tissue non-speciﬁc alkaline phosphatase is expressed in...

1487Development 121, 1487-1496 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Tissue non-specific alkaline phosphatase is expressed in both embryonic and

extraembryonic lineages during mouse embryogenesis but is not required for

migration of primordial germ cells

Grant R. MacGregor†, Brian P. Zambrowicz and Philippe Soriano*

Program in Molecular Medicine, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104, USA

*Author for correspondence†Present address: Department of Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA

Mouse primordial germ cells express tissue non-specificalkaline phosphatase (TNAP) during development, butthe widespread expression of another alkaline phos-phatase gene in the early embryo limits the potential useof this marker to trace germ cells. To attempt to identifygerm cells at all stages during embryonic developmentand to understand the role of TNAP in germ cellontogeny, mice carrying a βgeo (lacZ/neor) disruptedallele of the TNAP gene were generated by homologousrecombination in embryonic stem cells. Using β-galac-tosidase activity, the embryonic pattern of TNAPexpression was examined from the blastocyst stage to

embryonic day 14. Results indicate that primordial germcell progenitors do not express TNAP prior to gastrula-tion although at earlier times TNAP expression is foundin an extraembryonic lineage destined to form thechorion. In homozygous mutants, primordial germ cellsappear unaffected indicating that TNAP is not essentialfor their development or migration.

Key words : alkaline phosphatase, gene knock-out, primordial germcells, β-galactosidase, expression pattern, mouse, extraembryonicdevelopment

SUMMARY

INTRODUCTION

Primordial germ cells (PGCs) were first identified inembryonic day (E) 8.5 mouse embryos based on theirexpression of the enzyme alkaline phosphatase (AP)(Chiquoine, 1954). This correlation was reinforced by theobservation that the number of AP-positive cells was greatlyreduced in two sterile mouse mutants, White spotting and Steel,both known to lack germ cells in the gonads (McCoshen andMcCallion, 1975; Mintz and Russell, 1957). Recent work hasled to the identification of these cells during gastrulation, in acluster within the extraembryonic mesoderm just posterior tothe primitive streak (Ginsburg et al., 1990). However, demon-stration of a distinct population of AP-positive cells prior tothis stage is confounded by expression of another alkalinephosphatase gene throughout the embryo.

Alkaline phosphatases are found in a variety of species(McComb et al., 1979). In general, APs are located at the cellsurface, linked to the cell membrane via a phosphatidylinosi-tol glycan (PI-G) linkage (Low and Saltiel, 1988). Althoughthey are classified on the basis of their ability to hydrolyseorthophosphate monoesters at alkaline pH, their role undermore physiologic conditions remains largely unknown(McComb et al., 1979). Data from several developmentalsystems suggest that APs may play a role in the process of cell

migration (Chang et al., 1992; Kwong and Tam, 1984;Narisawa et al., 1992; Thibaudeau et al., 1993).

Alkaline phosphatases comprise a multi-gene family and atleast three distinct isoforms are known to be expressed in theadult mouse (Manes et al., 1990; Schurr et al., 1989; Terao etal., 1990; Weiss et al., 1988). Only two of these genes, theembryonic isoform (EAP) and the so-called tissue non-specificisoform (TNAP) are expressed during early mouse embryonicdevelopment (Hahnel et al., 1990). In the early stages of devel-opment there is approximately ten times more EAP transcriptthan TNAP transcript; however, a switch in the predominantisoform from EAP to TNAP occurs at around E7. Significantly,the TNAP isoform is expressed in mouse PGCs isolated fromthe genital ridge at E13 (Hahnel et al., 1990).

To understand the significance of TNAP expression inPGCs, we have created a null allele of the TNAP gene in mouseembryonic stem (ES) cells using homologous recombination.In doing so, TNAP coding sequences were replaced with a βgeo(lacZ/neor) reporter gene, to facilitate study of TNAPexpression during development independent of the EAPisoform. As the TNAP isoform is expressed at relatively lowlevels prior to gastrulation, the TNAP isoform might berestricted to PGCs, and the TNAPβgeo allele might thus enablelocalization of the PGCs during early stages of development.Here, we describe in detail the TNAPβgeo expression pattern

1488 G. R. MacGregor, B. P. Zambrowicz and P. Soriano

from E4.5 to E14.5 during mouse embryonic development withan emphasis on the expression within PGCs. We show thatTNAPβgeo expression can be first detected in prospective PGCsby mid primitive streak stage; however this marker is notexpressed by prospective PGCs prior to gastrulation. Further-more, PGCs appeared normally and migrated to the genitalridges in homozygous mutant embryos, indicating that TNAPexpression is not essential for PGC development.

