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Bmp4 is required for the generation of primordial germ cells in the mouse embryo

Bmp4 is required for the generation of primordial germ cells in the mouse embryo Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 is required for the generation of primordial germ cells in the mouse embryo 1,4 3 1 1 2 Kirstie A. Lawson, N. Ray Dunn, Bernard A.J. Roelen, Laura M. Zeinstra, Angela M. Davis, 3 1 2,3,4 Christopher V.E. Wright, Jeroen P.W.F.M. Korving, and Brigid L.M. Hogan 1 2 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands; Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2175 USA In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of b-galactosidase activity in lacZneo Bmp4 embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage. [Key Words: Primordial germ cells; allantois; mouse embryo; Bmp4; extraembryonic ectoderm; chimera] Received November 23, 1998; revised version accepted January 7, 1999. Before gastrulation, the mouse embryo consists of three lie scattered in a ring that extends up to three cell diam- distinct cell lineages which were established in the blas- eters from the junction with the extraembryonic ecto- tocyst during the peri-implantation period, that is, epi- derm (Lawson and Hage 1994). Early in gastrulation, they blast, extraembryonic endoderm, and trophectoderm. converge toward the primitive streak in the posterior of The epiblast, from which the entire fetus will form, as the embryo and translocate through it. Allocation to the well as the extraembryonic mesoderm and amnion ecto- germ cell lineage is thought to occur in ~ 45 cells around derm, is a cup-shaped epithelium apposed on its open E7.2, after the precursors have passed through the streak end to the extraembryonic ectoderm, a trophectoderm and have come to reside in the extraembryonic meso- derivative. Both epiblast and extraembryonic ectoderm derm (Lawson and Hage 1994). This is about the time are covered by visceral endoderm, which is part of the when the putative PGCs can first be identified morpho- extraembryonic endoderm lineage (Hogan et al. 1994). logically in a cluster posterior to the primitive streak in The primordial germ cells (PGCs) of the mouse em- a position that will later become the base of the allantois bryo are derived from part of the population of epiblast (Ginsburg et al. 1990). PGCs stain strongly in a charac- teristic pattern for alkaline phosphatase (AP) activity cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before (Chiquoine 1954), which by this stage is due to tissue gastrulation in the extreme proximal region of the epi- nonspecific AP (Hahnel et al. 1990; MacGregor et al. blast adjacent to the extraembryonic ectoderm, and have 1995). The PGCs continue to express AP during their descendants not only in the germ line, but also in extra- proliferation in the developing hindgut and migration embryonic structures, that is, the allantois, blood is- into the genital ridges (for review, see Buehr 1997). lands, and yolk sac mesoderm, as well as both layers of Transplantation studies have shown that genetically the amnion. At embryonic day (E) 6.0, these precursors marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to Corresponding authors. PGCs and extraembryonic mesoderm when grafted to E-MAIL brigid.hogan@mcmail.vanderbilt.edu; FAX (615) 343-2033. E-MAIL lawson@niob.knaw.nl; FAX 31 (30) 2516464. the proximal epiblast (Tam and Zhou 1996). These re- 424 GENES & DEVELOPMENT 13:424–436 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells sults raise the possibility that PGC precursors are in- were therefore assayed for the presence of PGCs by AP duced by extracellular factors and/or cell interactions staining. present locally at the junction between the extraembry- onic ectoderm and epiblast. tm1 Dosage effect of Bmp4 on PGC number Candidate genes encoding putative germ cell precursor tm1 inducing factors are predicted to be expressed in the Comparison of littermates of Bmp4 /+ intercrosses be- mouse embryo before and during gastrulation. One such tween E7.2 and E7.75 showed firstly that homozygous factor is Bone Morphogenetic Protein 4 (Bmp4), a mem- null mutants contained no PGCs [12 embryos from 7 ber of the TGFb superfamily of intercellular signaling (C57BL/6 × CBA) matings], and secondly that the inci- proteins (Hogan 1996; Waldrip et al. 1998). Most mouse dence of heterozygous embryos with recognizable PGCs embryos homozygous for a null mutation in Bmp4 die lagged behind that of the wild type until after the head- around gastrulation (~ E6.5) (Winnier et al. 1995). On fold stage on both the (C57BL/6 × CBA) and (129/ some genetic backgrounds, however, a proportion of the SvEv × Black Swiss) backgrounds (Fig. 2). More detailed mutant embryos survive until the early somite stage and quantitative analysis at E7.5 is not informative because show severe defects, particularly in the extraembryonic PGCs are still emerging from the cluster of AP-positive mesoderm (Winnier et al. 1995). In this paper, we exploit cells (Ginsburg et al. 1990), and the population is not yet this late phenotype to show that PGC formation abso- expanding exponentially (Lawson and Hage 1994). lutely requires Bmp4 signaling. In addition, the size of The whole-mount AP staining technique described the founding population of PGCs is significantly reduced here allows the quantitation of PGCs in situ in the em- in heterozygous mutant embryos. By using a Bmp4–lacZ bryo at more advanced stages. For example, as shown in reporter allele, we have definitively localized Bmp4 ex- Figure 3, PGCs are clearly present in the hindgut of wild- pression before gastrulation in the extraembryonic ecto- type and heterozygous embryos (Fig. 3A–C; see also Fig. derm and in mid- to late- primitive streak stage embryos 1E), but are completely absent from the homozygous in the extraembryonic mesoderm. Thus, Bmp4 is ex- mutants (Fig. 3D; see also Fig. 1F–H). This absence was pressed at the right time and in the right place to play a true for both genetic backgrounds (C57BL/6 × CBA: 29 role both in the quantitative induction of PGC precur- homozygous mutant embryos from 23 females; 129/ sors in the proximal epiblast and in their allocation to SvEv × Black Swiss: 8 homozygous mutant embryos the germ cell lineage in the extraembryonic mesoderm. from 5 females) and at all stages examined. The most Furthermore, by analyzing genetic chimeras, we have advanced mutant (C57BL/6 × CBA) embryo at E9.5 had clearly established a role for Bmp4 in the induction of 17 somites, and one (129/SvEv × Black Swiss) embryo PGC precursors and demonstrate for the first time that a was fully turned with 23 somites. secreted signal from the extraembryonic ectoderm is re- Heterozygous embryos, although indistinguishable quired for the normal development of the epiblast. from their wild-type littermates in terms of overall size and morphological features, including the allantois, had reduced numbers of PGCs on both genetic backgrounds Results (Fig. 3, cf. A with C; for the one exception concerning the allantois, see footnote to Fig. 4). In addition, PGCs were tm1 Phenotypic abnormalities in Bmp4 homozygous absent in 9% of the heterozygous (C57BL/6 × CBA) em- null mutants bryos (Fig. 4A). Although the heterozygous embryos had On both the (129/SvEv × Black Swiss) and (C57BL/ fewer PGCs, the regional distribution of PGCs in hetero- tm1 6 × CBA) genetic backgrounds, many Bmp4 homozy- zygous and wild-type littermates did not differ, with gous embryos develop up to and beyond the early somite PGCs spreading from the ventral hindgut through the stage. An example of a 20 somite (S) stage homozygous dorsal mesentery and into the genital ridges by E9.5. embryo is shown in Figure 1B. Among the late surviving To determine at which stage the difference in the size homozygous mutants, several consistent abnormalities of the PGC population arose, PGC number estimated on are observed. First, they are developmentally delayed in whole mounts was plotted against somite number. The comparison to their wild-type and heterozygous litter- regression line of log PGC number on somite number mates (Fig. 1A,B). Significantly for this study, all com- fitted to all values greater than zero for the heterozygotes was parallel to that for the wild type, but had reduced pletely lack an allantois (Fig. 1B,D), and many show se- vere posterior defects, including disorganized posterior elevation (P < 0.001) (Fig. 4A,B). The parallel regression ectoderm (Fig. 1G,H), overgrowth and endothelialization lines indicate that the rate of expansion of the PGC of the somatopleure (Fig. 1, cf. E with F and G), with population is the same in wild-type and heterozygous extension of endothelial cells into the amnion in the embryos. Assuming an average of one somite pair formed most severe mutant phenotypes (Fig. 1H), and small and every 90 min (Tam 1981), the slope gives a population poorly vascularized yolk sacs. doubling time of 15.8 hr, which is consistent with pre- The absence of an allantois in all homozygous null vious data (Tam and Snow 1981; Lawson and Hage 1994). Bmp4 mutants strongly suggested that they would also In contrast, the difference in elevation of the two regres- have a deficiency of PGCs, because the precursors of the sion lines suggests that the size of the founding popula- two cell types reside in similar positions in the proximal tion of PGCs is smaller in the heterozygotes. Wild-type epiblast before gastrulation. Embryos of different stages embryos on the (C57BL/6 × CBA) background have a GENES & DEVELOPMENT 425 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. tm1 Figure 1. Phenotypes of advanced Bmp4 (129/SvEv × Black tm1 Swiss) mutant embryos. (A) Bmp4 /+ embryo at the early forelimb bud stage showing wild-type morphology. (B) tm1 tm1 Bmp4 /Bmp4 , littermate of A showing delayed develop- ment, incomplete turning, irregular somites and kinked neural tube, uncharacteristic looping of the tail to the left, and absence of allantois. (C) Posterior of embryo in A (boxed region) with allantois (a). (D) No allantois in homozygous mutant (arrow). The broken line in B marks the level of dissection for D.(E–H) tm1 Sections of wild-type and homozygous null Bmp4 (129/ SvEv × Black Swiss) embryos showing posterior phenotype. AP and haemalum staining. (E) Wild type. Transverse section (TS) of posterior region of an E9.5 embryo with 27 somites. The umbilical vein (u) demarcates the junction between somato- pleure (sop) and amnion (am). PGCs (arrowhead) are migrating from the hindgut (hg) into the genital ridges (gr). (F)TSofthe posterior region of a −/− sibling to the embryo in E. This embryo had 23 somites and an external morphology similar to the em- bryo in B. There was no external allantois, but the region be- tween the amnion and the somatopleure was heavily endothe- lialized (e). (G) TS of the posterior region of another E9.5 −/− sibling with 14 somites and more severe posterior defects. The endothelialized somatopleure has reflected dorsally, forming a posterior pocket that becomes continuous with the amnion. A dorsal extension (n8) of the caudally disorganized neurectoderm (n) surrounded by surface ectoderm (se) is contained within the pocket. (H) Sagittal section of an E8.5 −/− embryo showing a severe mutant phenotype. The embryonic portion contains con- voluted ectoderm (ec) and limited mesoderm extending ros- trally from the primitive streak (ps). The amnion is normal ros- trally, but is filled caudally with mesoderm (arrow), which is continuous with the primitive streak. In addition, there is an accumulation of AP-positive amnion ectoderm (*). (A) Anterior; (da) dorsal aorta; (n) neural tube; (P) posterior; (ys) visceral yolk sac. Scale bars in C for A–D, 200 μm; in E for E–H, 200 μm. mean founding population of 45 (Lawson and Hage The extrapolation of the regression line of the wild 1994); extrapolation of the regression line for the wild type (C57BL/6 × CBA) in Figure 4A reaches the expected type to this value, and comparison with that for the het- founding population size of 45 at −1.2S, instead of the erozygotes at the same stage, gives a mean PGC founding expected allocation time equivalent to approximately population in the heterozygotes of 17 (a 62% reduction). −8S (12 hr before the 0S stage). This discrepancy suggests The slopes of the regression lines of PGCs numbers on that PGC number is consistently underestimated in the (129/SvEv × Black Swiss) (Fig. 4B) and (C57BL/ whole mounts, but it does not affect the relative differ- 6 × CBA) backgrounds were indistinguishable. However, ence in PGC number between wild type and heterozy- the elevation of the lines for both wild type and hetero- gotes, nor the inference that the size of the founding zygotes was higher in the (129/SvEv × Black Swiss) em- population is reduced by >50% in the heterozygotes. If bryos compared with their (C57BL/6 × CBA) counter- the lower number of PGCs in the heterozygotes were due parts (P < 0.001). This is consistent with a mean found- solely to a delay in PGC allocation or to delayed onset of ing population of 66 in the wild type and 30 in the PGC proliferation, rather than to a smaller founding heterozygote (129/SvEv × Black Swiss) (a 55% reduc- population, the length of the delay implied by the differ- tion). ence in elevation of the regression lines would be 22 hr 426 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells Figure 2. Incidence of wild-type and heterozygous embryos tm1 with recognizable PGCs at E7.2–E7.75 from Bmp4 /+ (C57BL/6 × CBA) intercrosses (7 females, 40 embryos) and lacZneo ICR × Bmp4 /+ matings (4 females, 38 embryos) examined in whole mount. (Open columns) Wild type; (hatched columns) heterozygotes; sample size in parentheses. The same trend was found in both groups (not shown): The pooled data show