gene expression in the axolotl germ line

8/13/2019 Gene Expression in the Axolotl Germ Line

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PATTERNS & PHENOTYPES

Gene Expression in the Axolotl Germ Line: Axdazl,Axvh, Axoct-4, and AxkitRosemary 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.

AxolotlOct-4has 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-kithomolog 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 231:871880, 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 twodistinct mechanisms governing thespecification of primordial germ cells

(PGCs) during embryonic develop-ment (Nieuwkoop and Sutasurya,

1979, 1981; Extavour and Akam,2003; Johnson et al., 2003b). In the

embryos of several common modelorganisms, such as those ofDrosoph-ila, Caenorhabditis elegans, Xeno-

pus, and zebrafish, germ plasm lo-

calized in the egg is segregated intoprimordial germ cells, and germ

plasm molecules specify PGCs by an

as yet unknown mechanism (for re-views 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

specification occurs through inter-

cellular signaling (for a review see

McLaren, 2003). Indeed, PGCs can

be induced ectopically from animal

caps or epiblast derived from

urodele or mouse embryos, respec-

tively, by exposure to appropriate

signals (Sutasurya and Nieuwkoop,

1974; Tam and Zhou, 1996; Johnson

et al., 2003a). Bone morphogeneticproteins (BMPs) are known to specify

ventral cell fates in vertebrates, and

an essential role for BMPs in the de-

velopment 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 presump-

tive posterior ventrolateral meso-

derm (Nieuwkoop, 1947), a region of

high BMP signaling. Based on these

observations, we have proposed

1Department of Cell Biology and Development, Weill Medical College of Cornell University, New York, New York2Department of Biological Science, Florida State University, Tallahassee, Florida3Division of Integrative Biology, University of Texas, Austin, Texas4Department of Biology, Purdue University, Lafayette, Indiana5Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, United KingdomGrant 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: [email protected]

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

DEVELOPMENTAL DYNAMICS 231:871880, 2004

2004 Wiley-Liss, Inc.


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that a common mechanism of germ

cell determination has been evolu-

tionarily conserved between urode-

les and mammals and is, in fact, the

basal mode among vertebrates

(Johnson et al., 2003a,b).

Relatively little is known about de-

termination of PGCs in urodeles.PGCs have been identified cytolog-

ically at mid-late tail bud stages lo-

cated at the dorsal edge of the lat-

eral 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). Nev-

ertheless, it remains unknown when

embryonic cells are committed to agerm cell fate in urodeles.

In earlier work, we examined ex-

pression of the dazl-like gene (Ax-

dazl) 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, consis-

tent with the absence of germ

plasm in urodele oocytes. By using

Axdazl as a molecular marker, wedefinitively identified PGCs in the

DLP of early larvae (stage 40). To fur-

ther characterize the development

of the germ line in axolotl embryos,

here we report the cloning and ex-

pression throughout the germ cell

cycle of three additional genes

known to be involved in develop-

ment of mouse germ cells as well as

further studies on the expression of

Axdazl.

Two of the genes, dazland vasa,

encode RNA binding proteins. Vh

(Vasa homolog) and Dazl-related

genes are expressed specifically 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 inverte-

brates (see Raz, 2003). Of interest, in

Xenopus their products are also

found in germ plasm and are re-

quired during early development of

PGCs (Ikenishi and Tanaka, 1997;

Houston et al., 1998; Houston andKing, 2000a).

A third gene,Oct-4, is of particularinterest, because homologs of the

mammalian genes have been diffi-cult to identify in lower animals.

Oct-4 encodes a class V POU-

domain transcription factor (Phillipsand 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 theblastocyst (Nichols et al., 1998) and

in embryonic stem (ES) cells to main-tain the pluripotent state (Niwa et

al., 2000; Pesce and Scholer, 2001).Mammalianc-kitis expressed in a

variety of tissues and is required for

development of several cell types,including germ cells, hematopoieticstem cells, and melanocytes (see

Morrison-Graham and Takahashi,1993).