MATERIALS AND METHODS

Construction of the TNAPβgeo targeting vectorA partial TNAP cDNA was generated by reverse transcriptionfollowed by the polymerase chain reaction (RT-PCR) using total RNAisolated from mouse liver as described (Sambrook et al., 1989).Primers used to amplify the cDNA (TTGGAATTCCGAGTGCCT-GCAGGATCGGAACGTC, spanning the exon 1-2 boundary; andCGCGGATCCGCGCTCAGAGCCTCTGGTGGCATCTCGT, exon6) were constructed based upon the published sequences (Hahnel andSchultz, 1989; Terao and Mintz, 1987). The approximately 700 bpproduct was subcloned into pTZ18 (Pharmacia) and its identityconfirmed by DNA sequencing. A northern analysis using total RNAindicated that the TNAP gene is active in ES cells (data not shown).A 129/Sv genomic library constructed in λDASH II (Stratagene) wasscreened with the partial cDNA and 5 phage were isolated andmapped by comparison with the published genomic structure (Teraoet al., 1990). The targeting vector was constructed in three steps. First,a BamHI-SstII fragment containing βgeo and the polyadenylylationsequence of the bovine growth hormone gene (bpA) was isolated frompPGKβgeobpA (Friedrich and Soriano, 1990) and cloned into the cor-responding sites of pBS (KS−) to generate pBSβgeobpA. Next, a 3.1kb ScaI genomic fragment extending from the 3′ portion of intron 1to the first 20 bp of exon 2 was cloned into a Sma site immediately5′ to the βgeo coding sequences. The ScaI site in exon 2 is locatedwithin the leader peptide sequence (Nair et al., 1987; Terao and Mintz,1987) whose processing in the TNAPβgeo allele is presumably lost.The sequence of the fusion construct was confirmed by DNA sequenc-ing. Although the murine TNAP locus has a second promoter locatedwithin intron 1, this promoter is located 5 kb upstream of this ScaIfragment (Studer et al., 1991). Finally, a 4 kb PvuI-NotI fragment con-taining approximately half of exon 6 through to intron 7 was cloned3′ to the bpA sequence. The total extent of homology was 7.1 kb. TheTNAPβgeo allele is 1.6 kb smaller than the wild-type allele and lacksthe active site, Ser 93, located in exon 5 (Hahnel and Schultz, 1989;Terao et al., 1990).

Derivation of mutant mice25 µg of the targeting vector were digested with NotI and introducedinto AB1 cells by electroporation as described (McMahon andBradley, 1990). Clones were selected with G418 (GIBCO; 300 µg/ml)and homologous recombinants detected by Southern analysis usingDNA probes derived from genomic sequences external to thetargeting construct. As neither of the two TNAP promoters is includedin the targeting vector, G418 resistance should occur only upon inte-gration downstream of an active cellular promoter. Targeted cloneswere injected into C57BL/6J blastocyst stage embryos and theresulting chimeras were bred to transmit the TNAPβgeo allele. Animalswere subsequently genotyped using a two allele, three primer PCR.The mutant allele has been designated Akp2 tm1Sor.

Localisation of β-galactosidase activity by X-gal stainingEmbryos were fixed in 4% paraformaldehyde in 100 mM sodiumphosphate (Na2PO4) pH 7.3 containing 0.01% Nonidet P-40 (NP-40)and 0.02% sodium deoxycholate (NaDC) on ice for varying periodsof time ranging from 5 minutes for blastocysts, 12 minutes for E5-

E7, 30 minutes for E8, 1 hour for E9-E11 and 2 hours for E12-E14embryos. Following four rinses in phosphate-buffered saline (PBS),embryos were stained for β-gal activity as previously described(Sanes et al., 1986) with inclusion of NP-40 and NaDC as for thefixation stage. Embryos were generally stained for 2 days at 37°C inthe dark with one change of staining solution. Where necessary,following ethanol dehydration, embryos were cleared in methyl sali-cylate (oil of wintergreen). The endoderm of E5 and E6 embryos wasremoved by incubation in 1.25 % pancreatin and 0.5 % trypsin incalcium- and magnesium-free PBS as described (Tam, 1990). Forfrozen sections, embryos were isolated and fixed in 2% paraformalde-hyde, 7.5% sucrose, 100 mM Na2PO4 pH 7.2 overnight at 4°C. Thefollowing day, they were washed in 3% sucrose, 100 mM Na2PO4 pH7.2 for 2 hours, after which they were cryoprotected in 30% sucrose,100 mM Na2PO4 pH 7.2 for 3 hours. Subsequently, they were frozenin OCT compound on dry ice and sectioned at 10-15 µm on a cryostat.Sections were attached to poly(L)lysine-coated slides and incubateddirectly in 5-Br-4-Cl-3-indolyl-β-D-galactoside (X-gal). The slideswere then rinsed in PBS, mounted with Hydromount (National Diag-nostics) and viewed using a microscope equipped with Nomarskioptics.

HistologyDecidua were obtained at E6, 7 and 8 and teased open leaving theembryo exposed but attached to the decidua. The embryo/deciduawere fixed in 2% formaldehyde, 0.2 % glutaraldehyde in PBS (10minutes for E6 and 7 and 12 minutes for E8), stained with X-gal asdescribed above then dehydrated in ethanol, cleared with Histoclear(National Diagnostics) and embedded in Paraplast (SherwoodMedical). Sections were cut at 6-8 µm using a microtome and coun-terstained with nuclear fast red. Testes were excised and fixed inBouin’s fluid. After embedding in paraffin blocks, sections were cutat 8 µm and stained with periodic acid-Schiff (PAS) reagent.