that the proportion of embryos with PGCs was smaller in the het- erozygotes in combined stages up to, and including, the head- fold (HF) stage (x test: P < 0.05). (ES/MS) Early streak/mid- streak; (LS) late streak; (NP) neural plate; (HF) headfold; (S) so- mite. (14.7 somite equivalents). The data do not support this interpretation. An alternative possibility, which cannot be distinguished from a direct effect on the number of cells allocated, is that more than half of the PGC founders in the heterozygotes die before the first division Figure 4. Linear regression analysis of PGC number (counted after allocation. in whole mount) vs. somite number in embryos from In summary, whereas one active allele of Bmp4 is suf- tm1 tm1 Bmp4 /+ intercrosses. (A) Bmp4 (C57BL/6 × CBA). (B) ficient for normal maintenance, proliferation, and the lacZneo Bmp4 (129/SvEv × Black Swiss). (s, broken line) Wild initiation of migration of the PGCs, the size of the type; (d, solid line) heterozygote; (n) homozygous null. The founding population, which is normally allocated at values in the regression equation, Y = a + bX, for log PGC (Y)on about E7.2 at the late midstreak/late streak stage, is somite (X) number at the mean values of X and Y with each set Bmp4 dosage dependent. of data were in A, wild type, 2.124 = 1.684 + 0.0286 (15.4); het- erozygote (PGC values >0), 1.647 = 1.268 + 0.0275 (13.8); B, wild type, 2.305 = 1.878 + 0.0288 (14.8); heterozygote, 2.089 = 1.541 + 0.0298 (18.4). Identification of genotype in B was by b-gal staining and phenotype. In A, the 25/26S heterozygote with 23 PGCs resembled an advanced homozygous null embryo (as in Fig. 1B) and completely lacked an allantois. Temporal and spatial pattern of Bmp4 expression during early mouse development Because Bmp4 is clearly important for PGC formation, it is essential to know its precise temporal and spatial ex- pression before and after gastrulation. To detect Bmp4 expression at this time with high sensitivity and single cell resolution, we used homologous recombination in ES cells to replace the first protein coding exon of the Figure 3. PGCs in posterior (hindgut) pieces from E8.5 sibling Bmp4 gene with a reporter cassette encoding b-galacto- tm1 embryos from a Bmp4 /+ (C57BL/6 × CBA) intercross mat- sidase (b-gal) with an amino-terminal nuclear localiza- ing. Alkaline phosphatase staining, dorsal view. (A) Wild type, tion signal (Fig. 5). Embryos homozygous for the 15S embryo. (B) High power of part of A showing individual lacZneo Bmp4 mutation on the (129/SvEv × Black Swiss) PGCs (arrow) in the hindgut. (C) Heterozygote, 15S embryo. tm1 background have the same phenotype as Bmp4 ho- There are fewer PGCs compared with the wild-type sibling in A. mozygotes (Fig. 6M,N). Moreover, removing the neo cas- (D) Homozygous null, 8S embryo. Although a hindgut is present sette has no effect on lacZ expression or mutant pheno- (hg), PGCs are entirely absent. Scale bars in A for A, C,and D, type (data not shown). 200 μm; in B, 100 μm. (+/+) Wild type; (+/−) heterozygote; (−/−) homozygous null. To determine the onset of Bmp4 expression in vivo, GENES & DEVELOPMENT 427 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. Figure 5. Targeted replacement of the Bmp4 gene with a lacZ reporter cassette. (A) Genomic organization of the wild-type and mutated al- leles and the structure of the targeting vector. Coding and noncoding exons are represented by solid and shaded rectangles, respectively. The lacZneo Bmp4 targeting vector contains 1.6 kb of 58 homology. The 6.1-kb 38 homology arm in- cludes the oligonucleotide-interrupted coding tm1blh exon 4 (open rectangle) from the Bmp4 tar- geting vector (Winnier et al. 1995) and is flanked by the herpes virus thymidine kinase cassette (HSV-tk) for negative selection (Soriano et al. 1991). Coding exon three is replaced with both lacZ and neo resistance cassettes; the arrow in- dicates the direction of neo transcription. loxP sites ( c) flank the neo cassette. The correctly recombined locus produces a fusion transcript between noncoding Bmp4 sequences and lacZ without disrupting the structure of neighboring introns. The 500-bp BamHI–BsmI fragment used as an external 58 probe is shown above the wild-type Bmp4 locus. In the 12C targeted ES cell line, recombination occurred in the intron between exons 3 and 4, as determined by the PCR strategy described in Winnier et al. (1995). (B) Southern blot analysis of progeny from a rep- lacZneo resentative backcross of the Bmp4 allele. By use of the 58 external probe and SpeI diges- tion, the wild-type and targeted loci generate 6.3 and 11.1 hybridizing bands, respectively. (B) BamHI; (Bs) BsmI; (C) ClaI; (E) EcoRI; (H) HindIII; (N) NotI; (P) PstI; (Sf) SfiI; (Sm) SmaI; (Sp) SpeI; (X) XbaI. (+/+) Wild type; (+/−) hetero- zygote. lacZneo Bmp4 heterozygous embryos were analyzed for tently larger in wild-type embryos than in heterozygotes b-gal activity from E3.5 onwards. Positive cells could not (Fig. 7A,B). be detected in intact blastocysts or in E4.5 embryos even Bmp4 produced by the extraembryonic ectoderm may after prolonged staining (data not shown). At E5.5, low be required for the induction of Bmp4 expression in the lacZneo levels of Bmp4 expression are first detected extraembryonic mesoderm derivatives of the proximal throughout the uncavitated extraembryonic ectoderm, epiblast. To explore this possibility, Bmp4 expression lacZneo including those cells that abut the epiblast (Fig. 6A). By was monitored in Bmp4 homozygous null em- ~ E6.0, just prior to overt streak formation, the highest bryos by b-gal staining. At the headfold stage, strong levels of lacZ expression become localized to the region b-gal activity is detected in the extraembryonic meso- of the extraembryonic ectoderm immediately adjacent to derm lining the exocoelom, as well as in cells accumu- the epiblast (Fig. 6B; see also Waldrip et al. 1998). As lating near the junction of the amnion with the posterior gastrulation begins, these b-gal-positive extraembryonic primitive streak, in the position normally occupied by cells are displaced proximally by the encroaching extra- the developing allantois (Fig. 6M,N). Bmp4 expression in embryonic mesoderm and subsequently contribute to the epiblast derivatives is therefore independent of Bmp4 lacZneo the chorion (Fig. 6C–F). Bmp4 expression is de- expression in the extraembryonic ectoderm. tected in newly formed extraembryonic mesoderm at the midstreak stage, as the exocoelom begins to form (Fig. Chimera analysis indicates a role for extraembryonic 6E). It is then expressed with increasing intensity in the ectoderm Bmp4 allantois and mesodermal components of the developing amnion, chorion, and visceral yolk sac (Fig. 6G–L). No The temporal and spatial expression pattern described expression is seen in the primitive streak at this time. above is compatible with a role in PGC allocation for Double staining for b-gal and AP activity shows that Bmp4 secreted by either the extraembryonic ectoderm, Bmp4 is expressed in cells in the vicinity of the PGCs, the extraembryonic mesoderm, or both. To distinguish but is clearly excluded from them (Fig. 7A,C,D). The area between these possibilities, we have exploited the fact posterior to the streak at the base of the initiating allan- that ES cells almost exclusively colonize the epiblast toic bud in which PGCs can be first identified is consis- when injected into blastocysts or aggregated with moru- 428 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells lacZneo Figure 6. Bmp4 expression in the early mouse embryo. (A) An E5.5 embryo viewed un- der Nomarski optics. Low levels of b-gal activ- ity are first detected throughout the uncavi- tated extraembryonic ectoderm (xe). (Arrow- head) Junction between embryonic and extraembryonic regions. (B) At the onset of gas- lacZneo trulation (early streak, ES), Bmp4 expres- sion continues in the extraembryonic ecto- derm, in a ring that abuts the epiblast (ep). (C,D) lacZneo As gastrulation proceeds, Bmp4 expres- sion within the extraembryonic ectoderm per- sists and is particularly evident between the mid-streak (MS) to late-streak (LS) stages within the posterior amniotic fold (paf). (E) Sag- ittal section through a MS/LS embryo. Low lev- els of b-gal activity within the extraembryonic mesoderm (arrow) are first detected at this stage, as the exocoelom (exo) begins to form. (F) Late-streak (LS) stage embryo. (G–L) Bmp4 ex- pression during allantois development. lacZ ex- pression is detected in the posterior accumula- tion of extraembryonic mesoderm that pre- cedes overt allantois formation (asterisk in G) and within the allantoic bud (ab) and allantois (a) as it extends through the exocoelomic cav- ity. Expression also persists in the extraembry- onic mesodermal components of the amnion (am), yolk sac (ysm), and chorion (cm) that line lacZneo the exocoelom. (M,N) Bmp4 homozygous null embryo at the headfold (HF) stage. (M) Whole mount, lateral view. (N) Parasagittal sec- tion of M. Strong b-gal activity is detected in the amnion and yolk sac mesoderm, as well as in the accumulation of extraembryonic mesoderm (*) posterior to the primitive streak (ps). Anterior (A) is to the left in B–N. (c) Chorion; (xn) extraembryonic endoderm; (ES) early streak; (OB) no bud; (EB) early bud; (NP) neural plate; (LNP) late neural plate. Scale bars in A, 100 μm; in B for B–J, 200 μm; in K for K and L, 100 μm; in M for M and N, 200 μm. lae (Beddington and Robertson 1989). In contrast, the re- expected from the fate map of the epiblast (Lawson et al. cipient embryo forms the trophectoderm and extraem- 1991) if there had been incomplete cell mingling in the bryonic endoderm derivatives and can contribute to the epiblast prior to gastrulation. Chimerism in the PGCs epiblast. A chimera with 100% ES-derived epiblast (Fig. 7E) was well correlated with the roughly estimated would then have ES-derived extraembryonic mesoderm degree of posterior somatic chimerism in both wild-type and PGCs and recipient-derived extraembryonic ecto- embryos and heterozygotes (Table 2), indicating that derm. In the experiment here, ROSA 26.1 ES cells that there was no positive or negative selection for germ cell are genetically marked with a ubiquitously expressed fate on the basis of the genotype of the recipient embryo lacZ reporter gene were either injected into blastocysts or on ES cell origin. tm1 or aggregated with morulae from Bmp4 heterozygous The number of PGCs in the heterozygotes was unaf- intercrosses. The resulting embryos were recovered at fected by the size of the wild-type population in the epi- nominal E8.5, genotyped, and analyzed for degree of chi- blast-derived tissues (Fig. 8A,B): There was no indication merism and PGC number on histological sections. in chimeras on either genetic background of an increase Between 32% (C57BL/6 × CBA; n = 72) and 50% (129/ in PGC number above the nonchimeric level towards the SvEv × Black Swiss; n = 80) of the embryos recovered wild-type level, even in chimeras with no detectable het- were chimeric: The ES cell contribution tended to be erozygous contribution to the epiblast-derived tissues. stronger in the aggregation chimeras, with 39% of the The smaller size of the PGC population in heterozygotes chimeric embryos showing >95% chimerism in the epi- is therefore due to reduced Bmp4 from the extraembry- blast derived tissues (Table 1). Generally, chimerism was onic ectoderm, and cannot be compensated by Bmp4 fine grained throughout the embryos, but in 12/40 blas- from wild-type extraembryonic mesoderm. tocyst injection chimeras and 5/23 aggregation chime- Wild-type ES cells in combination with homozygous ras, the extraembryonic mesoderm, the posterior part of null embryos were unable to rescue the mutant pheno- the embryo, and sometimes the anterior surface ecto- type: Neither allantois nor PGCs were present even derm were distinctly less chimeric. This result could be when the epiblast-derived component of the conceptus GENES & DEVELOPMENT 429 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. Figure 7. (A–D) Sections of embryos from lacZneo ICR × Bmp4 /+ matings stained for b-gal and AP activ- ity. (A) Heterozygote, late streak (LS) stage. Sagittal section of posterior region at embryonic/extraembryonic junction. b-gal staining (arrowhead), representing Bmp4 expression, is present in mesothelial cells lining the exocoelom. Three AP-positive PGCs (arrow) (of a total of seven in this embryo) lie internal to the b-gal staining region and do not stain blue. (B) Wild type, late streak stage, sibling of embryo in A, sagittal section as in A. The cluster of 11 PGCs (arrow) at the base of the incipient allantois (arrowhead) is larger than in the heterozygote. There were 33 identifiable PGCs in this embryo. (C) Heterozygote, headfold (hf) stage. Transverse section at the level of the head- fold (hf) and base of the allantois (a). (Dark field) b-Gal staining appears pink. There is strong b-gal activity in the periphery of the allantois but not in its core. (D) High power, bright-field view of part of C. b-Gal staining peripherally at the base of the allantois, but not in the AP-positive PGCs (arrow) lying more tm1 centrally. (E,F) Sections of R26.1 ES « Bmp4 / tm1 +× Bmp4 /+ chimeras stained for b-gal and AP activity: Wild-type ES cells stain blue. (E) Hindgut of a 75% chimeric wild-type embryo showing b-gal-positive PGCs (arrow) de- rived from the ES cells and a recipient-derived, b-gal-negative, PGC (arrowhead). (F) Sagittal section of 4S stage >95% chi- meric homozygous null embryo on the (C57BL/6 × CBA) background. The epiblast derived cells are of wild-type, ES cell origin and have no detectable contribution from the mutant cells that are confined to the chorion ectoderm (c) and visceral yolk sac endoderm (ys). The phenotype is characteristically homozygous null with no allantois (arrowhead), no PGCs, small visceral yolk sac and defective yolk sac vascularization. AP staining in the embryonic ectoderm and chorionic ecto- derm is independent of phenotype (cf. with A and B). (am) Amnion; (c) chorion; (h) heart; (ps) primitive streak; (vee) vis- ceral extraembryonic endoderm. Scale bars in A–E, 50 μm; in F, 100 μm. contained no detectable mutant cells (>95% wild-type the epiblast set aside at about the time of implantation. ES cell contribution) (Table 3; Fig. 7F). Therefore, Bmp4 The earlier allocated extraembryonic cell lineages, that produced by the extraembryonic ectoderm is required by is, trophectoderm and primitive endoderm, contribute the epiblast to generate an allantois and PGCs, and can- no descendants to the fetus, but provide the tissues re- not be substituted by Bmp4 produced by wild-type ex- quired for implantation and nutrition of the conceptus traembryonic mesoderm. (for review, see Rossant 1986). Evidence is now emerging that, in addition to their support functions, these extra- embryonic lineages play more intimate roles in embry- Discussion onic development. For example, early events in anterior It has been long established that all the fetal lineages, neural patterning require specific gene expression in the both somatic and germ line, are derived exclusively from adjacent visceral embryonic endoderm, a derivative of tm1 tm1 Table 1. Chimeras of R26.1 ES cells with embryos from Bmp4 × Bmp4 matings Percentage chimerism Recipient Total Mean somite genotype embryos number (range) 0 ø25 >25–50 >50–75 >75–95 >95 Morula aggregation (C57BL/6 × CBA) Wild type 19 8.4 (0–15) 14 0 1 0 2 2 +/− 40 8.1 (0–15) 30 3 1 2 0 4 −/− 13 2.8 (0–6) 5 0 1 2 2 3 Blastocyst injection (129/SvEv × Black Swiss) Wild type 13 13.7 (0–21) 10 1 1 1 0 0 +/− 43 15.4 (0–26) 15 10 7 8 2 1 −/− 24 5.3 (0–14) 15 5 1 3 0 0 430 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells Table 2. Percent chimerism in PGCs related to the extent of posterior somatic chimerism Percent chimerism (posterior) Genotype ø25 >25–50 >50–75 >75–95 >95 Wild type 1.6 (1) 35.2 ± 0.3 (2) 73.6 (1) 72.0 ± 32.5 (2) 94.5 ± 4.9 (2) tm1 Bmp4 /+ 4.1 ± 6.7 (12) 20.7 ± 12.8 (14) 47.1 ± 33.1 (5) 83.4 ± 6.3 (2) 95.0 ± 4.9 (5) Chimerism in PGCs [mean ± S.D.(n)]. the primitive endoderm (for review, see Beddington and Models for the specification of PGCs and allantois Robertson 1998). The results reported here demonstrate formation in the mouse embryo that the initiation of both the germ line and the allantois In this paper we report three independent sets of obser- is dependent on a signal from the first established extra- vations that together suggest possible models in which embryonic lineage, the trophectoderm. Bmp4 produced by extraembryonic cells quantitatively regulates the fate of PGC precursors in the epiblast and the size of the founding population of PGCs in the em- bryo. These models underscore the importance of cell– cell interactions in the formation of the mammalian germ line (Tam and Zhou 1996), and open up the mo- lecular analysis of the signaling pathways and genes in- volved. The first set of observations is that mouse embryos with no functional Bmp4 gene completely lack both PGCs and an allantois, cell types that arise from precur- sors located before gastrulation in the proximal epiblast (Lawson and Hage 1994). In addition, heterozygous tm1 Bmp4 embryos have fewer PGCs than wild type, al- though the allantois appears normal. From the regression analysis of PGC number against developmental stage (Fig. 4), this difference can be clearly attributed to a smaller founding population in the heterozygotes, and not to a lower proliferation rate. The second set of findings is that Bmp4 is expressed before gastrulation in the extraembryonic ectoderm, at highest levels in cells at the junction with the proximal epiblast. This expression pattern is particularly evident when assayed with a b-gal reporter inserted into the en- dogenous Bmp4 allele. Bmp4 is later expressed in the extraembryonic mesoderm, including the allantois, and in cells in the vicinity of the first identifiable PGCs. However, Bmp4 does not appear to be expressed in the PGCs themselves (Fig. 7A,D). In addition, the presence of b-gal activity in the extraembryonic mesoderm of ho- lacZneo mozygous Bmp4 embryos implies that Bmp4 in Figure 8. PGCs (estimated from histological sections) in chi- tm1 the extraembryonic ectoderm is not required to initiate meras of R26.1 ES cells with wild-type and Bmp4 /+ embryos. Bmp4 expression in the extraembryonic mesoderm (Fig. (A) Aggregation chimeras with (C57BL/6 × CBA) recipients. (B) 6M,N). Blastocyst injection chimeras with (129/SvEv × Black Swiss) re- The third set of observations is that the PGC-and-al- cipients. (Open symbols, broken line) Wild-type recipients; (solid symbols, solid line) heterozygous recipients; (circles) non- lantois-deficient phenotype of Bmp4 mutant embryos chimeric; (squares) ø25% chimeric; (triangles) >25%–50% chi- cannot be rescued by wild-type ES cells injected into meric; (diamond) >50%–75% chimeric; (four-pointed star) blastocysts or aggregated with morulae. In the resulting >75%–95% chimeric; (five pointed star) >95% chimeric. The chimeras, the wild-type ES cells contribute only to the number of PGCs in chimeric embryos falls within the distribu- epiblast-derived tissues, whereas the extraembryonic ec- tion of the nonchimeric embryos of the same genotype, irre- toderm and endoderm are derived from mutant cells. spective of the degree of chimerism. The plotted regression lines Even chimeras with apparently 100% wild-type cells in are for combined chimeric and nonchimeric embryos. The val- the epiblast derivatives show the mutant phenotype and ues in the regression equation (see legend to Fig. 4) are in A, lack PGCs. Similarly, the number of PGCs in chimeras (wild type) 2.165 = 1.876 + 0.0361 (8.0); (heterozygote) 1.631 = 1.205 + 0.0483 (8.8); B, (wild type) 2.364 = 1.976 + 0.0283 (13.7); with heterozygous embryos is not influenced by the de- (heterozygote) 2.155 = 1.775 + 0.0246 (15.4). gree of chimerism: Chimeras with only wild-type epi- GENES & DEVELOPMENT 431 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. −/− Table 3. Chimeras of R26.1 ES cells with Bmp4 embryos Percent chimerism (%) n Somites Allantois PGCs Morula aggregation 0 5 0–6 0 0 (C57BL/6 × CBA) <95 5 2–4 0 0 >95 3 0–4 0 0 Blastocyst injection 0 15 0–14 1 1? (129/SvEv × Black Swiss) ø75 9 0–10 1 0? This nonchimeric embryo had a severely abnormal headfold and a well elongated allantois. One dubious PGC was scored at the base of the allantois. This embryo was a normal looking 6/7 somite embryo with a well-developed allantois and was 75% chimeric. AP activity was virtually absent throughout the embryo, so no firm conclusion about the absence of PGCs can be drawn. blast cells have the smaller number of PGCs character- The availability of Bmp4 protein may also be regulated istic of heterozygous embryos. by the activity of proteases that cleave these binding proteins, for example proteases belonging to the astacin family that includes Bmp1 and tolloid (Cho and Blitz 1998; Mullins 1998). Model I: Extraembryonic ectoderm Bmp4 is Although the observations on PGCs are compatible the only signal with a morphogen gradient set up from the extraembry- The simplest model suggested by the data for the role of tm1 onic ectoderm, the rest of the Bmp4 phenotype is not. Bmp4 in regulating PGC formation is as follows: Bmp4 The presence and amount of yolk sac mesoderm and secreted by the extraembryonic ectoderm acts in a con- yolk sac vascularization vary in Bmp4 null embryos ac- centration dependent manner to regulate cell fate in the cording to the genetic background (Winnier et al. 1995), epiblast. Cells in the proximal epiblast that are nearest but the allantois fails to develop irrespective of the ge- to the extraembryonic ectoderm receive the highest netic background, and is also absent in chimeras with Bmp4 signal. Among these cells a proportion, <50%, be- wild-type ES cells, indicating that extraembryonic ecto- come precursors of both PGCs and part of the allantoic derm Bmp4 is an absolute requirement for allantois for- population (and other extraembryonic derivatives). Cells mation. The presence of a normal allantois in heterozy- more distant from the extraembryonic ectoderm receive gotes suggests that a lower threshold Bmp4 concentra- a lower Bmp4 signal and will contribute to all types of tion than that required for PGC formation allows the extraembryonic mesoderm, including allantois, but do development of an allantois. This is supported by lineage not contribute to PGCs. Only a few descendants of a analysis that has shown that the allantois is derived not PGC precursor in the epiblast actually become PGCs at only from the most proximal epiblast, but also from epi- the time of allocation (an average of 2.6 descendants after blast cells further from the junction with the extraem- 3.7 generations from an E6 precursor and 1.5 descendants bryonic ectoderm where the Bmp4 concentration would after 1.6 generations from an E6.5 precursor; Lawson and be expected to be lower (Lawson and Pedersen 1992; Hage 1994). The cell mingling that follows cell division Lawson and Hage 1994). In the heterozygotes, fewer epi- in the epiblast (Lawson et al. 1991; Gardner and Cock- blast cells would be exposed to this lower concentration croft 1998) could ensure that only some descendants re- than in the wild type, and the allantois would be ex- main close enough to the source of Bmp4 to receive suf- pected to be smaller, or the onset of its formation would ficient signal for PGC formation. Alternatively, the sig- be delayed. We have found no evidence of this. nal gradient could take time to establish. In either case, the critical concentration would only be achieved Model II: Two signals are required to generate PGCs shortly before PGC allocation. and allantois The precise local level of active Bmp4 protein and the time during which epiblast cells are exposed to it will As noted above, there is an inconsistency between the depend on multiple factors, for example the level of pro- apparently normal allantois phenotype of Bmp4 hetero- teins that can bind Bmp4 and prevent its interaction zygotes and the simple model of Bmp4 acting as a mor- with receptors such as BmpRII and BmpR1A (Alk3) pres- phogen and specifying PGCs and allantois at different ent in the epiblast (Mishina et al. 1995; Roelen et al. threshold concentrations. One explanation for this could 1997). The genes encoding the antagonists cerberus-like be that the allantois cells that are closely lineage related (mCer-1) and follistatin are first expressed respectively to the PGCs, that is, those that are descended from the in the anterior visceral endoderm (Belo et al. 1997; Biben same precursors most proximal in the epiblast, and clos- et al. 1998; Shawlot et al. 1998) and posterior streak (Al- est to the source of Bmp4, are crucial for initiating the bano et al. 1994; Feijen et al. 1994) early in gastrulation. process of allantoic bud formation. Therefore, we suggest 432 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells that the highest Bmp4 concentration is required not to to speculate that the response to the second signal oper- specify PGCs as in Model I, but rather to specify a group ates in favor of generating enough cells to initiate an of cells whose descendants, after traversing the streak, allantois, a structure that is absolutely required for the will either become the putative allantois initiator cells development of a placental mammal, and leaving the or PGCs (Fig. 9A,B). The size of this population will be remainder of the population available for allocation to related to both the number of cells in the most proximal the PGC lineage. The number of PGCs finally allocated epiblast and the strength and duration of the extraem- will therefore depend on the size of the precursor pool bryonic Bmp4 signal to which they are exposed. and that proportion of it directed into forming an allan- After the precursor population has been established, tois. All current findings on the PGCs are compatible the cells must be directed into either the allantois ini- with this model, that is, (1) the presence of a normal tiator pool or into the PGC lineage. This is most likely to allantois, but reduced numbers of PGCs in the heterozy- be in response to a second local signal, either before or gotes, (2) the absence of PGCs, but a normal allantois in after the cells have traversed the primitive streak (Fig. a small minority of heterozygotes on the (C57BL/ 9C,D). We currently favor the second scenario because 6 × CBA) background, (3) differences in the size of the previous clonal analysis suggests that the time of PGC PGC founding population between wild type (C57BL/ allocation is at ~ E7.2, when the PGCs first become iden- 6 × CBA) and (129/SvEv × Black Swiss), and (4) delayed tifiable in a cluster at the base of the incipient allantois. appearance of PGCs in heterozygotes (Fig. 2) together Allocation is said to have occurred when cells no longer with the smaller size of the region in which PGCs are enter or leave the population (McLaren 1976), that is, the first identifiable (Fig. 7A,B). The two-signal model does population has become lineage restricted and self-per- not require that the extraembryonic ectoderm Bmp4 is petuating. Allocation is unlikely to occur in the epiblast functional at the time of PGC allocation. because, on average, 1.6 cell cycles of 6.8 hr in the early If this two-signal model is correct, a prediction is that streak embryo, and 3.7 cycles in the prestreak embryo, embryos bearing a mutation in a gene that affects allan- intervene before PGC lineage restriction (Lawson and toic phenotype, but that allows the development of an Hage 1994). abnormal allantois, may