RESULTS

Axolotl vasa and dazl Are First

Expressed Specifically in Germ

Cells of Late Tail Bud Stage

Embryos

A full-length axolotl vasa (Axvh)

cDNA (accession no. AY542375) wasobtained by screening a stage 18embryo cDNA library (Busse and

Seguin, 1993) using a polymerasechain reaction (PCR) fragment am-

plified from testes RNA by degener-ate reverse transcriptase (RT) -PCR

(see Experimental Procedures sec-tion). The amino acid sequence ob-

tained shares greater similarity withthe mammalianvasahomolog than

with any other sequence in the da-tabase. It has an overall amino acid

identity of 61% to human, 59% tomouse, 56% to Xenopus, 55% to ze-

brafish, and 52% to chickenvasaho-mologs.

A survey of adult tissues by RT-PCR

found expression of Axvh only inovary and testis (Fig. 1A) similar to

Axdazl(Johnson et al., 2001). In de-veloping embryos, Axvh RNA is

present from cleavage to gastrulaand declines gradually after stage

12, with very little present by stage 40(Fig. 1B). This presumably reflects

maternal RNA, because it is wide-

spread in the dissected parts of em-bryos (Fig. 1EG), and no specific ex-

pression could be detected fromstage 8 35 by in situ hybridization

(ISH; not shown). Maternal Axdazl

RNA also showed a wide distributionat these stages (Johnson et al.,

2001). By stage 40, AxvhRNA is pre-dominantly in the posterior and dor-

sal region (data not shown) of theembryo, and by stage 45, is local-

ized in the posterior trunk, which in-cludes the germ cells (Fig. 1H). By

ISH, specific expression of Axvhwasobserved starting at a low level in

PGCs in the DLP at approximatelystage 38 and then at a higher level

in germ cells in the forming gonadsat stage 45 (Fig. 2G,H).

Expression of Axdazl RNA in em-

bryos and ovary has been described(Johnson et al., 2001). In more recentexperiments, Axdazl RNA was first

detected in PGC at late tail bud(stage 33, earlier than previously re-

ported). Labeled cells are locatedin the DLP in the posterior half of

the trunk (Fig. 2A,B), where previ-ous workers have placed PGCs

(see above). Axscl (accession no.AF313414), a marker of hemangio-

blast precursors (for a review see Be-gley and Green, 1999) is expressed

in the ventral blood island and DLP in

a pattern similar to that of Xscl inXenopus (Ciau-Uitz et al., 2000). Atstage 35, Axscl RNA is declining in

the DLP as Axdazl expression is in-

creasing, and the two genes are ap-parently 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 screen-

ing an axolotl ovary cDNA library

with a PCR fragment obtained bydegenerate RT-PCR (see Experimen-

tal Procedures section). The 3111-base pair sequence contains the

conserved POU-specific and POUhomeodomains (comprising the

DNA binding domain) found in allPOU proteins (see Spaniol et al.,

1996; Remenyi et al., 2000; Phillipsand Luisi, 2000). Based on the se-

quences available in the database

872 BACHVAROVA ET AL.


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course of expression was obtained by

RT-PCR (data not shown).Analysis of expression in dissected

embryonic parts (Fig. 1F,H) shows Ax-oct-4RNA is present in all regions at

stage 10.75, and significantly ele-vated 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 Ax-oct-4expression is first seen at stage8 in the perinuclear cytoplasm of

cells in the animal half (not shown).At stages 1011, cells of the surface

layer of ectoderm and presumptivemesoderm express Axoct-4 as they

extend vegetally toward the blas-topore; expression decreases as the

cells round the lips of the blastoporeto a very low level in the ingressed

dorsal mesoderm (Fig. 3A). At stage12, this pattern continues with strong

expression in the ectoderm on thedorsal side from the posterior to the

anterior pole (Fig. 3CF). The lateringressing posterior mesoderm ex-

presses Axoct-4 strongly (Fig. 3C),with expression diminishing more an-

teriorly (Fig. 3D,E). Some endoderm

adjacent to the archenteron is alsolabeled (Fig. 3E). Little or no signifi-

cant expression was seen afterstage 15. Importantly, no expression

was seen in PGCs through early lar-val stages (see below for gonadal

stages).