Detection of alkaline phosphatase enzyme activityFrozen sections of genital ridges from E11.5 wild-type and homozy-gous embryos were stained for AP activity using the Sigma Diag-nostics Kit # 86 (with fast red violet) for a period of 45 minutes at37°C as instructed by the manufacturer. Following staining, slideswere rinsed briefly by dipping in PBS, mounted with Hydromount,coverslipped and viewed as before.

Staging of embryonic development around gastrulation.The classification of Ginsburg (Ginsburg et al., 1990) was adopted tostage the day 7 embryos.

RESULTS

Derivation of mutant miceTo facilitate an analysis of the pattern of TNAP expressionduring early embryonic development and to generate a nullallele of this gene, a LacZ/neomycin amino-phosphotrans-ferase (G418) resistance fusion gene, (βgeobpA)(Friedrich andSoriano, 1990), was ligated in frame to TNAP codingsequences 20 nucleotides downstream of the initiator ATGcodon in exon 2 (see Materials and Methods). The structure ofthe targeting construct is depicted in Fig. 1A.

Following electroporation into ES cells, nineteen of twentyfive clones (76%) screened by Southern analysis were shownto have undergone homologous recombination (Fig. 1B). Thisrelatively high frequency is likely due to factors that includethe use of isogenic DNA (teRiele et al., 1992) and the use ofa ‘promoter-less’ targeting construct. Two clones, 1A1 and1B2, were used to derive germ-line chimaeras, and gave rise

1489Alkaline phosphatase and PGCs

TNAPβgeo allele by homologous recombination. (A) The upperwild-type 129/Sv TNAP locus, the middle the targeting construct andructure of the TNAPβgeo allele. Exons are depicted by numbered boxes.smid vector. Sizes of diagnostic restriction fragments (in kb) ofalleles are indicated by arrows. The 5′ and 3′ probes used in theoted by shaded boxes. B, BfrI; S, SstI; N, NotI. (B) Southern analysisd-type mice and two ES clones 1A1 and 1B2. The restriction digest andis is indicated below the panel. The extreme right-hand panel is ageo heterozygous inter-cross showing the presence of offspring of all

to the TNAP 1 and TNAP 3 lines, respectively. Heterozygousanimals on either an inbred 129/Sv or a hybrid B6/129 back-ground did not display an abnormal phenotype. Heterozygoteswere intercrossed and progeny were genotyped using PCR at8 days of age. Approximately one quarter (39/168) of theprogeny were homozygous for the mutation, indicating thatTNAP is not essential for embryonic development. However,homozygotes succumb to seizures at around two weeks afterbirth due to metabolic defects associated with the loss of TNAPfunction. This phenotype will bedescribed elsewhere (MacGregor etal., unpublished data).

To demonstrate that we hadcreated a null mutation, embryosderived from heterozygousTNAPβgeo parents were isolated atE11.5, fixed and embedded, andfrozen sections were prepared in theregion of the genital ridges.Genotypes were determined usingPCR on total DNA extracted fromyolk sacs. Sections from wild-typeand homozygous mutant embryoswere stained for βgal and APactivity. Staining of sections fromhomozygous mutant embryos withX-gal demonstrated the presence ofPGCs (Fig. 2A). In serial sectionsfrom the same embryo, no APactivity can be detected (Fig. 2B). Incontrast, PGCs can easily be identi-fied by their intense red staining insections from a wild-type embryo(Fig. 2C). In addition, RT-PCR wasperformed on total RNA isolatedfrom E13 gonads of wild-type, het-erozygous and homozygous TNAPβ-

geo embryos, using oligonucleotidesfrom TNAP exons 11 and 12, whichwere not deleted in the mutant allele.This analysis showed that there is notranscriptional read through of thepoly(A) signal in the β-geobpAcassette in the TNAPβ-geo allele (datanot shown), consistent with aprevious study (Chen et al., 1994).These results suggest that theβgeobpA insertion has created a nullallele and there is no other APexpressed in germ cells.

Expression pattern ofTNAPβgeo duringembryogenesisTo assess the pattern of expressionof the TNAP gene during embryoge-nesis and specifically within thePGCs, heterozygous males weremated with wild-type females andembryos were isolated at differentstages of gestation. The embryos

Fig. 1. Generation of the schematic represents the the lower the predicted stpKS is Bluescript, the plaendogenous and targeted Southern analysis are denon DNA from 129/Sv wilprobe used in each analyspedigree blot of a TNAPβ

genotypes.

were fixed and stained with X-gal to localize β-galactosidase(βgeo) activity associated with the expression of the TNAPβgeo

allele.