have PGCs, but that pheno- The precise nature and location of the second signal types specifically lacking an allantois will not. In sup- are unknown. However, several lines of evidence lead us port of this prediction, homozygous eed (Faust et al. 1995), T (Brachyury) (V. Wilson and R. Beddington, pers. comm.) and Otx2 mutants (K. Lawson, unpubl.) all show abnormal allantois development but have cells with an AP-staining pattern characteristic of PGCs. Nothing is known of the nature or source of the second signal. The possibility that it is Bmp4 produced by the early extra- embryonic mesoderm will, in the future, be tested by chimera analysis with homozygous Bmp4 mutant ES cells. Bmp4 may act indirectly and/or in synergy with other signaling factors In the model proposed above, we have assumed that Figure 9. Two-signal model of Bmp4 regulation of PGC allo- Bmp4 secreted by the extraembryonic ectoderm acts di- cation. (A) Preprimitive streak stage. Bmp4 is produced by the rectly on the epiblast. However, we cannot at this time extraembryonic ectoderm (xe) adjacent to the proximal epiblast exclude the possibility that Bmp4 acts indirectly, for ex- (ep) and a gradient (blue arrows) is set up to which the proximal ample by regulating the production of another signaling epiblast cells respond and become directed toward an allantois molecule(s) by the extraembryonic ectoderm and/or en- initiator/PGC fate (s). (A) Anterior; (P) posterior; (Pr) proximal; doderm. It is also possible that Bmp4 acts in synergy (D) distal. (B) Early primitive streak stage. Bmp4 continues to be with another factor(s) made by the extraembryonic ecto- produced by the extraembryonic ectoderm. The gradient can be derm or epiblast and that PGC precursor fate, is deter- steepened by the presence of the extracellular antagonists mCer-1 (red) anteriorly and follistatin (green) posteriorly. These mined by a combination of factors. In the future, these may also limit the temporal window within which Bmp4 can possibilities may be tested by incubating distal epiblast act in the epiblast. Along with other epiblast cells, the allantois in culture with different combinations of extraembry- initiator/PGC progenitors divide with a generation time of 6.5– onic tissues or signaling factors and assaying for the ap- 7.0 hr, and the progeny align toward and begin to move through pearance of PGCs in vitro. Moreover, if Bmp4 acts in the posterior streak. (C) Midstreak stage. Part of the newly synergy with other obligatory factors to specify PGC cell formed extraembryonic mesoderm (xm) is derived from the fate, it is possible that null mutants in genes encoding most proximal epiblast and is specified to respond to the puta- these factors will have a complete deficiency, or reduced tive second signal (black arrows). This coincides with the onset number, of PGCs. In contrast, embryos lacking genes of Bmp4 expression in the extraembryonic mesoderm. (D) Mid- that antagonize Bmp4 function might be expected to streak/late streak stage. By ~ E7.2, precursors have separated into allantois initiator (m) and PGC (d) lineages. have more PGCs and extraembryonic tissues. In this re- GENES & DEVELOPMENT 433 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. will generate a fusion transcript between 58 UTR of Bmp4 and gard, it would be interesting to assay for PGC number in lacZ. Note that this construction was deliberately designed to null mutants of Smad2 that have extraembryonic meso- maintain all potential regulatory elements within the Bmp4 derm but little embryonic mesoderm (Waldrip et al. locus, including introns. 1998). Electroporation, selection, and identification of targeted Model III: Bmp4 controls growth and cell movement ES cells An alternative possibility to the above models is that TL1 ES cells were electroporated with 150 μg A total of 19 × 10 Bmp4, rather than regulating PGC/allantois precursor of NotI-digested targeting vector DNA and subjected to positive cell fate, instead controls the growth of proximal epiblast and negative selection (Winnier et al. 1995). DNA from doubly cells and their translocation toward and through the pos- resistant clones was digested with SpeI for Southern blot analy- terior streak into the extraembryonic region. According sis using the 500-bp 58 external BsmI–BamHI probe (Fig. 5A,B) to this model, epiblast precursors of the PGCs and their and internal lacZ and neo probes (data not shown). One cor- rectly targeted line, 12C, in which the 38 homologous recombi- descendants in Bmp4 null embryos may translocate ab- nation occurred within the intron between coding exons 3 and normally from the proximal epiblast into the anterior 4, was injected into C57BL/6NHsd (Harlan Sprague Dawley) part of the streak. If already fully specified, they would blastocysts; resulting male chimeras were mated with outbred then become PGCs ectopically: These have not been lacZneo Black Swiss (Taconic) females. F1 Bmp4 heterozygotes found. If specification and allocation normally occur af- lacZneo were serially backcrossed onto Black Swiss. Bmp4 hetero- ter traversing the streak, the originally proximal cells zygotes were routinely identified by PCR analysis for the neo could be influenced by signals in the anterior part of the (Dunn et al. 1997) or lacZ sequences (see below) within the streak and contribute to the embryonic mesoderm being targeted allele. formed at that time. We cannot exclude this possibility because, although development anterior to the node can be relatively normal in Bmp4 null embryos, interpreta- Chimera generation and retrospective genotyping tion is confounded by the general overall reduction in Injection chimeras Injection chimeras were generated as de- growth that becomes apparent at the onset of gastrula- scribed (Bradley 1987; Hogan et al. 1994). Blastocysts from tion in the mutants. One way of testing this model in the tm1 Bmp4 /+ (129/SvEv × Black Swiss) intercrossings were in- future would be clonal analysis of the proximal epiblast jected with 12–15 ROSA26.1 (R26.1) ES cells (kindly provided in the mutants. by Elizabeth Robertson; Varlet et al. 1997). Following transfer, the embryos were recovered between E8.5 and 9.5, fixed, and processed for double b-gal and AP staining. The genotype of the Materials and methods host blastocyst was determined retrospectively by PCR analysis of enzymatically isolated yolk sac endoderm (Hogan et al. 1994). Mouse strains Potentially contaminating mesoderm was monitored in the en- tm1blh The following genetic backgrounds for the Bmp4 null mu- doderm DNA preparations by PCR analysis for the lacZ gene tation (Winnier et al. 1995) were used in the present study. with the following primer sequences: lacZ 1, 58-TCTGCT- tm1 C57BL/6–Bmp4 males (Dunn et al. 1997) were mated with TCAATCAGCGTGCC-38 and lacZ 2, 58-GCCGTCTGAATTT- (C57BL/6 × CBA) females. The progeny were intercrossed for GACCTGA-38. F1 one generation and the line maintained by backcrossing hetero- zygous males to (C57BL/6 × CBA) females. This line was used Aggregation chimeras Aggregation of eight-cell stage embryos F1 for PGC analysis and for morula aggregation chimeras. For gen- with ES cells was basically as described (Nagy and Rossant tm1 erating embryos for blastocyst injection, the Bmp4 mutation 1993). Briefly, R26.1 ES cells were cultured on mouse embry- was maintained on a (129/SvEv × Black Swiss) background by onic fibroblast feeder cells. For aggregation, ES cells were tryp- lacZneo intercrossing. Bmp4 was maintained on the (129/ sinized and the fibroblasts were allowed to reattach to the tissue SvEv × Black Swiss) background and embryos for PGC and ex- culture plastic for 30 min. Subsequently, the ES cells were pression pattern analysis obtained from heterozygous matings transferred to a smaller volume for 2 hr to form aggregation and matings with ICR females. clumps. Eight-cell stage embryos were collected at E2.5 from tm1 Bmp4 /+ matings on the (C57BL/6 × CBA) background. The zona pellucida was removed with acid Tyrode’s solution and single embryos aggregated with clumps of 10–15 ES cells in Construction of the lacZ knock-in targeting vector aggregation wells in 20 μl droplets of M16 medium and cultured lacZneo Detailed information on the construction of the Bmp4 overnightat37°Cin5%CO (Zwijsen et al. 1999). Embryos targeting vector and Cre-mediated excision of the neo cassette were transferred the following day into E2.5 pseudopregnant can be found at http://www.mc.vanderbilt.edu/vumcdept/cell- (C57BL/6 × CBA) recipients. The embryos were recovered at F1 bio/hogan.html. Briefly, most of exon 3, from nucleotides 6807– nominal E8.5 and further processed and genotyped as described 7178 (Kurihara et al. 1993), including the translation initiation above. Very retarded embryos were genotyped on the parietal ATG and sequences encoding amino acids 29–124 of the pro endoderm of Reichert’s membrane. region, is entirely replaced with a b-gal cassette from pPD1.27, which encodes bacterial b-gal with SV40 nuclear localization and polyadenylation signals (Fire et al. 1990) (Fig. 5A). The cas- Developmental index sette also contains a loxP-site-flanked positive selection MC1- neo cassette (a gift from Steve O’Gorman, Salk Institute, La The stage of embryo development as judged by a score of mor- lacZneo Jolla, CA). After recombination, the targeted Bmp4 allele phological features (modified from Brown 1990), or the number 434 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells of somite pairs, was very variable between and within litters of 8100) for 1–2 hr. The plastic was then polymerized at 4°C. Serial the same nominal gestational age. Morphological score was lin- sections cut at 7 μm were stained for AP activity with ASMX/ early correlated with somite number up to 20 somite pairs, and Fast Red TR (Sigma) for 30–90 min according to the manufac- the relationship was the same in wild type and heterozygotes on turer’s instructions. The sections were counterstained with the (C57BL/6 × CBA) background (data not shown). Somite Mayer’s haemalum and mounted in Aquamount (Gurr). PGCs pairs are laid down on average every 90 min during normal were counted on the basis of the densely staining AP-positive development, at least up to 30 somites (Tam 1981). Somite cytoplasmic spot, using a 25× objective lens (Ginsburg et al. number was therefore used as a measure of the developmental 1990; Lawson and Hage 1994). age of individual embryos. Combined b-gal and AP staining Embryos were fixed in 4% paraformaldehyde for 2 hr as above and stained for b-galat37°C lacZneo for 4 hr (chimeras with R26.1 ES cells) or 8 hr (Bmp4 b-Gal staining embryos). They were then dehydrated, embedded in Technovit lacZneo Bmp4 embryos and decidua were fixed in 4% parafor- 8100, sectioned, and stained for AP activity as above without maldehyde in PBS at 4°C for 30 min with rocking, washed twice counterstaining. for 10 min in PBS at 4°C, transferred into freshly prepared X-gal solution and stained overnight (or longer) at 37°C (Hogan et al. 1994). After rinsing with PBS, embryos were post-fixed in 4% Statistics paraformaldehyde. Some embryos were cleared in 80% glycerol Regression analysis and comparison of regression lines were in PBS. performed as described, using the F test to compare variances For histological analysis, stained decidua were dehydrated (Snedecor and Cochran 1967). into 100% isopropanol, washed twice with 1:1 isopropanol/par- affin wax, embedded in wax, and 7-μm sections lightly counter- stained with eosin. Acknowledgments We thank Drs. Jacqueline Deschamps, David Greenstein, An- Detecting and counting PGCs thony Mahowald, Christine Mummery, David Threadgill, and Lilianna Solnica-Krezel for discussions and critical comments Whole mounts Embryos between E7.5 and E9.5 were dissected on the manuscript; Dr. Lucy Liaw and Linda Hargett for help in from the decidua and Reichert’s membrane reflected. The yolk lacZneo establishing the Bmp4 line; Marie-Jose ´ Goumans, Marga sac was separated from the ectoplacental cone of embryos that van Rooijen, and Dr. Elizabeth Robertson for invaluable advice were in the process of, or had completed, turning. Both yolk sac on making chimeras. We also thank Angela D. Land-Dedrick for and amnion were reflected but left attached to the embryo. The assistance in manuscript preparation and Dominic Doyle for the embryos were fixed in 4% paraformaldehyde in PBS for 2 hr at illustration of Figure 9. N.R.D. was supported by a grant from 4°C. The embryos were washed three times in PBS, during the National Institutes of Health (HD 28955) to B.L.M.H., who which time they were further dissected according to size. Em- is an Investigator of the Howard Hughes Medical Institute. bryos up to ~ 6S (early hind gut) were left intact; embryos be- The publication costs of this article were defrayed in part by tween ~ 7S and ~ 11S were transected at the level of S4/5 into an payment of page charges. This article must therefore be hereby anterior and posterior portion; the yolk sac of still older em- marked ‘advertisement’ in accordance with 18 USC section bryos was trimmed and the embryos then transected at the level 1734 solely to indicate this fact. of the anterior intestinal portal or, when forelimb buds were present, at the level of S10. The posterior portion of these older embryos was then split longitudinally along the line of the dor- References sal aorta to yield two curved strips, one consisted of the hindgut, allantois, intermediate, and lateral plate mesoderm, and re- Albano, R.M., R. Arkell, R.S.P. Beddington, and J.C. 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Tis- sue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse em- bryogenesis but is not required for migration of primordial 436 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 is required for the generation of primordial germ cells in the mouse embryo Kirstie A. Lawson, N. Ray Dunn, Bernard A.J. Roelen, et al. Genes Dev. 1999, 13: This article cites 30 articles, 14 of which can be accessed free at: References http://genesdev.cshlp.org/content/13/4/424.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Cold Spring Harbor Laboratory Press http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes & Development Unpaywall