Fig. 2.

Fig. 3. Expression of Axoct-4 in gastrula stage embryos, using in situ hybridization tosections. 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 astage 12 embryo. Dorsal is up in all sections.C:A section at the yolk plug.DF:Successivelymore anterior sections. arch, archenteron. Scale bar 250 m in A (applies to all panels).

Fig. 4. Axkitexpression in embryos and larvae, as shown by in situ hybridization to cross-sections. A: Stage 35 embryo, showing expression in isolated cells in the skin (arrow shows oneof the four). Note also expression in two cells within the notochord (n). B: High-magnificationview of a labeled cell in the skin of the stage 35 embryo. At the arrow, it can be seen that thelabeled cytoplasm is associated with pigment. C: Stage 44 embryo, showing scattered la-beled cells in the skin, strong staining in the cells of the notochord, and scattered stained cellson 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 stemor 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).

Fig. 2. Expression ofAxdazl,Axscl, andAxvhin axolotl embryos visualized cross-sectionsof the posterior region. A: Mid-posteriortrunk of a stage 33 embryo, showing Axdazlexpression (arrows) in clusters of cells in thedorsal 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 em-bryo hybridized to the Axdazland the Axsclprobes, respectively. Both genes are ex-pressed at relatively low levels in the dorsallateral plate (arrows). Overlays reveal thatthe Axscland Axdazlstained cells overlap.Axscl is strongly expressed in the ventralblood island at the ventral tip of the embryo(arrowhead).E,F: A similar pair of sectionsfrom a different stage 35 embryo. In D andF, nuclei were stained red with safranin, andthe sections were dehydrated andmounted in Permount. G: Axvhexpressionin a stage 38 embryo (arrows). H: Axvhex-pression in cells in the forming gonads of a

stage 45 embryo. n, notochord. Scale barin A 200 m in A,B, 150 m in CF, 100 min G, 75 m in H.



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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 screen-

ing a larval library with an RT-PCRproduct obtained using nested de-

generate PCR primers. The 950amino acid sequence obtained

showed 39% identity to mouse c-kit,44% to a Xenopuskit-related kinase

(Xkrk), and 32% to zebrafish kit.

A probe made from the AxkitcDNA was used to examine its ex-

pression in embryos by ISH. In axolotlas in mammals (Bernex et al., 1996),

c-kitRNA is found in a variety of tis-sues, and only key results will be pre-

sented here. No expression was ob-served in cleavage, gastrula, and

neurula stages. Axkit expression isfound in all three of the tissues shown

in classic studies to be affected by

c-kitmutations: melanocytes, hema-topoietic cells, and germ cells (see

Morrison-Graham and Takahashi,1993). It is expressed in scattered

cells in the skin (Fig. 4A,B); these areidentified as melanocytes, because

they are associated with pigmentand labeling is initiated at stage 35

when melanocytes are first evident

on the surface of the larvae (Bordz-

ilovskaya et al., 1989). Unlike mice,

Axkit is not expressed in unpig-mented melanoblasts migrating out

of the neural crest. Individual la-beled cells in the larval liver (Fig. 4D)

