of individual tissue-specic regulatory elements present within the promoter of the ancestral gene. Such a mechanism may be responsible for the presence of multiple class V POU genes in Xenopus and their overlap with the expression pattern of Axoct-4. In addition to its role in the early embryo, the expression pattern in mice (Pesce et al., 1998) and axolotl suggests that Oct-4 may also func-

tion in female gonadal germ cells, not during meiotic progression but later in growing diplotene oocytes. Our results reveal a signicant difference in the expression pattern of oct-4 orthologs in the pregonadal PGCs of axolotl and mouse. In mouse embryos, the germ cells are segregated by late gastrulation at E7.2 (Lawson and Hage, 1994) and mouse oct-4 is expressed in PGCs from this point throughout migration to the gonad, and in early gonadal oogonia (Yeom et al., 1996; Pesce et al., 1998). In contrast, Axoct-4 expression is absent during corresponding stages in the axolotl, even when germ cells are marked by expression of Axvh and Axdazl, from approximately stage 35 onward. The enhancer that drives expression of oct-4 in the mouse epiblast (the homolog of the amphibian animal hemisphere) is distinct from that which drives expression in the inner cell mass and migrating PGCs (Yeom et al., 1996). If axolotl represents the basal condition, then expression in migratory PGCs is a new function for Oct-4, perhaps evolving as a new enhancer was added to a gene whose original role was during the segregation of embryonic and extraembryonic tissues of early embryos (Nichols et al., 1998).

Axkit
Unlike axolotl, in mice, c-kit expression appears in germ cells at approximately E7.5, shortly after they are segregated in the late gastrula, and continues during germ cell migration to the gonad (Manova and Bachvarova, 1991). This expression is required for normal survival and proliferation of PGCs (Sette et al., 2000). In axolotl, Axkit RNA does not appear until the germ cells have become gonia in the gonads of feeding larvae.

lotl, neither gene is expressed until the germ cells have resided in the gonad for at least a week. Expression of dazl and vasa homologs is up-regulated at roughly the same stage in mouse and axolotl at late tail bud before or as the gonad is formed. In axolotl, these are in fact the earliest known markers of a restricted germ cell lineage. Thus, if axolotl PGCs are segregated before the late tail bud stage, they do not express two important genes characteristic of pregonadal germ cells in mouse. Alternatively, germ cells may be segregated signicantly later in axolotl than in the mouse (any time up to late tail bud, corresponding to approximately E9 E9.5 in the mouse). The overlap of expression of Axscl and Axdazl at stages 3335 suggests that lineage restriction may not have occurred by this time, and vasa expression at stage 40 would mark the rst segregated germ cells in axolotl. It should be noted that, unlike most vertebrates, in axolotl germ cells originate at a site close to the site of origin of gonadal cells; thus a long migration to the gonad is not required. Moreover, unlike mouse PGCs, axolotl PGCs do not proliferate before they reach the gonad (Humphrey, 1925). Therefore, they may not need to activate a germ cell-specic program operative in the migrating and proliferating germ cells of mice. This view predicts that the receptor CXCR4, which is required for normal migration of germ cells in other vertebrates (see Raz, 2003), may play little role in axolotl. In conclusion, assuming that urodeles represent the basal state, then the evidence suggests that, during the evolutionary path between amphibians and mammals, germ cells became segregated earlier and instituted a distinctive pregonadal germ cell program.

Concluding Remarks
In mouse embryos, the germ cells are segregated by late gastrulation, and during the next 3 days during their residence in the gut and migration to the gonad, they express both c-kit and oct-4, expression that continues in gonadal germ cells. In axo-

EXPERIMENTAL PROCEDURES Embryos and Larvae

Embryos and larvae were obtained from the Axolotl Colony at Indiana University or from spawnings of locally maintained adults. Embryos were staged according to Bordzilovskaya et al. (1989) and xed in 4%

GENE EXPRESSION IN AXOLOTL GERM LINE 879

paraformaldehyde in phosphate buffered saline. Gonads from feeding larvae 2.5 cm to 8 cm in length were collected and xed in 4% paraformaldehyde or Bouins xative. The stage of development of the gonad did not correlate strictly with length or age, so they were staged and sexed by size, position, and cytology of the germ cells.

described (Masi et al., 2000). Ten micrograms of total RNA was used for adult tissue Northern blots while onehalf embryo equivalents of total RNA was used for developmental Northern blots.

ISH
ISH was carried out on sections of embryos and larvae, and adult ovaries using digoxigenin-labeled probes as described (Johnson et al., 2001).

Extraction of RNA and RT-PCR

RNA was extracted from adult tissues or embryos, cDNA was prepared, and semiquantitative RT-PCR was carried out as described (Johnson et al., 2001).

ACKNOWLEDGMENTS
We thank Carl Seguin for the stage 18 cDNA library. We also thank Garry Morgan for helpful discussion of lampbrush chromosomes and for providing some of the axolotl larvae.

Cloning of Axolotl cDNAs

Axvh and Axoct-4 PCR fragments were amplied from either testes or ovary cDNA, respectively, using degenerate primers to conserved motifs (LMACAQT and YLVLDEAD for Axvh; QTTICREA and VVRVWFCN for Axoct-4) and cloned as described (Johnson et al., 2001). After sequence verication, the Axvh PCR fragment was then used to screen a lambda stage 18 embryo library (Busse and Seguin, 1993) to obtain a full-length cDNA. Inserts were isolated and cloned as described (Johnson et al., 2001). The Axoct-4 PCR fragment was used to screen an ovary cDNA library. A partial cDNA was retrieved, and a 5RACE kit (Gibco/BRL) was used to obtain the entire open reading frame. Both clones were sequenced to completion by primer extension. An Axkit PCR fragment was amplied from larval RNA using nested degenerate PCR primers chosen based on alignments of c-kit from multiple species, avoiding the kinase and transmembrane domains. This approach yielded a 500-base pair fragment that was labeled and used to screen a larval lambda zap library. Two clones where obtained and produced identical restriction maps. One clone, kit 5, was selected for further sequencing.

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