Bmp4 is required for the generation of primordial germ cells in the mouse embryo

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Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 is required for the generation of primordial germ cells in the mouse embryo 1,4 3 1 1 2 Kirstie A. Lawson, N. Ray Dunn, Bernard A.J. Roelen, Laura M. Zeinstra, Angela M. Davis, 3 1 2,3,4 Christopher V.E. Wright, Jeroen P.W.F.M. Korving, and Brigid L.M. Hogan 1 2 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands; Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2175 USA In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of b-galactosidase activity in lacZneo Bmp4 embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage. [Key Words: Primordial germ cells; allantois; mouse embryo; Bmp4; extraembryonic ectoderm; chimera] Received November 23, 1998; revised version accepted January 7, 1999. Before gastrulation, the mouse embryo consists of three lie scattered in a ring that extends up to three cell diam- distinct cell lineages which were established in the blas- eters from the junction with the extraembryonic ecto- tocyst during the peri-implantation period, that is, epi- derm (Lawson and Hage 1994). Early in gastrulation, they blast, extraembryonic endoderm, and trophectoderm. converge toward the primitive streak in the posterior of The epiblast, from which the entire fetus will form, as the embryo and translocate through it. Allocation to the well as the extraembryonic mesoderm and amnion ecto- germ cell lineage is thought to occur in ~ 45 cells around derm, is a cup-shaped epithelium apposed on its open E7.2, after the precursors have passed through the streak end to the extraembryonic ectoderm, a trophectoderm and have come to reside in the extraembryonic meso- derivative. Both epiblast and extraembryonic ectoderm derm (Lawson and Hage 1994). This is about the time are covered by visceral endoderm, which is part of the when the putative PGCs can first be identified morpho- extraembryonic endoderm lineage (Hogan et al. 1994). logically in a cluster posterior to the primitive streak in The primordial germ cells (PGCs) of the mouse em- a position that will later become the base of the allantois bryo are derived from part of the population of epiblast (Ginsburg et al. 1990). PGCs stain strongly in a charac- teristic pattern for alkaline phosphatase (AP) activity cells that will give rise mainly to the extraembryonic mesoderm. Precursors of the PGCs are located before (Chiquoine 1954), which by this stage is due to tissue gastrulation in the extreme proximal region of the epi- nonspecific AP (Hahnel et al. 1990; MacGregor et al. blast adjacent to the extraembryonic ectoderm, and have 1995). The PGCs continue to express AP during their descendants not only in the germ line, but also in extra- proliferation in the developing hindgut and migration embryonic structures, that is, the allantois, blood is- into the genital ridges (for review, see Buehr 1997). lands, and yolk sac mesoderm, as well as both layers of Transplantation studies have shown that genetically the amnion. At embryonic day (E) 6.0, these precursors marked distal epiblast cells from pre- and early-primitive streak-stage embryos, which would normally contribute to neuroectoderm and never to the PGCs, can give rise to Corresponding authors. PGCs and extraembryonic mesoderm when grafted to E-MAIL brigid.hogan@mcmail.vanderbilt.edu; FAX (615) 343-2033. E-MAIL lawson@niob.knaw.nl; FAX 31 (30) 2516464. the proximal epiblast (Tam and Zhou 1996). These re- 424 GENES & DEVELOPMENT 13:424–436 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells sults raise the possibility that PGC precursors are in- were therefore assayed for the presence of PGCs by AP duced by extracellular factors and/or cell interactions staining. present locally at the junction between the extraembry- onic ectoderm and epiblast. tm1 Dosage effect of Bmp4 on PGC number Candidate genes encoding putative germ cell precursor tm1 inducing factors are predicted to be expressed in the Comparison of littermates of Bmp4 /+ intercrosses be- mouse embryo before and during gastrulation. One such tween E7.2 and E7.75 showed firstly that homozygous factor is Bone Morphogenetic Protein 4 (Bmp4), a mem- null mutants contained no PGCs [12 embryos from 7 ber of the TGFb superfamily of intercellular signaling (C57BL/6 × CBA) matings], and secondly that the inci- proteins (Hogan 1996; Waldrip et al. 1998). Most mouse dence of heterozygous embryos with recognizable PGCs embryos homozygous for a null mutation in Bmp4 die lagged behind that of the wild type until after the head- around gastrulation (~ E6.5) (Winnier et al. 1995). On fold stage on both the (C57BL/6 × CBA) and (129/ some genetic backgrounds, however, a proportion of the SvEv × Black Swiss) backgrounds (Fig. 2). More detailed mutant embryos survive until the early somite stage and quantitative analysis at E7.5 is not informative because show severe defects, particularly in the extraembryonic PGCs are still emerging from the cluster of AP-positive mesoderm (Winnier et al. 1995). In this paper, we exploit cells (Ginsburg et al. 1990), and the population is not yet this late phenotype to show that PGC formation abso- expanding exponentially (Lawson and Hage 1994). lutely requires Bmp4 signaling. In addition, the size of The whole-mount AP staining technique described the founding population of PGCs is significantly reduced here allows the quantitation of PGCs in situ in the em- in heterozygous mutant embryos. By using a Bmp4–lacZ bryo at more advanced stages. For example, as shown in reporter allele, we have definitively localized Bmp4 ex- Figure 3, PGCs are clearly present in the hindgut of wild- pression before gastrulation in the extraembryonic ecto- type and heterozygous embryos (Fig. 3A–C; see also Fig. derm and in mid- to late- primitive streak stage embryos 1E), but are completely absent from the homozygous in the extraembryonic mesoderm. Thus, Bmp4 is ex- mutants (Fig. 3D; see also Fig. 1F–H). This absence was pressed at the right time and in the right place to play a true for both genetic backgrounds (C57BL/6 × CBA: 29 role both in the quantitative induction of PGC precur- homozygous mutant embryos from 23 females; 129/ sors in the proximal epiblast and in their allocation to SvEv × Black Swiss: 8 homozygous mutant embryos the germ cell lineage in the extraembryonic mesoderm. from 5 females) and at all stages examined. The most Furthermore, by analyzing genetic chimeras, we have advanced mutant (C57BL/6 × CBA) embryo at E9.5 had clearly established a role for Bmp4 in the induction of 17 somites, and one (129/SvEv × Black Swiss) embryo PGC precursors and demonstrate for the first time that a was fully turned with 23 somites. secreted signal from the extraembryonic ectoderm is re- Heterozygous embryos, although indistinguishable quired for the normal development of the epiblast. from their wild-type littermates in terms of overall size and morphological features, including the allantois, had reduced numbers of PGCs on both genetic backgrounds Results (Fig. 3, cf. A with C; for the one exception concerning the allantois, see footnote to Fig. 4). In addition, PGCs were tm1 Phenotypic abnormalities in Bmp4 homozygous absent in 9% of the heterozygous (C57BL/6 × CBA) em- null mutants bryos (Fig. 4A). Although the heterozygous embryos had On both the (129/SvEv × Black Swiss) and (C57BL/ fewer PGCs, the regional distribution of PGCs in hetero- tm1 6 × CBA) genetic backgrounds, many Bmp4 homozy- zygous and wild-type littermates did not differ, with gous embryos develop up to and beyond the early somite PGCs spreading from the ventral hindgut through the stage. An example of a 20 somite (S) stage homozygous dorsal mesentery and into the genital ridges by E9.5. embryo is shown in Figure 1B. Among the late surviving To determine at which stage the difference in the size homozygous mutants, several consistent abnormalities of the PGC population arose, PGC number estimated on are observed. First, they are developmentally delayed in whole mounts was plotted against somite number. The comparison to their wild-type and heterozygous litter- regression line of log PGC number on somite number mates (Fig. 1A,B). Significantly for this study, all com- fitted to all values greater than zero for the heterozygotes was parallel to that for the wild type, but had reduced pletely lack an allantois (Fig. 1B,D), and many show se- vere posterior defects, including disorganized posterior elevation (P < 0.001) (Fig. 4A,B). The parallel regression ectoderm (Fig. 1G,H), overgrowth and endothelialization lines indicate that the rate of expansion of the PGC of the somatopleure (Fig. 1, cf. E with F and G), with population is the same in wild-type and heterozygous extension of endothelial cells into the amnion in the embryos. Assuming an average of one somite pair formed most severe mutant phenotypes (Fig. 1H), and small and every 90 min (Tam 1981), the slope gives a population poorly vascularized yolk sacs. doubling time of 15.8 hr, which is consistent with pre- The absence of an allantois in all homozygous null vious data (Tam and Snow 1981; Lawson and Hage 1994). Bmp4 mutants strongly suggested that they would also In contrast, the difference in elevation of the two regres- have a deficiency of PGCs, because the precursors of the sion lines suggests that the size of the founding popula- two cell types reside in similar positions in the proximal tion of PGCs is smaller in the heterozygotes. Wild-type epiblast before gastrulation. Embryos of different stages embryos on the (C57BL/6 × CBA) background have a GENES & DEVELOPMENT 425 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. tm1 Figure 1. Phenotypes of advanced Bmp4 (129/SvEv × Black tm1 Swiss) mutant embryos. (A) Bmp4 /+ embryo at the early forelimb bud stage showing wild-type morphology. (B) tm1 tm1 Bmp4 /Bmp4 , littermate of A showing delayed develop- ment, incomplete turning, irregular somites and kinked neural tube, uncharacteristic looping of the tail to the left, and absence of allantois. (C) Posterior of embryo in A (boxed region) with allantois (a). (D) No allantois in homozygous mutant (arrow). The broken line in B marks the level of dissection for D.(E–H) tm1 Sections of wild-type and homozygous null Bmp4 (129/ SvEv × Black Swiss) embryos showing posterior phenotype. AP and haemalum staining. (E) Wild type. Transverse section (TS) of posterior region of an E9.5 embryo with 27 somites. The umbilical vein (u) demarcates the junction between somato- pleure (sop) and amnion (am). PGCs (arrowhead) are migrating from the hindgut (hg) into the genital ridges (gr). (F)TSofthe posterior region of a −/− sibling to the embryo in E. This embryo had 23 somites and an external morphology similar to the em- bryo in B. There was no external allantois, but the region be- tween the amnion and the somatopleure was heavily endothe- lialized (e). (G) TS of the posterior region of another E9.5 −/− sibling with 14 somites and more severe posterior defects. The endothelialized somatopleure has reflected dorsally, forming a posterior pocket that becomes continuous with the amnion. A dorsal extension (n8) of the caudally disorganized neurectoderm (n) surrounded by surface ectoderm (se) is contained within the pocket. (H) Sagittal section of an E8.5 −/− embryo showing a severe mutant phenotype. The embryonic portion contains con- voluted ectoderm (ec) and limited mesoderm extending ros- trally from the primitive streak (ps). The amnion is normal ros- trally, but is filled caudally with mesoderm (arrow), which is continuous with the primitive streak. In addition, there is an accumulation of AP-positive amnion ectoderm (*). (A) Anterior; (da) dorsal aorta; (n) neural tube; (P) posterior; (ys) visceral yolk sac. Scale bars in C for A–D, 200 μm; in E for E–H, 200 μm. mean founding population of 45 (Lawson and Hage The extrapolation of the regression line of the wild 1994); extrapolation of the regression line for the wild type (C57BL/6 × CBA) in Figure 4A reaches the expected type to this value, and comparison with that for the het- founding population size of 45 at −1.2S, instead of the erozygotes at the same stage, gives a mean PGC founding expected allocation time equivalent to approximately population in the heterozygotes of 17 (a 62% reduction). −8S (12 hr before the 0S stage). This discrepancy suggests The slopes of the regression lines of PGCs numbers on that PGC number is consistently underestimated in the (129/SvEv × Black Swiss) (Fig. 4B) and (C57BL/ whole mounts, but it does not affect the relative differ- 6 × CBA) backgrounds were indistinguishable. However, ence in PGC number between wild type and heterozy- the elevation of the lines for both wild type and hetero- gotes, nor the inference that the size of the founding zygotes was higher in the (129/SvEv × Black Swiss) em- population is reduced by >50% in the heterozygotes. If bryos compared with their (C57BL/6 × CBA) counter- the lower number of PGCs in the heterozygotes were due parts (P < 0.001). This is consistent with a mean found- solely to a delay in PGC allocation or to delayed onset of ing population of 66 in the wild type and 30 in the PGC proliferation, rather than to a smaller founding heterozygote (129/SvEv × Black Swiss) (a 55% reduc- population, the length of the delay implied by the differ- tion). ence in elevation of the regression lines would be 22 hr 426 