are likely to correspond to hemato-poietic stem cells and progenitor

cells that express and requirec-kitat

Fig. 5. Expression ofAxdazl,Axvh,Axoct-4, and Axkit in gonadal germ cells. The first fourrows show gonads of feeding larvae with oocytes at the indicated stages. The labelmeiosis indicates that the larger oocytes are in the leptotenepachytene stages ofmeiosis, 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 germcells 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, witha small growing oocyte, and labeled oogonia (arrows). FJ:Axvh.F:Gonia.G:Leptotenepachytene oocytes. Later oocytes have a distinct nuclear spot (arrows).H:Early diploteneoocytes with a large pale nucleus and a thin rim of pale cytoplasm that is distorted uponfixation. Three oocytes show a prominent nuclear spot. I: Three intact oocytes with a highexpression in the cytoplasm and in a prominent nuclear spot; in two the spots appearspartially double (arrows). J: A small growing oocyte from adult ovary with two adjacentspots (arrow) in the nucleus. K: Axoct-4 probe, no expression in gonia. L: Gonad fixed inBouin's solution and stained with hematoxylin and eosin (H&E) with leptotenepachyteneoocytes 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: Slightlylarger oocytes. O: A large vitellogenic oocyte of 800 m from an adult ovary. c, perinu-clear cytoplasm, showing a moderate level of transcripts; y, outer yolky layer showsbackground expression. PT: Axkit. P: Gonia. Q: Little expression in leptotenepachyteneoocytes. Gonia are still expressing. R: Little expression in early diplotene oocytes. Strong

spots of expression represent gonia. S: Small growing oocytes. T: A large vitellogenicoocyte 880 m in diameter. Scale bars 100 m in A (applies to A,B,F,G,K,P,Q), 100 m inC (applies to CE,HJ,M,N,R,S), 25 m in L, 200 m in O (applies to O,T).

TABLE 1. Percentage Amino Acid

Identity and Similarity of AxOct-4

and Mouse Oct-4to Other

Vertebrate Class V POU Domain

Sequencesa

Organism Identity Similarity

AxolotlOct-4compared to othervertebrates

Human 75% 88%Mouse 73% 88%Zpou2 73% 86%Xoct-25 67% 86%Xoct-79 66% 83%Xoct-91 66% 83%XLPOU60 60% 80%

MouseOct-4compared to othervertebrates

Human 92% 98%

Axolotl 73% 88%Zpou2 63% 85%Xoct-91 63% 80%Xoct-79 63% 78%Xoct-25 61% 84%XLPOU60 53% 78%

aData were calculated by CLUSTALanalysis over the amino acidscorresponding to 152 amino acidsof the mouse sequence (aminoacids 131282), including the POUSand POUH DNA binding domains(Spaniol et al., 1996; Remenyi et al.,2001).

GENE EXPRESSION IN AXOLOTL GERM LINE 875


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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 correspondto a fourth cell type more recently

discovered to be affected by c-kitmutations, the interstitial cells of Ca-

jal (ICC) located in the gut (Maedaet al., 1992).

c-kit is expressed in gonadal germcells as in mice (see below). How-

ever, in mouse embryos,c-kitexpres-sion appears in germ cells shortly af-

ter they are segregated in the lategastrula, continues during germ cell

migration to the gonad (Manovaand Bachvarova, 1991), and is re-

quired for normal survival and prolif-eration of PGCs (see Sette et al.,

2000). Expression was not found in

PGCs in tail bud embryos or earlylarvae; it was also absent in earlygonadal germ cells (not shown).

In frog, a kit-related gene, Xkrk, isexpressed in adult ovary, presum-

ably in growing oocytes, but proba-bly not in PGCs (Baker et al., 1995),

nor has it been reported that kit-re-lated genes are expressed in mela-

nocytes, or blood precursors (Bakeret 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 anotherkit-related geneofXenopus,Xkl1(Kao and Bernstein,

1995).The c-kitortholog of zebrafish, en-

coded at sparse, is expressed andhas a role in developing melano-

cytes but apparently not in germcells, blood cell precursors, or ICC

(Parichy et al., 1999). Chicken kit isexpressed in gonadal germ cells,

ICC, hematopoietic cells, and mela-nocytes, and has a role in at least

the latter two cell types (Sasaki et al.,

1993; Lahav et al., 1994; Lecoin etal., 1995; Siatskas and Boyd, 2000;Reedy et al., 2003), similar to mice.

Thus, thekitexpression pattern in ax-olotl resembles that in amniotes

more closely than does that of thefrog or zebrafish.