Embryonic day 14 and 13Staining of embryos at E14 and 13 revealed expression of theTNAPβgeo allele in the developing skeletal system, theembryonic gonad, the placenta and the intestine (Fig. 3A,B).The activity within the developing skeleton is associated with


Fig. 2. Alkaline phosphatase staining in germ cells of TNAPβgeo embryos. Serial frozen sections of genital ridges from a homozygous mutantTNAPβgeo embryo were stained for β-geo activity (A) or AP enzyme activity (B). Sections from a wild-type embryo stained for AP enzymeactivity (C). Note the absence of AP activity in the mutant but the presence of PGCs in the genital ridge revealed by X-Gal staining forTNAPβgeo activity. The X-gal staining is weak in A as all the sections were incubated for a similar period of time (45 minutes). Longer stainingin X-Gal reveals the presence of many more PGCs (data not shown).

the expression of AP in chondrocytes and osteoblasts both ofwhich synthesize matrix vesicles thought to play a centralrole in the process of bone calcification in humans (Ali,1986). The staining in the embryonic gonad is due toexpression in the germ cells. AP expression in the intestinalepithelium at this developmental stage has previously beenshown to be from the same isoform as that expressed in thePGCs (Merchant-Larios et al., 1985). In mouse, the TNAPisoform is known to be expressed in the placenta during fetaldevelopment (Terao and Mintz, 1987). Again, as describedpreviously, the TNAP expression within the primitiveplacenta appears to be localized to the labyrinthine region(Smith, 1973). In summary, the data demonstrate that at thisdevelopmental stage the pattern of X-gal staining faithfullyrecapitulates the previously determined pattern of TNAP geneexpression.

Embryonic day 11At E11, TNAPβgeo expression was detected in the PGCslocalized within the genital ridge, the intestinal epithelium andthe spinal cord (Kwong and Tam, 1984) (Fig. 3C). X-galstaining of frozen sections confirmed these findings (Fig. 3D).Double staining of frozen sections of the genital ridge fromheterozygous embryos indicated that the majority (over 90%)of the alkaline phosphatase cells also stained with X-gal (datanot shown). At low levels of expression, the staining patternwas observed as a subcellular cytoplasmic granule. The lackof absolute concordance for both stains is likely due to thethickness of the sections (10-15 µm) which would precludeobserving both stains in a proportion of the cells.

Embryonic day 8At E8, high level expression of TNAPβgeo is found in theposterior primitive streak (Fig. 4A). β-galactosidase activity atthis time is also observed within the chorion. Histologicalsections at E8 demonstrated that paraxial mesoderm, neurec-toderm and PGCs in the hindgut pocket express TNAPβgeo (Fig.5C,D).

Embryonic day 7Analysis of TNAPβgeo expression at the late and early head foldstages (Fig. 4B,C; respectively) revealed expression in threelocations, at the base of the allantois where the emerging PGCsare located, the primitive streak and the chorion (Smith, 1973).The number of TNAPβgeo-expressing cells found at the base ofthe allantois at this stage identified by X-gal staining ofdissected, squashed embryos was estimated to be between 80and 100 (data not shown). Three embryos at approximatelystage 7-III, 7-II and 7-I (Ginsburg et al., 1990) are shown (Fig.4D-F, respectively). At stage 7-III, staining was observed inthe chorion, allantois and amnion. Histological sections ofembryos at this stage (Fig. 5B) demonstrated the chorion andallantois staining and indicated that the staining within theamnion was restricted to the ectodermal component. At stage7-II, staining was found only in the allantoic bud and in cellssurrounding the ectoplacental cavity. At stages prior to devel-opment of the allantoic bud, expression was observed withinthe extraembryonic mesoderm just posterior to the primitivestreak and in cells surrounding the ectoplacental cavity (Fig.4F). This mid- to late-streak stage was the earliest time atwhich staining could be observed in the prospective PGCs.


tern of TNAPβgeo in transgenic embryos and embryonic gonad. terozygous E14 TNAPβgeo embryo stained for βgeo activity. Theithin the developing skeletal system. (B) Dissected homozygous E13strating the expression in the developing skeleton, the gonad (g), theta (p). Note the localization of expression within the primitive placenta

ion. Staining in the placenta outside of the labyrinthine region was embryos (data not shown) and is non-specific. (C) A heterozygousyo showing staining in spinal cord (n), genital ridge (g) and intestine showing PGCs migrating into the genital ridge (g) of a homozygousryo; mesonephric duct (m).

Embryonic day 6X-gal staining of E6.5 prestreak stage embryos revealed thepresence of β-geo-positive cells in a cluster within theextraembryonic ectoderm (Fig. 4G-I). To determine moreprecisely the location of these cells, theparietal endoderm was removed byenzymatic treatment prior to staining.This location was further confirmedusing histological sections (Fig. 5A).Examination of embryos between E6and E8 suggests that the β-geo-positivecells of chorion are derived from cellsoriginating within the extraembryonicectoderm.

Embryonic day 5In egg cylinder stage embryos, theTNAPβgeo-expressing cells werelocalized within the extraembryonicectoderm (Fig. 4J-L). Gentle squashingof the embryos resulted in detachment ofthe extraembryonic ectoderm from therest of the embryo and revealed a sig-nificant number of blue cells at theborder (Fig. 4L). Counts of TNAPβgeo-expressing cells at this stage range from10 to 20.

Embryonic day 4.5In expanded blastocyst at E4.5, in mostcases, the majority of TNAPβgeo-expressing cells were located withinthe mural trophectoderm (Fig. 4M,N)with an occasional expressing cellfound in the inner cell mass (ICM).Between two and four blue cells wereobserved at this stage. In E4.5 blasto-cysts flushed at about the time ofimplantation, TNAPβgeo expression wasfound in cells of the trophectoderm aswell as in cells of the ICM (data notshown). In outgrowths of E3.5 blasto-cysts cultured for 3 days, TNAPβgeo

expression was found in giant cells aswell as in ICM derivatives. The ICMstaining was always found in cells onthe edge of the ICM clump abutting thetrophectoderm-derived cells (data notshown). Staining was never observed inE3.5 blastocysts.