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells Figure 2. Incidence of wild-type and heterozygous embryos tm1 with recognizable PGCs at E7.2–E7.75 from Bmp4 /+ (C57BL/6 × CBA) intercrosses (7 females, 40 embryos) and lacZneo ICR × Bmp4 /+ matings (4 females, 38 embryos) examined in whole mount. (Open columns) Wild type; (hatched columns) heterozygotes; sample size in parentheses. The same trend was found in both groups (not shown): The pooled data show that the proportion of embryos with PGCs was smaller in the het- erozygotes in combined stages up to, and including, the head- fold (HF) stage (x test: P < 0.05). (ES/MS) Early streak/mid- streak; (LS) late streak; (NP) neural plate; (HF) headfold; (S) so- mite. (14.7 somite equivalents). The data do not support this interpretation. An alternative possibility, which cannot be distinguished from a direct effect on the number of cells allocated, is that more than half of the PGC founders in the heterozygotes die before the first division Figure 4. Linear regression analysis of PGC number (counted after allocation. in whole mount) vs. somite number in embryos from In summary, whereas one active allele of Bmp4 is suf- tm1 tm1 Bmp4 /+ intercrosses. (A) Bmp4 (C57BL/6 × CBA). (B) ficient for normal maintenance, proliferation, and the lacZneo Bmp4 (129/SvEv × Black Swiss). (s, broken line) Wild initiation of migration of the PGCs, the size of the type; (d, solid line) heterozygote; (n) homozygous null. The founding population, which is normally allocated at values in the regression equation, Y = a + bX, for log PGC (Y)on about E7.2 at the late midstreak/late streak stage, is somite (X) number at the mean values of X and Y with each set Bmp4 dosage dependent. of data were in A, wild type, 2.124 = 1.684 + 0.0286 (15.4); het- erozygote (PGC values >0), 1.647 = 1.268 + 0.0275 (13.8); B, wild type, 2.305 = 1.878 + 0.0288 (14.8); heterozygote, 2.089 = 1.541 + 0.0298 (18.4). Identification of genotype in B was by b-gal staining and phenotype. In A, the 25/26S heterozygote with 23 PGCs resembled an advanced homozygous null embryo (as in Fig. 1B) and completely lacked an allantois. Temporal and spatial pattern of Bmp4 expression during early mouse development Because Bmp4 is clearly important for PGC formation, it is essential to know its precise temporal and spatial ex- pression before and after gastrulation. To detect Bmp4 expression at this time with high sensitivity and single cell resolution, we used homologous recombination in ES cells to replace the first protein coding exon of the Figure 3. PGCs in posterior (hindgut) pieces from E8.5 sibling Bmp4 gene with a reporter cassette encoding b-galacto- tm1 embryos from a Bmp4 /+ (C57BL/6 × CBA) intercross mat- sidase (b-gal) with an amino-terminal nuclear localiza- ing. Alkaline phosphatase staining, dorsal view. (A) Wild type, tion signal (Fig. 5). Embryos homozygous for the 15S embryo. (B) High power of part of A showing individual lacZneo Bmp4 mutation on the (129/SvEv × Black Swiss) PGCs (arrow) in the hindgut. (C) Heterozygote, 15S embryo. tm1 background have the same phenotype as Bmp4 ho- There are fewer PGCs compared with the wild-type sibling in A. mozygotes (Fig. 6M,N). Moreover, removing the neo cas- (D) Homozygous null, 8S embryo. Although a hindgut is present sette has no effect on lacZ expression or mutant pheno- (hg), PGCs are entirely absent. Scale bars in A for A, C,and D, type (data not shown). 200 μm; in B, 100 μm. (+/+) Wild type; (+/−) heterozygote; (−/−) homozygous null. To determine the onset of Bmp4 expression in vivo, GENES & DEVELOPMENT 427 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. Figure 5. Targeted replacement of the Bmp4 gene with a lacZ reporter cassette. (A) Genomic organization of the wild-type and mutated al- leles and the structure of the targeting vector. Coding and noncoding exons are represented by solid and shaded rectangles, respectively. The lacZneo Bmp4 targeting vector contains 1.6 kb of 58 homology. The 6.1-kb 38 homology arm in- cludes the oligonucleotide-interrupted coding tm1blh exon 4 (open rectangle) from the Bmp4 tar- geting vector (Winnier et al. 1995) and is flanked by the herpes virus thymidine kinase cassette (HSV-tk) for negative selection (Soriano et al. 1991). Coding exon three is replaced with both lacZ and neo resistance cassettes; the arrow in- dicates the direction of neo transcription. loxP sites ( c) flank the neo cassette. The correctly recombined locus produces a fusion transcript between noncoding Bmp4 sequences and lacZ without disrupting the structure of neighboring introns. The 500-bp BamHI–BsmI fragment used as an external 58 probe is shown above the wild-type Bmp4 locus. In the 12C targeted ES cell line, recombination occurred in the intron between exons 3 and 4, as determined by the PCR strategy described in Winnier et al. (1995). (B) Southern blot analysis of progeny from a rep- lacZneo resentative backcross of the Bmp4 allele. By use of the 58 external probe and SpeI diges- tion, the wild-type and targeted loci generate 6.3 and 11.1 hybridizing bands, respectively. (B) BamHI; (Bs) BsmI; (C) ClaI; (E) EcoRI; (H) HindIII; (N) NotI; (P) PstI; (Sf) SfiI; (Sm) SmaI; (Sp) SpeI; (X) XbaI. (+/+) Wild type; (+/−) hetero- zygote. lacZneo Bmp4 heterozygous embryos were analyzed for tently larger in wild-type embryos than in heterozygotes b-gal activity from E3.5 onwards. Positive cells could not (Fig. 7A,B). be detected in intact blastocysts or in E4.5 embryos even Bmp4 produced by the extraembryonic ectoderm may after prolonged staining (data not shown). At E5.5, low be required for the induction of Bmp4 expression in the lacZneo levels of Bmp4 expression are first detected extraembryonic mesoderm derivatives of the proximal throughout the uncavitated extraembryonic ectoderm, epiblast. To explore this possibility, Bmp4 expression lacZneo including those cells that abut the epiblast (Fig. 6A). By was monitored in Bmp4 homozygous null em- ~ E6.0, just prior to overt streak formation, the highest bryos by b-gal staining. At the headfold stage, strong levels of lacZ expression become localized to the region b-gal activity is detected in the extraembryonic meso- of the extraembryonic ectoderm immediately adjacent to derm lining the exocoelom, as well as in cells accumu- the epiblast (Fig. 6B; see also Waldrip et al. 1998). As lating near the junction of the amnion with the posterior gastrulation begins, these b-gal-positive extraembryonic primitive streak, in the position normally occupied by cells are displaced proximally by the encroaching extra- the developing allantois (Fig. 6M,N). Bmp4 expression in embryonic mesoderm and subsequently contribute to the epiblast derivatives is therefore independent of Bmp4 lacZneo the chorion (Fig. 6C–F). Bmp4 expression is de- expression in the extraembryonic ectoderm. tected in newly formed extraembryonic mesoderm at the midstreak stage, as the exocoelom begins to form (Fig. Chimera analysis indicates a role for extraembryonic 6E). It is then expressed with increasing intensity in the ectoderm Bmp4 allantois and mesodermal components of the developing amnion, chorion, and visceral yolk sac (Fig. 6G–L). No The temporal and spatial expression pattern described expression is seen in the primitive streak at this time. above is compatible with a role in PGC allocation for Double staining for b-gal and AP activity shows that Bmp4 secreted by either the extraembryonic ectoderm, Bmp4 is expressed in cells in the vicinity of the PGCs, the extraembryonic mesoderm, or both. To distinguish but is clearly excluded from them (Fig. 7A,C,D). The area between these possibilities, we have exploited the fact posterior to the streak at the base of the initiating allan- that ES cells almost exclusively colonize the epiblast toic bud in which PGCs can be first identified is consis- when injected into blastocysts or aggregated with moru- 428 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells lacZneo Figure 6. Bmp4 expression in the early mouse embryo. (A) An E5.5 embryo viewed un- der Nomarski optics. Low levels of b-gal activ- ity are first detected throughout the uncavi- tated extraembryonic ectoderm (xe). (Arrow- head) Junction between embryonic and extraembryonic regions. (B) At the onset of gas- lacZneo trulation (early streak, ES), Bmp4 expres- sion continues in the extraembryonic ecto- derm, in a ring that abuts the epiblast (ep). (C,D) lacZneo As gastrulation proceeds, Bmp4 expres- sion within the extraembryonic ectoderm per- sists and is particularly evident between the mid-streak (MS) to late-streak (LS) stages within the posterior amniotic fold (paf). (E) Sag- ittal section through a MS/LS embryo. Low lev- els of b-gal activity within the extraembryonic mesoderm (arrow) are first detected at this stage, as the exocoelom (exo) begins to form. (F) Late-streak (LS) stage embryo. (G–L) Bmp4 ex- pression during allantois development. lacZ ex- pression is detected in the posterior accumula- tion of extraembryonic mesoderm that pre- cedes overt allantois formation (asterisk in G) and within the allantoic bud (ab) and allantois (a) as it extends through the exocoelomic cav- ity. Expression also persists in the extraembry- onic mesodermal components of the amnion (am), yolk sac (ysm), and chorion (cm) that line lacZneo the exocoelom. (M,N) Bmp4 homozygous null embryo at the headfold (HF) stage. (M) Whole mount, lateral view. (N) Parasagittal sec- tion of M. Strong b-gal activity is detected in the amnion and yolk sac mesoderm, as well as in the accumulation of extraembryonic mesoderm (*) posterior to the primitive streak (ps). Anterior (A) is to the left in B–N. (c) Chorion; (xn) extraembryonic endoderm; (ES) early streak; (OB) no bud; (EB) early bud; (NP) neural plate; (LNP) late neural plate. Scale bars in A, 100 μm; in B for B–J, 200 μm; in K for K and L, 100 μm; in M for M and N, 200 μm. lae (Beddington and Robertson 1989). In contrast, the re- expected from the fate map of the epiblast (Lawson et al. cipient embryo forms the trophectoderm and extraem- 1991) if there had been incomplete cell mingling in the bryonic endoderm derivatives and can contribute to the epiblast prior to gastrulation. Chimerism in the PGCs epiblast. A chimera with 100% ES-derived epiblast (Fig. 7E) was well correlated with the roughly estimated would then have ES-derived extraembryonic mesoderm degree of posterior somatic chimerism in both wild-type and PGCs and recipient-derived extraembryonic ecto- embryos and heterozygotes (Table 2), indicating that derm. In the experiment here, ROSA 26.1 ES cells that there was no positive or negative selection for germ cell are genetically marked with a ubiquitously expressed fate on the basis of the genotype of the recipient embryo lacZ reporter gene were either injected into blastocysts or on ES cell origin. tm1 or aggregated with morulae from Bmp4 heterozygous The number of PGCs in the heterozygotes was unaf- intercrosses. The resulting embryos were recovered at fected by the size of the wild-type population in the epi- nominal E8.5, genotyped, and analyzed for degree of chi- blast-derived tissues (Fig. 8A,B): There was no indication merism and PGC number on histological sections. in chimeras on either genetic background of an increase Between 32% (C57BL/6 × CBA; n = 72) and 50% (129/ in PGC number above the nonchimeric level towards the SvEv × Black Swiss; n = 80) of the embryos recovered wild-type level, even in chimeras with no detectable het- were chimeric: The ES cell contribution tended to be erozygous contribution to the epiblast-derived tissues. stronger in the aggregation chimeras, with 39% of the The smaller size of the PGC population in heterozygotes chimeric embryos showing >95% chimerism in the epi- is therefore due to reduced Bmp4 from the extraembry- blast derived tissues (Table 1). Generally, chimerism was onic ectoderm, and cannot be compensated by Bmp4 fine grained throughout the embryos, but in 12/40 blas- from wild-type extraembryonic mesoderm. tocyst injection chimeras and 5/23 aggregation chime- Wild-type ES cells in combination with homozygous ras, the extraembryonic mesoderm, the posterior part of null embryos were unable to rescue the mutant pheno- the embryo, and sometimes the anterior surface ecto- type: Neither allantois nor PGCs were present even derm were distinctly less chimeric. This result could be when the epiblast-derived component of the conceptus GENES & DEVELOPMENT 429 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. Figure 7. (A–D) Sections of embryos from lacZneo ICR × Bmp4 /+ matings stained for b-gal and AP activ- ity. (A) Heterozygote, late streak (LS) stage. Sagittal section of posterior region at embryonic/extraembryonic junction. b-gal staining (arrowhead), representing Bmp4 expression, is present in mesothelial cells lining the exocoelom. Three AP-positive PGCs (arrow) (of a total of seven in this embryo) lie internal to the b-gal staining region and do not stain blue. (B) Wild type, late streak stage, sibling of embryo in A, sagittal section as in A. The cluster of 11 PGCs (arrow) at the base of the incipient allantois (arrowhead) is larger than in the heterozygote. There were 33 identifiable PGCs in this embryo. (C) Heterozygote, headfold (hf) stage. Transverse section at the level of the head- fold (hf) and base of the allantois (a). (Dark field) b-Gal staining appears pink. There is strong b-gal activity in the periphery of the allantois but not in its core. (D) High power, bright-field view of part of C. b-Gal staining peripherally at the base of the allantois, but not in the AP-positive PGCs (arrow) lying more tm1 centrally. (E,F) Sections of R26.1 ES « Bmp4 / tm1 +× Bmp4 /+ chimeras stained for b-gal and AP activity: Wild-type ES cells stain blue. (E) Hindgut of a 75% chimeric wild-type embryo showing b-gal-positive PGCs (arrow) de- rived from the ES cells and a recipient-derived, b-gal-negative, PGC (arrowhead). (F) Sagittal section of 4S stage >95% chi- meric homozygous null embryo on the (C57BL/6 × CBA) background. The epiblast derived cells are of wild-type, ES cell origin and have no detectable contribution from the mutant cells that are confined to the chorion ectoderm (c) and visceral yolk sac endoderm (ys). The phenotype is characteristically homozygous null with no allantois (arrowhead), no PGCs, small visceral yolk sac and defective yolk sac vascularization. AP staining in the embryonic ectoderm and chorionic ecto- derm is independent of phenotype (cf. with A and B). (am) Amnion; (c) chorion; (h) heart; (ps) primitive streak; (vee) vis- ceral extraembryonic endoderm. Scale bars in A–E, 50 μm; in F, 100 μm. contained no detectable mutant cells (>95% wild-type the epiblast set aside at about the time of implantation. ES cell contribution) (Table 3; Fig. 7F). Therefore, Bmp4 The earlier allocated extraembryonic cell lineages, that produced by the extraembryonic ectoderm is required by is, trophectoderm and primitive endoderm, contribute the epiblast to generate an allantois and PGCs, and can- no descendants to the fetus, but provide the tissues re- not be substituted by Bmp4 produced by wild-type ex- quired for implantation and nutrition of the conceptus traembryonic mesoderm. (for review, see Rossant 1986). Evidence is now emerging that, in addition to their support functions, these extra- embryonic lineages play more intimate roles in embry- Discussion onic development. For example, early events in anterior It has been long established that all the fetal lineages, neural patterning require specific gene expression in the both somatic and germ line, are derived exclusively from adjacent visceral embryonic endoderm, a derivative of tm1 tm1 Table 1. Chimeras of R26.1 ES cells with embryos from Bmp4 × Bmp4 matings Percentage chimerism Recipient Total Mean somite genotype embryos number (range) 0 ø25 >25–50 >50–75 >75–95 >95 Morula aggregation (C57BL/6 × CBA) Wild type 19 8.4 (0–15) 14 0 1 0 2 2 +/− 40 8.1 (0–15) 30 3 1 2 0 4 −/− 13 2.8 (0–6) 5 0 1 2 2 3 Blastocyst injection (129/SvEv × Black Swiss) Wild type 13 13.7 (0–21) 10 1 1 1 0 0 +/− 43 15.4 (0–26) 15 10 7 8 2 1 −/− 24 5.3 (0–14) 15 5 1 3 0 0 430 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells Table 2. Percent chimerism in PGCs related to the extent of posterior somatic chimerism Percent chimerism (posterior) Genotype ø25 >25–50 >50–75 >75–95 >95 Wild type 1.6 (1) 35.2 ± 0.3 (2) 73.6 (1) 72.0 ± 32.5 (2) 94.5 ± 4.9 (2) tm1 Bmp4 /+ 4.1 ± 6.7 (12) 20.7 ± 12.8 (14) 47.1 ± 33.1 (5) 83.4 ± 6.3 (2) 95.0 ± 4.9 (5) Chimerism in PGCs [mean ± S.D.(n)]. the primitive endoderm (for review, see Beddington and Models for the specification of PGCs and allantois Robertson 1998). The results reported here demonstrate formation in the mouse embryo that the initiation of both the germ line and the allantois In this paper we report three independent sets of obser- is dependent on a signal from the first established extra- vations that together suggest possible models in which embryonic lineage, the trophectoderm. Bmp4 produced by extraembryonic cells quantitatively regulates the fate of PGC precursors in the epiblast and the size of the founding population of PGCs in the em- bryo. These models underscore the importance of cell– cell interactions in the formation of the mammalian germ line (Tam and Zhou 1996), and open up the mo- lecular analysis of the signaling pathways and genes in- volved. The first set of observations is that mouse embryos with no functional Bmp4 gene completely lack both PGCs and an allantois, cell types that arise from precur- sors located before gastrulation in the proximal epiblast (Lawson and Hage 1994). In addition, heterozygous tm1 Bmp4 embryos have fewer PGCs than wild type, al- though the allantois appears normal. From the regression analysis of PGC number against developmental stage (Fig. 4), this difference can be clearly attributed to a smaller founding population in the heterozygotes, and not to a lower proliferation rate. The second set of findings is that Bmp4 is expressed before gastrulation in the extraembryonic ectoderm, at highest levels in cells at the junction with the proximal epiblast. This expression pattern is particularly evident when assayed with a b-gal reporter inserted into the en- dogenous Bmp4 allele. Bmp4 is later expressed in the extraembryonic mesoderm, including the allantois, and in cells in the vicinity of the first identifiable PGCs. However, Bmp4 does not appear to be expressed in the PGCs themselves (Fig. 7A,D). In addition, the presence of b-gal activity in the extraembryonic mesoderm of ho- lacZneo mozygous Bmp4 embryos implies that Bmp4 in Figure 8. PGCs (estimated from histological sections) in chi- tm1 the extraembryonic ectoderm is not required to initiate meras of R26.1 ES cells with wild-type and Bmp4 /+ embryos. Bmp4 expression in the extraembryonic mesoderm (Fig. (A) Aggregation chimeras with (C57BL/6 × CBA) recipients. (B) 6M,N). Blastocyst injection chimeras with (129/SvEv × Black Swiss) re- The third set of observations is that the PGC-and-al- cipients. (Open symbols, broken line) Wild-type recipients; (solid symbols, solid line) heterozygous recipients; (circles) non- lantois-deficient phenotype of Bmp4 mutant embryos chimeric; (squares) ø25% chimeric; (triangles) >25%–50% chi- cannot be rescued by wild-type ES cells injected into meric; (diamond) >50%–75% chimeric; (four-pointed star) blastocysts or aggregated with morulae. In the resulting >75%–95% chimeric; (five pointed star) >95% chimeric. The chimeras, the wild-type ES cells contribute only to the number of PGCs in chimeric embryos falls within the distribu- epiblast-derived tissues, whereas the extraembryonic ec- tion of the nonchimeric embryos of the same genotype, irre- toderm and endoderm are derived from mutant cells. spective of the degree of chimerism. The plotted regression lines Even chimeras with apparently 100% wild-type cells in are for combined chimeric and nonchimeric embryos. The val- the epiblast derivatives show the mutant phenotype and ues in the regression equation (see legend to Fig. 4) are in A, lack PGCs. Similarly, the number of PGCs in chimeras (wild type) 2.165 = 1.876 + 0.0361 (8.0); (heterozygote) 1.631 = 1.205 + 0.0483 (8.8); B, (wild type) 2.364 = 1.976 + 0.0283 (13.7); with heterozygous embryos is not influenced by the de- (heterozygote) 2.155 = 1.775 + 0.0246 (15.4). gree of chimerism: Chimeras with only wild-type epi- GENES & DEVELOPMENT 431 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. −/− Table 3. Chimeras of R26.1 ES cells with Bmp4 embryos Percent chimerism (%) n Somites Allantois PGCs Morula aggregation 0 5 0–6 0 0 (C57BL/6 × CBA) <95 5 2–4 0 0 >95 3 0–4 0 0 Blastocyst injection 0 15 0–14 1 1? (129/SvEv × Black Swiss) ø75 9 0–10 1 0? This nonchimeric embryo had a severely abnormal headfold and a well elongated allantois. One dubious PGC was scored at the base of the allantois. This embryo was a normal looking 6/7 somite embryo with a well-developed allantois and was 75% chimeric. AP activity was virtually absent throughout the embryo, so no firm conclusion about the absence of PGCs can be drawn. blast cells have the smaller number of PGCs character- The availability of Bmp4 protein may also be regulated istic of heterozygous embryos. by the activity of proteases that cleave these binding proteins, for example proteases belonging to the astacin family that includes Bmp1 and tolloid (Cho and Blitz 1998; Mullins 1998). Model I: Extraembryonic ectoderm Bmp4 is Although the observations on PGCs are compatible the only signal with a morphogen gradient set up from the extraembry- The simplest model suggested by the data for the role of tm1 onic ectoderm, the rest of the Bmp4 phenotype is not. Bmp4 in regulating PGC formation is as follows: Bmp4 The presence and amount of yolk sac mesoderm and secreted by the extraembryonic ectoderm acts in a con- yolk sac vascularization vary in Bmp4 null embryos ac- centration dependent manner to regulate cell fate in the cording to the genetic background (Winnier et al. 1995), epiblast. Cells in the proximal epiblast that are nearest but the allantois fails to develop irrespective of the ge- to the extraembryonic ectoderm receive the highest netic background, and is also absent in chimeras with Bmp4 signal. Among these cells a proportion, <50%, be- wild-type ES cells, indicating that extraembryonic ecto- come precursors of both PGCs and part of the allantoic derm Bmp4 is an absolute requirement for allantois for- population (and other extraembryonic derivatives). Cells mation. The presence of a normal allantois in heterozy- more distant from the extraembryonic ectoderm receive gotes suggests that a lower threshold Bmp4 concentra- a lower Bmp4 signal and will contribute to all types of tion than that required for PGC formation allows the extraembryonic mesoderm, including allantois, but do development of an allantois. This is supported by lineage not contribute to PGCs. Only a few descendants of a analysis that has shown that the allantois is derived not PGC precursor in the epiblast actually become PGCs at only from the most proximal epiblast, but also from epi- the time of allocation (an average of 2.6 descendants after blast cells further from the junction with the extraem- 3.7 generations from an E6 precursor and 1.5 descendants bryonic ectoderm where the Bmp4 concentration would after 1.6 generations from an E6.5 precursor; Lawson and be expected to be lower (Lawson and Pedersen 1992; Hage 1994). The cell mingling that follows cell division Lawson and Hage 1994). In the heterozygotes, fewer epi- in the epiblast (Lawson et al. 1991; Gardner and Cock- blast cells would be exposed to this lower concentration croft 1998) could ensure that only some descendants re- than in the wild type, and the allantois would be ex- main close enough to the source of Bmp4 to receive suf- pected to be smaller, or the onset of its formation would ficient signal for PGC formation. Alternatively, the sig- be delayed. We have found no evidence of this. nal gradient could take time to establish. In either case, the critical concentration would only be achieved Model II: Two signals are required to generate PGCs shortly before PGC allocation. and allantois The precise local level of active Bmp4 protein and the time during which epiblast cells are exposed to it will As noted above, there is an inconsistency between the depend on multiple factors, for example the level of pro- apparently normal allantois phenotype of Bmp4 hetero- teins that can bind Bmp4 and prevent its interaction zygotes and the simple model of Bmp4 acting as a mor- with receptors such as BmpRII and BmpR1A (Alk3) pres- phogen and specifying PGCs and allantois at different ent in the epiblast (Mishina et al. 1995; Roelen et al. threshold concentrations. One explanation for this could 1997). The genes encoding the antagonists cerberus-like be that the allantois cells that are closely lineage related (mCer-1) and follistatin are first expressed respectively to the PGCs, that is, those that are descended from the in the anterior visceral endoderm (Belo et al. 1997; Biben same precursors most proximal in the epiblast, and clos- et al. 1998; Shawlot et al. 1998) and posterior streak (Al- est to the source of Bmp4, are crucial for initiating the bano et al. 1994; Feijen et al. 1994) early in gastrulation. process of allantoic bud formation. Therefore, we suggest 432 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells that the highest Bmp4 concentration is required not to to speculate that the response to the second signal oper- specify PGCs as in Model I, but rather to specify a group ates in favor of generating enough cells to initiate an of cells whose descendants, after traversing the streak, allantois, a structure that is absolutely required for the will either become the putative allantois initiator cells development of a placental mammal, and leaving the or PGCs (Fig. 9A,B). The size of this population will be remainder of the population available for allocation to related to both the number of cells in the most proximal the PGC lineage. The number of PGCs finally allocated epiblast and the strength and duration of the extraem- will therefore depend on the size of the precursor pool bryonic Bmp4 signal to which they are exposed. and that proportion of it directed into forming an allan- After the precursor population has been established, tois. All current findings on the PGCs are compatible the cells must be directed into either the allantois ini- with this model, that is, (1) the presence of a normal tiator pool or into the PGC lineage. This is most likely to allantois, but reduced numbers of PGCs in the heterozy- be in response to a second local signal, either before or gotes, (2) the absence of PGCs, but a normal allantois in after the cells have traversed the primitive streak (Fig. a small minority of heterozygotes on the (C57BL/ 9C,D). We currently favor the second scenario because 6 × CBA) background, (3) differences in the size of the previous clonal analysis suggests that the time of PGC PGC founding population between wild type (C57BL/ allocation is at ~ E7.2, when the PGCs first become iden- 6 × CBA) and (129/SvEv × Black Swiss), and (4) delayed tifiable in a cluster at the base of the incipient allantois. appearance of PGCs in heterozygotes (Fig. 2) together Allocation is said to have occurred when cells no longer with the smaller size of the region in which PGCs are enter or leave the population (McLaren 1976), that is, the first identifiable (Fig. 7A,B). The two-signal model does population has become lineage restricted and self-per- not require that the extraembryonic ectoderm Bmp4 is