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 sexualdifferentiation of the gonads occurs,

expression continues in females in

gonia and in meiotic oocytes (Fig.5B). Meiotic cells (in leptotene, zygo-

tene, or pachytene stages of meio-

sis) were identified in hematoxylinand eosin (H&E) -stained sectionsof 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 oo-cytes (diameter, 3075 m; Fig. 5C),

although it reappeared in larger oo-

cytes (Fig. 5D). From this point, ex-pression was examined in adult ova-

ries. Cytoplasmic staining continues

in growing oocytes and graduallydeclines during vitellogenesis as de-scribed (Johnson et al., 2001), al-

though significant amounts of ma-

ternal RNA remain in the egg asmeasured by RT-PCR (Johnson et al.,

2001). Stained oogonia were ob-

served in adult ovaries (Fig. 5E). Ex-pression was seen in testes of all lar-

vae examined up to 8 cm in length,

but was not analyzed in detail.Axvh RNA is expressed continu-

ously in gonadal germ cells. Expres-sion is strong at the gonial stage (Fig.

5F), whereas at meiotic stages, lep-totene to pachytene, most of theAxvhRNA is concentrated in a nu-

clear spot (Fig. 5G; see below). Thespot is even more striking in small dip-

lotene oocytes (Fig. 5H). Then Axvh

RNA begins to accumulate in the cy-toplasm of diplotene growing oo-

cytes up to 200 m in diameter (Fig.5I,J) and decreases in concentra-

tion thereafter (not shown, similar to

that described for Axdazl; Johnsonet al., 2001). As in Xenopus(Ikenishi

and Tanaka, 2000),Axvhis not local-ized within the oocyte cytoplasm at

any stage. In full-grown oocytes, the

signal has decreased to back-ground levels (not shown). In males,

expression was observed in testes oflarvae up to 8 cm in length (not

shown).

No expression of Axoct-4was ob-served in PGCs, or in gonadal germ

cells from the gonial stages in feed-

ing female larvae through lepto-tene-pachytene meiotic oocytes

(Fig. 5K and not shown). Axoct-4

mRNA first appears in germ cells dur-

ing early diplotene in growing oo-

cytes approximately 70 m in diam-

eter (Fig. 5M). From this point, it is

highly concentrated in the cyto-

plasm of small growing oocytes (Fig.

5N) and is still present in the perinu-

clear cytoplasm during yolk deposi-tion (Fig. 5O). It then declines but is

still detectable in a large region of

inner cytoplasm in full-grown oo-

cytes (not shown). Expression was

not observed in larval testes (not

shown).

Axkit expression first 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 unde-

tectable levels in meiotic oocytes(Fig. 5Q) and reappears in growing

diplotene oocytes (Fig. 5RT) at ap-

proximately the same point that Ax-

oct-4mRNA appears, a pattern sim-

ilar 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 612 m in diameter was ob-

served within the nucleus (Fig. 5HJ).

Most or all oocytes up to 500 m in

diameter contained at least onespot; larger oocytes were not exam-

ined carefully because many sec-

tions are required to survey one oo-

cyte. For Axdazl, such spots were

also observed (Fig. 5D), but they

were smaller and not consistently

present. For Axkit, they were re-

duced even more, and few if any

were detected for Axoct-4. These

spots most likely represent the lamp-

brush loops on which the gene is

transcribed. This interpretation is sup-

ported by the finding 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 Axvhspot 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 fluffy loops.



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DISCUSSION

Urodeles represent an importantmodel system, in addition to the

mouse, for the study of the inductivemode of germ cell formation in ver-

tebrates. We and others have pro-

posed that this mode is basalamong vertebrates (Extavour andAkam, 2003; Johnson et al., 2003b),

and urodeles most likely reflect thebasal mode among tetrapods. Thus,

further study of this process in urode-les may provide important insights

into the basic biology of germ cell

development.The gene expression patterns we

have described are summarized inTable 2. The patterns found in go-

nadal germ cells of axolotl are similarto those in the mouse, but we find

significant differences in pregonadalgerm cells. In mice, germ cells are

segregated from somatic lineagesat embryonic day (E) 7.2, late gas-

trulation (Lawson and Hage, 1994),whereas in axolotl, germ cell segre-

gation may occur significantly later.