To ensure that patterns of X-galstaining represented expression ofTNAPβgeo and not non-specific hydroly-sis of the substrate, wild-type controlembryos were stained at each stage indevelopment examined. With theexception of modest, diffuse back-ground activity observed in the placentaat E13-14, non-specific staining wasnever observed.

Fig. 3. Expression pat(A) Lateral view of hestaining is localized wTNAPβgeo embryo illuintestine (i) and placento the labyrinthine regobserved in wild-typeE11.5 TNAPβgeo embr(i). (D) Frozen sectionE10.5 TNAP βgeo emb

Germ cell development in mutant miceIn order to determine if TNAP is essential for germ cell devel-opment between E11.5 and prepubertal stages, 14 day old het-erozygous and homozygous TNAPβgeo male litter mates were


Fig. 4. TNAPβgeo expression during early embryogenesis. (A) Embryonic day 8. A heterozygous embryo exhibiting stainingin the chorion (arrow), hindgut and primitive streak. (B-F)Embryonic day 7-8. Five heterozygous embryos between mid tolate streak and late headfold stages exhibiting staining inpresumptive PGCs at the base of the allantois (arrowhead), in theneural ectoderm in the vicinity of the primitive streak and in cellssurrounding the ectoplacental cavity (arrow). (G-I) Embryonic day6. (G) TNAPβgeo-expressing cells in a day 6 egg cylinder stageembryo are located proximal to the embryonic ectoderm. (H) Earlystreak embryo in which the parietal endoderm was removed byenzymatic digestion to provide a clear view of the embryonic andextraembryonic ectoderm, viewed under Nomarski optics. Note theposition of these cells relative to the constriction (open arrow)demarcating the boundary between the embryonic andextraembryonic ectoderm. Estimates of the numbers of blue cells inembryos at this stage range between 25 and 50. (I) Bright-fieldphotograph of a similar stage embryo demonstrating the presenceof some blue cells close to the boundary (open arrow) betweenembryonic and extraembryonic ectoderm. (J-L) Embryonic day 5.(J) An early egg cylinder stage embryo viewed under Nomarskioptics, from which the parietal endoderm has been removed. TheTNAPβgeo-expressing cells are again found within theextraembryonic ectoderm above the constriction (open arrow)demarcating the boundary between embryonic and extraembryonicectoderm. Squashing embryos at this stage enabled an estimate tobe made of the number of blue cells ranging from 10 to 20. K) Thesame embryo viewed under bright-field optics. (L) Gentlesquashing of the embryo resulted in detachment of theextraembryonic ectoderm from the embryonic ectoderm. (M-N) Embryonic day 4. In this preimplantation expandedblastocyst stage embryo, the TNAPβgeo-expressing cells are locatedin the trophectoderm. The number of blue cells per blastocystobserved at this stage ranged from 2 to 4. The same blastocyst hasbeen photographed at two different angles and focal planes (M,N)to demonstrate the position of the blue cells relative to the ICM(arrowheads). Staining of wild-type blastocysts for extendedperiods failed to reveal any blue cells. (A-F) ×100. (G,K,M,N) ×200, (L) ×400.

killed and testes examined histologically for the presence ofgerm cells. At 14 days of age the most advanced male germcell type is the pachytene stage primary spermatocyte (Nebelet al., 1961). Equivalent numbers of cells at this stage werefound in testes of both the heterozygous and homozyogusTNAPβgeo males (Fig. 6).

No gross defect can be detected in the migration of PGCsduring embryogenesis in homozygous TNAPβgeo individuals(see Fig. 3C,D). In addition, we estimated numbers of PGCsin the gonadal ridges of E10.5 wild-type and heterozygous andhomozygous mutant TNAPβ-geo embryos by counting AP-positive PGCs (wild-type) or β-geo-positive PGCs (heterozy-gous and homozygous mutant) in histologic frozen sectionscontaining genital ridges. No difference in the numbers ofPGCs was observed in mutant compared to heterozygous orwild-type littermates (data not shown). In summary, it appearsthat PGC migration and development does not require TNAPactivity.

DISCUSSION

The study of PGCs at early stages of mouse embryonic devel-

opment has been confounded by the lack of suitable lineagemarkers. Although several gene products other than TNAP areknown to be expressed in PGCs during development, none ofthese are expressed exclusively in the PGCs (Cooke et al.,1994; Scholer et al., 1990) and the earliest marker restricted toPGCs is TNAP. By alkaline phosphatase staining, McLarenand co-workers (Ginsburg et al., 1990) identified a group ofAP-positive cells within the extraembryonic mesoderm justposterior to the primitive streak. However, widespreadexpression of EAP in earlier stage embryos (Ginsburg et al.,1990; Hahnel et al., 1990) precluded using alkaline phos-phatase activity as a discreet marker for PGCs. In an attemptto circumvent this problem, we tagged the TNAP gene withβgeo by gene targeting.