petuating. Allocation is unlikely to occur in the epiblast functional at the time of PGC allocation. because, on average, 1.6 cell cycles of 6.8 hr in the early If this two-signal model is correct, a prediction is that streak embryo, and 3.7 cycles in the prestreak embryo, embryos bearing a mutation in a gene that affects allan- intervene before PGC lineage restriction (Lawson and toic phenotype, but that allows the development of an Hage 1994). abnormal allantois, may have PGCs, but that pheno- The precise nature and location of the second signal types specifically lacking an allantois will not. In sup- are unknown. However, several lines of evidence lead us port of this prediction, homozygous eed (Faust et al. 1995), T (Brachyury) (V. Wilson and R. Beddington, pers. comm.) and Otx2 mutants (K. Lawson, unpubl.) all show abnormal allantois development but have cells with an AP-staining pattern characteristic of PGCs. Nothing is known of the nature or source of the second signal. The possibility that it is Bmp4 produced by the early extra- embryonic mesoderm will, in the future, be tested by chimera analysis with homozygous Bmp4 mutant ES cells. Bmp4 may act indirectly and/or in synergy with other signaling factors In the model proposed above, we have assumed that Figure 9. Two-signal model of Bmp4 regulation of PGC allo- Bmp4 secreted by the extraembryonic ectoderm acts di- cation. (A) Preprimitive streak stage. Bmp4 is produced by the rectly on the epiblast. However, we cannot at this time extraembryonic ectoderm (xe) adjacent to the proximal epiblast exclude the possibility that Bmp4 acts indirectly, for ex- (ep) and a gradient (blue arrows) is set up to which the proximal ample by regulating the production of another signaling epiblast cells respond and become directed toward an allantois molecule(s) by the extraembryonic ectoderm and/or en- initiator/PGC fate (s). (A) Anterior; (P) posterior; (Pr) proximal; doderm. It is also possible that Bmp4 acts in synergy (D) distal. (B) Early primitive streak stage. Bmp4 continues to be with another factor(s) made by the extraembryonic ecto- produced by the extraembryonic ectoderm. The gradient can be derm or epiblast and that PGC precursor fate, is deter- steepened by the presence of the extracellular antagonists mCer-1 (red) anteriorly and follistatin (green) posteriorly. These mined by a combination of factors. In the future, these may also limit the temporal window within which Bmp4 can possibilities may be tested by incubating distal epiblast act in the epiblast. Along with other epiblast cells, the allantois in culture with different combinations of extraembry- initiator/PGC progenitors divide with a generation time of 6.5– onic tissues or signaling factors and assaying for the ap- 7.0 hr, and the progeny align toward and begin to move through pearance of PGCs in vitro. Moreover, if Bmp4 acts in the posterior streak. (C) Midstreak stage. Part of the newly synergy with other obligatory factors to specify PGC cell formed extraembryonic mesoderm (xm) is derived from the fate, it is possible that null mutants in genes encoding most proximal epiblast and is specified to respond to the puta- these factors will have a complete deficiency, or reduced tive second signal (black arrows). This coincides with the onset number, of PGCs. In contrast, embryos lacking genes of Bmp4 expression in the extraembryonic mesoderm. (D) Mid- that antagonize Bmp4 function might be expected to streak/late streak stage. By ~ E7.2, precursors have separated into allantois initiator (m) and PGC (d) lineages. have more PGCs and extraembryonic tissues. In this re- GENES & DEVELOPMENT 433 Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Lawson et al. will generate a fusion transcript between 58 UTR of Bmp4 and gard, it would be interesting to assay for PGC number in lacZ. Note that this construction was deliberately designed to null mutants of Smad2 that have extraembryonic meso- maintain all potential regulatory elements within the Bmp4 derm but little embryonic mesoderm (Waldrip et al. locus, including introns. 1998). Electroporation, selection, and identification of targeted Model III: Bmp4 controls growth and cell movement ES cells An alternative possibility to the above models is that TL1 ES cells were electroporated with 150 μg A total of 19 × 10 Bmp4, rather than regulating PGC/allantois precursor of NotI-digested targeting vector DNA and subjected to positive cell fate, instead controls the growth of proximal epiblast and negative selection (Winnier et al. 1995). DNA from doubly cells and their translocation toward and through the pos- resistant clones was digested with SpeI for Southern blot analy- terior streak into the extraembryonic region. According sis using the 500-bp 58 external BsmI–BamHI probe (Fig. 5A,B) to this model, epiblast precursors of the PGCs and their and internal lacZ and neo probes (data not shown). One cor- rectly targeted line, 12C, in which the 38 homologous recombi- descendants in Bmp4 null embryos may translocate ab- nation occurred within the intron between coding exons 3 and normally from the proximal epiblast into the anterior 4, was injected into C57BL/6NHsd (Harlan Sprague Dawley) part of the streak. If already fully specified, they would blastocysts; resulting male chimeras were mated with outbred then become PGCs ectopically: These have not been lacZneo Black Swiss (Taconic) females. F1 Bmp4 heterozygotes found. If specification and allocation normally occur af- lacZneo were serially backcrossed onto Black Swiss. Bmp4 hetero- ter traversing the streak, the originally proximal cells zygotes were routinely identified by PCR analysis for the neo could be influenced by signals in the anterior part of the (Dunn et al. 1997) or lacZ sequences (see below) within the streak and contribute to the embryonic mesoderm being targeted allele. formed at that time. We cannot exclude this possibility because, although development anterior to the node can be relatively normal in Bmp4 null embryos, interpreta- Chimera generation and retrospective genotyping tion is confounded by the general overall reduction in Injection chimeras Injection chimeras were generated as de- growth that becomes apparent at the onset of gastrula- scribed (Bradley 1987; Hogan et al. 1994). Blastocysts from tion in the mutants. One way of testing this model in the tm1 Bmp4 /+ (129/SvEv × Black Swiss) intercrossings were in- future would be clonal analysis of the proximal epiblast jected with 12–15 ROSA26.1 (R26.1) ES cells (kindly provided in the mutants. by Elizabeth Robertson; Varlet et al. 1997). Following transfer, the embryos were recovered between E8.5 and 9.5, fixed, and processed for double b-gal and AP staining. The genotype of the Materials and methods host blastocyst was determined retrospectively by PCR analysis of enzymatically isolated yolk sac endoderm (Hogan et al. 1994). Mouse strains Potentially contaminating mesoderm was monitored in the en- tm1blh The following genetic backgrounds for the Bmp4 null mu- doderm DNA preparations by PCR analysis for the lacZ gene tation (Winnier et al. 1995) were used in the present study. with the following primer sequences: lacZ 1, 58-TCTGCT- tm1 C57BL/6–Bmp4 males (Dunn et al. 1997) were mated with TCAATCAGCGTGCC-38 and lacZ 2, 58-GCCGTCTGAATTT- (C57BL/6 × CBA) females. The progeny were intercrossed for GACCTGA-38. F1 one generation and the line maintained by backcrossing hetero- zygous males to (C57BL/6 × CBA) females. This line was used Aggregation chimeras Aggregation of eight-cell stage embryos F1 for PGC analysis and for morula aggregation chimeras. For gen- with ES cells was basically as described (Nagy and Rossant tm1 erating embryos for blastocyst injection, the Bmp4 mutation 1993). Briefly, R26.1 ES cells were cultured on mouse embry- was maintained on a (129/SvEv × Black Swiss) background by onic fibroblast feeder cells. For aggregation, ES cells were tryp- lacZneo intercrossing. Bmp4 was maintained on the (129/ sinized and the fibroblasts were allowed to reattach to the tissue SvEv × Black Swiss) background and embryos for PGC and ex- culture plastic for 30 min. Subsequently, the ES cells were pression pattern analysis obtained from heterozygous matings transferred to a smaller volume for 2 hr to form aggregation and matings with ICR females. clumps. Eight-cell stage embryos were collected at E2.5 from tm1 Bmp4 /+ matings on the (C57BL/6 × CBA) background. The zona pellucida was removed with acid Tyrode’s solution and single embryos aggregated with clumps of 10–15 ES cells in Construction of the lacZ knock-in targeting vector aggregation wells in 20 μl droplets of M16 medium and cultured lacZneo Detailed information on the construction of the Bmp4 overnightat37°Cin5%CO (Zwijsen et al. 1999). Embryos targeting vector and Cre-mediated excision of the neo cassette were transferred the following day into E2.5 pseudopregnant can be found at http://www.mc.vanderbilt.edu/vumcdept/cell- (C57BL/6 × CBA) recipients. The embryos were recovered at F1 bio/hogan.html. Briefly, most of exon 3, from nucleotides 6807– nominal E8.5 and further processed and genotyped as described 7178 (Kurihara et al. 1993), including the translation initiation above. Very retarded embryos were genotyped on the parietal ATG and sequences encoding amino acids 29–124 of the pro endoderm of Reichert’s membrane. region, is entirely replaced with a b-gal cassette from pPD1.27, which encodes bacterial b-gal with SV40 nuclear localization and polyadenylation signals (Fire et al. 1990) (Fig. 5A). The cas- Developmental index sette also contains a loxP-site-flanked positive selection MC1- neo cassette (a gift from Steve O’Gorman, Salk Institute, La The stage of embryo development as judged by a score of mor- lacZneo Jolla, CA). After recombination, the targeted Bmp4 allele phological features (modified from Brown 1990), or the number 434 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 and primordial germ cells of somite pairs, was very variable between and within litters of 8100) for 1–2 hr. The plastic was then polymerized at 4°C. Serial the same nominal gestational age. Morphological score was lin- sections cut at 7 μm were stained for AP activity with ASMX/ early correlated with somite number up to 20 somite pairs, and Fast Red TR (Sigma) for 30–90 min according to the manufac- the relationship was the same in wild type and heterozygotes on turer’s instructions. The sections were counterstained with the (C57BL/6 × CBA) background (data not shown). Somite Mayer’s haemalum and mounted in Aquamount (Gurr). PGCs pairs are laid down on average every 90 min during normal were counted on the basis of the densely staining AP-positive development, at least up to 30 somites (Tam 1981). Somite cytoplasmic spot, using a 25× objective lens (Ginsburg et al. number was therefore used as a measure of the developmental 1990; Lawson and Hage 1994). age of individual embryos. Combined b-gal and AP staining Embryos were fixed in 4% paraformaldehyde for 2 hr as above and stained for b-galat37°C lacZneo for 4 hr (chimeras with R26.1 ES cells) or 8 hr (Bmp4 b-Gal staining embryos). They were then dehydrated, embedded in Technovit lacZneo Bmp4 embryos and decidua were fixed in 4% parafor- 8100, sectioned, and stained for AP activity as above without maldehyde in PBS at 4°C for 30 min with rocking, washed twice counterstaining. for 10 min in PBS at 4°C, transferred into freshly prepared X-gal solution and stained overnight (or longer) at 37°C (Hogan et al. 1994). After rinsing with PBS, embryos were post-fixed in 4% Statistics paraformaldehyde. Some embryos were cleared in 80% glycerol Regression analysis and comparison of regression lines were in PBS. performed as described, using the F test to compare variances For histological analysis, stained decidua were dehydrated (Snedecor and Cochran 1967). into 100% isopropanol, washed twice with 1:1 isopropanol/par- affin wax, embedded in wax, and 7-μm sections lightly counter- stained with eosin. Acknowledgments We thank Drs. Jacqueline Deschamps, David Greenstein, An- Detecting and counting PGCs thony Mahowald, Christine Mummery, David Threadgill, and Lilianna Solnica-Krezel for discussions and critical comments Whole mounts Embryos between E7.5 and E9.5 were dissected on the manuscript; Dr. Lucy Liaw and Linda Hargett for help in from the decidua and Reichert’s membrane reflected. The yolk lacZneo establishing the Bmp4 line; Marie-Jose ´ Goumans, Marga sac was separated from the ectoplacental cone of embryos that van Rooijen, and Dr. Elizabeth Robertson for invaluable advice were in the process of, or had completed, turning. Both yolk sac on making chimeras. We also thank Angela D. Land-Dedrick for and amnion were reflected but left attached to the embryo. The assistance in manuscript preparation and Dominic Doyle for the embryos were fixed in 4% paraformaldehyde in PBS for 2 hr at illustration of Figure 9. N.R.D. was supported by a grant from 4°C. The embryos were washed three times in PBS, during the National Institutes of Health (HD 28955) to B.L.M.H., who which time they were further dissected according to size. 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Tis- sue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse em- bryogenesis but is not required for migration of primordial 436 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 21, 2021 - Published by Cold Spring Harbor Laboratory Press Bmp4 is required for the generation of primordial germ cells in the mouse embryo Kirstie A. Lawson, N. Ray Dunn, Bernard A.J. Roelen, et al. Genes Dev. 1999, 13: This article cites 30 articles, 14 of which can be accessed free at: References http://genesdev.cshlp.org/content/13/4/424.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Cold Spring Harbor Laboratory Press

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