Axvh and Axdazl

As in other animals,Axvhis expressed

throughout the life cycle of gonadalgerm cells and Axdazlthroughout al-

most all of this period. Mutational andexpression studies in mice and other

animals (Raz, 2000; Tanaka et al.,

2000; Saunders et al., 2003) suggestthat the principal role for Dazl and

Vasaproteins is during the mitotic pro-liferation of gonadal germ cells and

the early phases of meiosis, and thatthis role may be a conserved feature

in all metazoans. In addition, productsof these genes may play another role

in early germ cells of those animalswith germ plasm. For example, mater-

nal gene products of vasaand dazl

orthologs are associated with thegerm plasm inXenopusembryos, and

they play essential roles during earlymigration of PGCs to the genital ridge

(Ikenishi and Tanaka, 1997; Houstonand King, 2000a).

In mice, Mvhis first expressed or is

greatly up-regulated as the germcells enter the gonad in response tosignals from gonadal cells (Toyooka

et al., 2000). In axolotl, Axvh is firstexpressed at a level detectable by

ISH in the early larva at stage 38, butit is not clear whether this is in re-

sponse to signals from gonadal cells.Germ cells may be located from an

early stage adjacent to precursorsof the gonad. Cytologically distinc-

tive gonadal cells first appear in thelarva at approximately stage 43.

Perhaps there is a functional matu-ration of the gonadal precursor cellsaround stage 40, which induces a

response in the germ cells similar tothat seen in mouse, i.e., up-regula-

tion of vasa.In axolotl Axdazlexpression is clearly

detectable at late tail bud, stage 3335, earlier thanAxvh. Thus, PGCs must

undergo some developmental matu-ration during the pregonadal phase

of their development. In mice, dazltranscripts are apparently present at

a higher level than Mvhtranscripts in

pregonadal germ cells at E10.5(Molyneaux et al., 2004), and in fact,

dazlis expressed in ES cells and then

at a low level in germ cells differenti-ating from them (Geijsen et al., 2004).

It is not clear whether dazl may beup-regulated in mouse germ cells at a

point corresponding to this event inaxolotl.

Axoct-4

In mammals, the POU domain tran-

scription factor Oct-4 is considered

to be a marker of pluripotentiality. It

is expressed in the ICM of blasto-

cysts, 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 difficult to identify close ho-

mologs of Oct-4 in lower verte-

brates. Thus far, of the several class V

POU domain transcription factors

identified in nonmammalian spe-

cies, zebrafish Pou2(Zpou2) has the

highest amino acid similarity to

mammalian Oct-4 (Table 1). More-

over, it is present in the early embryo

as maternal RNA, and injection of

high concentrations of morpholino

directed against zpou2 mRNA ar-

rests development at the early gas-

trula stage (Burgess et al., 2002). For

these reasons, it has been proposedthat Zpou2 is a true ortholog of

mammalian Oct-4 (Burgess et al.,

2002).

Here, we report the sequence and

expression profile 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 gas-

trula in a pattern similar to that of the

mouse. Significantly, Axoct-4 is ex-

pressed 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, in-

cluding the two POU domains and

the linker region. By comparison,

Zpou2has 63% identity (Table 1). We

propose that axolotl and mouse

Oct-4proteins represent a subclass

of class V POU factors that is char-

acteristic of organisms whose germ

cells are formed by induction, as in

axolotls and mice. In this regard, we

have isolated genes with significant

TABLE 2. Summary of Axdazl, Axvh, Axoct-4, and AxkitExpression in Germ Cells

Expression in blastulagastrula PGC expressiona Expression during oogenesis

Axdazl Widespread maternal Yes, from stage 33 All stages except very early diploteneAxvh Widespread maternal Yes, from stage 38 All stagesAxoct-4 Widespread maternal; zygotic

expression in ectoderm andposterior mesoderm of gastrula

No From early diplotene

Axkit None No In gonia and diplotene oocytes

aPGC, primordial germ cell.