It was important to compare the pattern of expression of theTNAPβgeo allele with its wild-type counterpart, to verify thatpotential cis-regulatory elements of the TNAP locus had notbeen displaced or deleted during construction of the TNAPβgeo

allele. The expression of the TNAPβgeo allele reported here istemporally and spatially identical to patterns of AP expressionpreviously described in the developing skeletal system(Kaufman, 1992; Rugh, 1968), the labyrinthine region of theplacenta (Smith, 1973), the primordial germ cells (Chiquoine,1954; Ginsburg et al., 1990), the primitive streak (Smith,1973), the developing gut (Merchant-Larios et al., 1985) andthe neural tube (Kwong and Tam, 1984). In no case was ectopicTNAPβgeo expression detected where AP activity had not pre-viously been described. Thus, the TNAPβgeo allele appears torepresent faithfully the pattern of expression of its wild-typecounterpart during embryogenesis.

The βgeo-marked allele allowed us to examine TNAPexpression at developmental stages when widespread EAPexpression had previously prevented observation of TNAP-expressing cells (Ginsburg et al., 1990). Prior to gastrulation,βgeo was expressed exclusively within the extraembryonicectoderm of E6 and E5 embryos. In expanded blastocysts orblastocysts flushed at about the time of implantation, βgeo-expressing cells were found mostly in the trophectoderm, butoccasionally in the ICM. No expression was found in blasto-cysts prior to E4.5. These results are consistent with theincrease in TNAP transcription previously observed in blasto-cysts (Hahnel et al., 1990). As the ICM has the capacity to giverise to polar trophectoderm (Dyce et al., 1987), and polar tro-phectoderm gives rise to extraembryonic ectoderm (Copp,1979), the TNAP-positive cells in late blastocysts may be pre-cursors of the blue cells found in the extraembryonic ectoderm.However, the mural location of TNAP-positive cells in manyblastocysts suggests there may be no lineage relationshipbetween these two TNAP-positive cell populations. Theextraembryonic cells expressing TNAP at E5 and early E6likely contribute to the formation of the chorion by day 8,which gives rise to the fetal placenta by E9.5 and ultimatelythe labryinthine region of the placenta by E13. It is unlikelythat these extraembryonic βgeo-expressing cells contribute tothe germ lineage.

To ascertain whether the PGC lineage expresses TNAP priorto gastrulation, embryos were examined at multiple stagesbetween E6 and E7. No expression prior to early E7 wasapparent in any cells other than the extraembryonic cells previously described. At early E7, a small group of cellslocated within the extraembryonic mesoderm posterior to the


activity in E6,7 and 8 embryos. (A) Sagittal section of an E6 embryobryonic ectoderm (ee). (B) Sagittal section of an E7 embryo with β-galllantois (a) and ectodermal component of the amnion (am). E8 embryo and higher magnification of the hindgut region (D) showing (n), paraxial mesoderm (m) and germ cells (pgc) located in the hindgut

primitive streak activated TNAP expression. The timing,number and location of these cells is in agreement with thepattern of AP expression in PGC precursors previously deter-mined using a modifiedenzymatic stain for AP (Ginsburget al., 1990). The number of thesecells increased and were laterfound at the base of the allantois,in the developing hindgut andeventually migrated into thegenital ridges. These resultsindicate that the PGC lineagedoes not activate expression ofTNAP prior to gastrulation.

A recent study (Lawson andHage, 1994) described fatemapping experiments using thetechnique of iontophoreticinjection of lysinated rhodaminedextran into individual epiblastcells of E6.0 and E6.5 embryos.After a period of in vitro culture,embryos were both stained for APactivity and examined for thepresence of the injected dye inorder to determine retrospectivelythe location of prospective PGCsat these stages in development.These studies indicated that thePGC precursors were found in theproximal embryonic ectodermwithin 3 cell diameters of theborder between embryonic andextraembryonic ectoderm. Thesecells are not committed to becomeonly PGCs as they always giverise to other extraembryonicmesodermal derived structures.Our expression studies demon-strate the presence of blue cells inthe entire allantois as well as inother cells of extraembryonicmesodermal origin, specificallythe yolk sac and the ectodermalcomponent of the amnion (datanot shown). As the PGCs are onlya subpopulation of extraembry-onic mesodermal cells, theseresults suggest that the daughtercells of the E6 PGC precursorsdescribed by Lawson and Hagelikely express TNAP whetherthey become PGCs or otherextraembryonic mesodermalcomponents.

It has been hypothesized thatalkaline phosphatases may play arole in the processes of cellguidance and migration in avariety of non-mammalianspecies. Several studies have

Fig. 5. Localization of βgal with staining in the extraemstaining in the chorion (c), a(C) transverse section of an staining in the neurectodermdiverticulum.

demonstrated a potential involvement of AP expression bothin the migration of the pronephric duct during development inthe salamander (Thibaudeau et al., 1993; Zackson and


Fig. 6. Germ cells in prepubertal homozygous TNAPβgeo males.Histology of testes from heterozygous (A) and homozygous (B) 14day postnatal TNAPβgeo males. Note the presence of pachytene stagespermatocytes (arrows) in both sections (Magnification ×160).