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sequence identity to mammalian

Oct-4 from primitive fish (sturgeonand lungfish) as well as turtles (Masi

and Johnson, unpublished observa-

tions), species selected on the basisof a prediction that they use regula-

tive germ cell specification (see

Johnson et al., 2003b). Within thiscontext, it is striking that a close ho-molog of Oct-4has not been found

in chick (Soodeen-Karamath and

Gibbins, 2001; confirmed by searchof Sanger expressed sequence tag

database), an amniote with germ

plasm (Tsunekawa et al., 2000).

Xenopus contains several mem-

bers of the class V POU domain tran-

scription factors (Xoct-25, Xoct-79,

Xoct-91,XLPOU60), showing an over-all identity of between 53% and 63%

to the conserved region of murineOct-4, whose transcripts are presentin oocytes or early embryos (Frank

and Harland, 1992; Hinkley et al.,

1992; Whitfield et al., 1993). We wereunable to detect equivalent genes

in axolotls. In fact, a search for ma-

ternally expressed POU proteinsyielded only one sequence,Axbrn-1(Masi and Johnson, 2001), whose

later embryonic expression is re-stricted primarily to the central ner-

vous system. It is interesting that,when the individual expression pat-

terns of each of the Xenopusclass VPOU genes are considered to-gether, the additive expression

pattern resembles that of Axoct-4.One possibility is that the several

class V POU genes inXenopushave

arisen as a result of multiple dupli-cations of a single ancestral Oct-4related gene. In this regard, the du-

plication-degeneration-comple-mentation (DDC) model (Prince and

Pickett, 2002) predicts that a com-plex pattern of ancestral gene func-

tions might be subfunctionalizedafter duplications, followed by lossof individual tissue-specific regula-

tory elements present within thepromoter of the ancestral gene.

Such a mechanism may be respon-

sible for the presence of multipleclass V POU genes in Xenopusand

their overlap with the expressionpattern of Axoct-4.

In addition to its role in the early

embryo, the expression pattern inmice (Pesce et al., 1998) and axolotl

suggests that Oct-4 may also func-

tion in female gonadal germ cells,

not during meiotic progression butlater in growing diplotene oocytes.

Our results reveal a significant dif-ference in the expression pattern of

oct-4 orthologs in the pregonadalPGCs of axolotl and mouse. In

mouse embryos, the germ cells aresegregated by late gastrulation at

E7.2 (Lawson and Hage, 1994) andmouse oct-4 is expressed in PGCs

from this point throughout migrationto the gonad, and in early gonadal

oogonia (Yeom et al., 1996; Pesce etal., 1998). In contrast, Axoct-4 ex-

pression is absent during corre-sponding stages in the axolotl, even

when germ cells are marked by ex-pression of Axvh and Axdazl, from

approximately stage 35 onward. The

enhancer that drives expression ofoct-4in the mouse epiblast (the ho-molog of the amphibian animal

hemisphere) is distinct from thatwhich drives expression in the inner

cell mass and migrating PGCs

(Yeom et al., 1996). If axolotl repre-sents the basal condition, then ex-

pression in migratory PGCs is a newfunction for Oct-4, perhaps evolving

as a new enhancer was added to agene whose original role was during

the segregation of embryonic andextraembryonic tissues of early em-

bryos (Nichols et al., 1998).

Axkit

Unlike axolotl, in mice, c-kitexpres-sion appears in germ cells at approx-

imately E7.5, shortly after they aresegregated in the late gastrula, and

continues during germ cell migrationto the gonad (Manova and Bach-

varova, 1991). This expression is re-quired for normal survival and prolif-

eration of PGCs (Sette et al., 2000).

In axolotl, Axkit RNA does not ap-pear until the germ cells have be-come gonia in the gonads of feed-

ing larvae.

Concluding Remarks

In mouse embryos, the germ cellsare segregated by late gastrulation,

and during the next 3 days duringtheir residence in the gut and migra-

tion to the gonad, they express bothc-kitand oct-4, expression that con-

tinues in gonadal germ cells. In axo-

lotl, neither gene is expressed until

the germ cells have resided in thegonad for at least a week. Expres-

sion of dazl and vasa homologs isup-regulated at roughly the same

stage in mouse and axolotl at latetail bud before or as the gonad is

formed. In axolotl, these are in factthe earliest known markers of a re-

stricted germ cell lineage. Thus, if ax-olotl PGCs are segregated before

the late tail bud stage, they do notexpress two important genes char-

acteristic of pregonadal germ cellsin mouse. Alternatively, germ cells

may be segregated significantlylater in axolotl than in the mouse

(any time up to late tail bud, corre-sponding to approximately E9E9.5

in the mouse). The overlap of expres-

sion of Axscl and Axdazl at stages3335 suggests that lineage restric-tion may not have occurred by this

time, and vasaexpression at stage40 would mark the first segregated

germ cells in axolotl.It should be noted that, unlike

most vertebrates, in axolotl germcells originate at a site close to the

site of origin of gonadal cells; thus along migration to the gonad is not

required. Moreover, unlike mousePGCs, axolotl PGCs do not prolifer-

ate before they reach the gonad

(Humphrey, 1925). Therefore, theymay not need to activate a germ

cell-specific program operative inthe migrating and proliferating germ

cells of mice. This view predicts thatthe receptor CXCR4, which is re-

quired for normal migration of germcells in other vertebrates (see Raz,

2003), may play little role in axolotl. Inconclusion, assuming that urodeles

represent the basal state, then theevidence suggests that, during the

evolutionary path between amphib-

ians and mammals, germ cells be-came segregated earlier and insti-tuted a distinctive pregonadal germ

cell program.

EXPERIMENTAL PROCEDURES

Embryos and Larvae

Embryos and larvae were obtained

from the Axolotl Colony at IndianaUniversity or from spawnings of lo-

cally maintained adults. Embryoswere staged according to Bordz-

ilovskaya et al. (1989) and fixed in 4%



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paraformaldehyde in phosphate

buffered saline. Gonads from feed-ing larvae 2.5 cm to 8 cm in length

were collected and fixed in 4% para-formaldehyde or Bouins fixative. The

stage of development of the gonaddid not correlate strictly with length

or age, so they were staged andsexed by size, position, and cytology

of the germ cells.

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 etal., 2001).

Cloning of Axolotl cDNAs

Axvh and Axoct-4 PCR fragmentswere amplified from either testes or

ovary cDNA, respectively, using de-generate primers to conserved mo-

tifs (LMACAQT and YLVLDEAD for

Axvh; QTTICREA and VVRVWFCN for

Axoct-4) and cloned as described

(Johnson et al., 2001). After se-quence verification, the Axvh PCR

fragment was then used to screen alambda stage 18 embryo library

(Busse and Seguin, 1993) to obtain afull-length cDNA. Inserts were iso-

lated and cloned as described(Johnson et al., 2001). The Axoct-4

PCR fragment was used to screenan ovary cDNA library. A partial

cDNA was retrieved, and a 5RACEkit (Gibco/BRL) was used to obtain

the entire open reading frame. Bothclones were sequenced to comple-

tion by primer extension.AnAxkitPCR fragment was ampli-

fied from larval RNA using nested de-generate PCR primers chosen based

on alignments of c-kit from multiple

species, avoiding the kinase andtransmembrane domains. This ap-proach yielded a 500-base pair frag-

ment that was labeled and used toscreen a larval lambda zap library.

Two clones where obtained andproduced identical restriction maps.

One clone, kit 5, was selected forfurther sequencing.

Northern Blots

Tissue and developmental Northern

blots were performed as previously

described (Masi et al., 2000). Ten mi-

crograms of total RNA was used foradult tissue Northern blots while one-

half embryo equivalents of total RNAwas used for developmental North-

ern blots.

ISH

ISH was carried out on sections of em-

bryos and larvae, and adult ovariesusing digoxigenin-labeled probes as

described (Johnson et al., 2001).

ACKNOWLEDGMENTSWe thank Carl Seguin for the stage18 cDNA library. We also thank Garry

Morgan for helpful discussion oflampbrush chromosomes and for

providing some of the axolotl larvae.

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