Steinberg, 1988), and in growth cone guidance during axonaloutgrowth from the distal limb in the developing grasshopper(Chang et al., 1993). In addition, expression of AP is oftenrestricted both temporally and spatially to populations of cellsthat are involved with migration or morphogenesis. Despite anabsence of TNAP expression in the TNAPβgeo homozygousembryos, PGC migration appeared normal at all stagesexamined. The absence of any residual AP activity in theTNAP mutant germ cells suggests that another AP isozyme isnot compensating for the lack of TNAP, in agreement with aprevious molecular analysis (Hahnel et al., 1990). In addition,the ability of a surviving homozygous TNAP male to produceoffspring (data not shown) further indicates that TNAP is notrequired to produce functional gametes in the male. Theseresults demonstrate that TNAP is not essential for PGCmigration or development and there appears to be no obviouseffect of a null mutation of the TNAP gene on any othermigratory or morphologic process during murine embryogen-esis.

The TNAPβgeo line provides a convenient marker for earlygerm cells to study their fate both in culture or in chimaeras.For example, it will be of interest to determine whether PGCsmutant for TNAP are at a competitive disadvantage with PGCs

that express TNAP. In addition, it should be possible to purifyPGCs early in development using vital stains for β-galactosi-dase such as fluorescein-di-galactoside (FDG) (MacGregor etal., 1991). This will facilitate the isolation of new markersrestricted to germ cells which could be of value to help under-stand those factors that govern allocation of the germ-celllineage.

We thank Elizabeth Thomas for excellent care of the animals andGlenda Froelick and Katrina Waymire for advice and preparation offrozen sections. We are very grateful to Kirstie Lawson for commu-nicating data prior to publication, and to Kirstie Lawson, Rosa Bed-dington and Anne McLaren for helpful discussions. We thank MarkRoth and our laboratory colleagues for critical reading of the manu-script. This work was supported by a grant from the National Instituteof Child Health and Human Development (NICHD) to P. S.

REFERENCES

Ali, S. Y. (1986). Cell Mediated Calcification and Matrix Vesicles. New York:Elsevier.

Chang, W. S., Serikawa, K., Allen, K. and Bently, D. (1992). Disruption ofpioneer growth cone guidance in vivo by removal of glycosyl-phosphatidylinositol-anchored cell surface proteins. Development 114, 507-519.

Chang, W. S., Zachow, K. R. and Bentley, D. (1993). Expression of epithelialalkaline phosphatase in segmentally iterated bands during grasshopper limbmorphogenesis. Development 118, 651-663.

Chen, Z., Friedrich, G. A. and Soriano, P. (1994). Transcriptional enhancerfactor 1 disruption by a retroviral gene trap leads to heart defects andembryonic lethality in mice. Genes Dev. 8, 2293-2301.

Chiquoine, A. D. (1954). The identification, origin, and migration of theprimordial germ cells in the mouse embryo. Anat. Rec. 118, 135-146.

Cooke, J. E., Godin, I., ffrench-Constant, C., Heasman, J. and Wylie, C. C.(1994). Culture and manipulation of primordial germ cells. In Guide toTechniques in Mouse Development (ed. P. M. Wassarman and M. L.DePamphilis). vol. 225 pp. 37-58. San Diego: Academic Press.

Copp, A. J. (1979). Interaction between inner cell mass and trophectoderm ofthe mouse blastocyst, II. the fate of the polar trophectoderm. J. Embryol. Exp.Morph. 51, 109-120.

Dyce, J., George, M., Goodall, H. and Fleming, T. P. (1987). Dotrophectoderm and inner cell mass cells in the mouse blastocyst maintaindiscrete lineages? Development 110, 685-698.

Friedrich, G. A. and Soriano, P. (1990). Promoter traps in embryonic stemcells: A genetic screen to identify and mutate developmental genes in mice.Genes Dev. 5, 1513-1523.

Ginsburg, M., Snow, M. H. L. and McLaren, A. (1990). Primordial germcells in the mouse during gastrulation. Development 110, 521-528.

Hahnel, A. C., Rappolee, D. A., Millan, J. L., Manes, T., Ziomek, C. A.,Theodosiou, N. G., Werb, Z., Pederson, R. A. and Schultz, G. A. (1990).Two alkaline phosphatase genes are expressed during early development inthe mouse embryo. Development 110, 555-564.

Hahnel, A. C. and Schultz, G. A. (1989). Cloning and characterization of acDNA encoding alkaline phosphatase in mouse embryonal carcinoma cells.Clinica Chimica Acta 186, 171-174.

Kaufman, M. H. (1992). The Atlas of Mouse Development. San Diego:Academic Press.

Kwong, W. H. and Tam, P. P. L. (1984). The pattern of alkaline phosphataseactivity in the developing mouse spinal cord. J. Embryol. Exp. Morph. 82,241-251.

Lawson, K. and Hage, W. (1994). Clonal analysis of the origin of primordialgerm cells in the mouse. In Germline Development, Ciba FoundationSymposium 182, pp. 68-91. Chichester: Wiley.

Low, M. G. and Saltiel, A. R. (1988). Structural and functional roles ofglycosyl-phosphatidylinositol in membranes. Science 239, 268-275.

MacGregor, G. R., Nolan, G. P., Fiering, S., Roederer, M. and Herzenberg,L. A. (1991). Use of E.coli lacZ (beta-galactosidase) as a reporter gene. InGene Transfer and Expression Protocols 7 (ed. E. J. Murray) Clifton:Humana Press.

Manes, T., Glade, K., Ziomek, C. A. and Millan, J. L. (1990). Genomic


structure and comparison of mouse tissue-specific alkaline phosphatasegenes. Genomics 8, 541-554.

McComb, R. B., Bowers Jr, G. N. and Posen, S. (1979). AlkalinePhosphatase. New York, NY: Plenum Press.

McCoshen, J. A. and McCallion, D. J. (1975). A study of the primordial germcells during their migratory phase in Steel mutant mice. Experimentia 31,589-590.

McMahon, A. P. and Bradley, A. (1990). The Wnt-1 (int-1) proto-oncogene isrequired for development of a large region of the mouse brain. Cell 62, 1073-1085.

Merchant-Larios, H., Mendlovic, F. and Alvarez-Buyulla, A. (1985).Characterisation of alkaline phosphatase from primordial germ cells andontogenesis of this enzyme in the mouse. Differentiation 29, 145-151.

Mintz, B. and Russell, E. S. (1957). Gene induced embryologicalmodifications of primordial germ cells in the mouse. J. Exp. Zool. 134, 207-237.

Nair, B. C., Johnson, D. E., Majeska, R. J., Rodkey, J. A., Bennet, C. D. andRodan, G. A. (1987). Rat alkaline phosphatase II. Structural similaritiesbetween the osteosarcoma, bone, kidney and placental isoenzymes. Arch.Biochem. Biophys. 254, 28-34.

Narisawa, S., Hofmann, M.-C., Ziomek, C. A. and Millan, J. L. (1992).Embryonic alkaline phosphatase is expressed at M-phase in thespermatogenic lineage of the mouse. Development 116, 159-165.

Nebel, B. R., Amarose, A. P. and Hackett, E. M. (1961). Calendar ofgametogenic development in the prepuberal male mouse. Science 134, 832-833.

Rugh, R. (1968). The Mouse: its Reproduction and Development. New York:Oxford University Press.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning.2nd Edition. Cold Spring Harbor: CSHL Press.

Sanes, J. R., Rubenstein, J. L. R. and Nicolas, J. F. (1986). Use of arecombinant retrovirus to study post-implantation cell lineage in mouseembryos. EMBO J. 5, 3133-3142.

Scholer, H. R., Dressler, G. R., Balling, R., Rohdewohld, H. and Gruss, P.

(1990). Oct-4: a germline-specific transcription factor mapping to the mouset-complex. EMBO J. 9, 2185-2195.

Schurr, E., Henthorn, P. S., Harris, H., Skamene, E. and Gros, P. (1989).Localisation of two alkaline phosphatase genes to the proximal region ofmouse chromosome 1. Cytogenet. Cell Genet. 52, 65-67.

Smith, M. S. R. (1973). Changes in distribution of alkaline phosphatase duringearly implantation and development of the mouse. Austr. J. Biol. Soc. 26,209-217.

Studer, M., Terao, M., Gianni, M. and Garattini, E. (1991). Characterizationof a second promoter for the mouse liver/bone/kidney-type alkalinephosphatase gene : cell and tissue specific expression. Biochem. Biophys.Res. Comm. 179, 1352-1360.

Tam, P. (1990). Studying development in embryo fragments. InPostimplantation Mammalian Embryos: a Practical Approach, pp. 317-337.(ed. A. Copp and D. Cockcroft) Oxford: IRL Press.

Terao, M. and Mintz, B. (1987). Cloning and characterisation of a cDNAcoding for mouse placental alkaline phosphatase. Proc. Natl. Acad. Sci. USA84, 7051-7055.

Terao, M., Studer, M., Gianni, M. and Garattini, F. (1990). Isolation andcharacterization of the mouse liver/bone/kidney-type alkaline phosphatasegene. Biochem. J. 268, 641-648.

teRiele, H., Maandag, E. R. and Berns, A. (1992). Highly efficient targetingin embryonic stem cells through homologous recombination with isogenicDNA constructs. Proc. Natl. Acad. Sci. USA 89, 5128-5132.

Thibaudeau, G., Drawbridge, J., Dollarhide, A. W., Haque, T. andSteinberg, M. S. (1993). Three populations of migrating amphibianembryonic cells utilize different guidance cues. Dev. Biol. 159, 657-668.

Weiss, M. J., Ray, K., Henthorn, P. S., Lamb, B., Kadesch, T. and Harris,H. (1988). Structure of the human liver/bone/kidney alkaline phosphatasegene. J. Biol. Chem. 263, 12002-12010.

Zackson, S. L. and Steinberg, M. S. (1988). A molecular marker for cellguidance information in the axolotl embryo. Dev. Biol. 127, 435-442.

(Accepted 4 January 1995)

tissue non-speciﬁc alkaline phosphatase is expressed in...

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