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PERSPECTIVE

Signaling for germ cells

Anne McLaren1

Wellcome/Cancer Research Campaign Institute, Cambridge, CB2 1QR, UK

Shakespeare advises us that some men are born great, rescent lineage marker, so that after culturing for some some achieve greatness, and some have greatness thrust 40 hours they could identify the descendants of the in- upon them. So it is with germ cells. In Drosophila, the jected cell and ascertain their fate. They found that the very first cells to be formed in the , the pole cells, only cells giving rise to primordial germ cells (recogniz- are germ cells whose descendants have no fate other than able again by their high alkaline phosphatase activity) to give rise to gametes. In Caenorhabditis and in zebra were those located around the rim of the epiblast cup fish, determinants present in the egg are asym- (i.e., proximal), immediately adjacent to the inverted ex- metrically segregated at each subsequent cell division traembryonic cup. At 6.0 days, the germ cell until a definitive germ cell lineage is achieved. But in progenitors were distributed all round the rim; by 6.5 mice, and by extrapolation in all , it seems that days they were found nearer to the side of the cup where cells have germ cell status thrust upon them. the had formed. This entire proximal population of cells moves, perhaps under pressure from cell proliferation, through the primitive streak and up- Establishment of the germ cell lineage wards, into the territory of the extraembryonic ecto- What is the evidence? The earliest that primordial germ derm. Some of the cells settle down, to form the initial cells have been identified in the mouse is midway cluster of primordial germ cells; others nearby will give through , ∼7.25 days after fertilization (Gins- rise to the allantois; the majority continue to migrate, burg et al. 1970). Recognizable by their high alkaline forming extraembryonic and separating the phosphatase activity, they are seen as a cluster of cells cavities of the two cups. located in the extraembryonic region posterior to the Crucially, Lawson and Hage found that none of their primitive streak, and can subsequently be tracked from injected cells gave rise only to germ cells, proving that that location, along their migratory route and into the even at this late stage no exclusive germ cell lineage had future gonads. All attempts to identify germ cells or been established. By counting the clonal descendants germ cell determinants (e.g., germplasm, ‘nuage’) earlier and the marked and unmarked germ cells, they were able in development have failed. Experiments in which a ge- to carry out a clonal analysis. This indicated that germ netically marked cell was introduced into the embryo cell lineage restriction was taking place in a group of ∼ either at the 4-cell (Kelly 1977) or at the mid- or late- some 45 cells at 7.2 hours after fertilization, by which stage (Gardner 1977) established that cells time the migrating cells would have reached the extra- with primordial germ cells among their descendants also embryonic location in which the initial cluster of pri- gave rise to somatic cells, thus no exclusive germ cell mordial germ cells had earlier been seen. lineage existed in the preimplantation embryo. More recently, this approach has been extended to the Ancestors or neighbors? postimplantation period by Lawson and Hage (1994). Im- mediately before gastrulation and during the early stages So far so good. The germ cell lineage was founded in the (6.0 and 6.5 days after fertilization), the mouse embryo place and at the time when primordial germ cells first can be visualized as a thick-walled cup of tissue (the could be identified. But were those cells programmed to epiblast or embryonic ectoderm), which will give rise to gather in that location and become germ cells, as a result the entire fetus as well as contributing to some of the of some asymmetric segregation of determinants at each placental membranes. On top of this is inverted a second of the many cell divisions since fertilization? Given the thick-walled cup of tissue (the extraembryonic ecto- extensive cell mingling that occurs in the epiblast before derm, derived from the trophectoderm) which will give gastrulation (Gardner and Cockcroft 1998), the likeli- rise to the main part of the placenta. Both cups are en- hood of such a happening seemed small. It was reduced closed in a thin bag of primitive . Lawson and to zero by a recent experiment, simple in design but very Hage removed the from the uterus, then in- demanding in execution (Tam and Zhou 1996). jected a single epiblast cell in each embryo with a fluo- Tam and Zhou knew from the work of Lawson and Hage that the ancestors of the germ cells were normally located in the proximal region of the epiblast, around the 1E-MAIL [email protected]; FAX +44 1223 334089. rim of the epiblast cup. In contrast, cells at the distal tip

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McLaren of the epiblast, at the bottom of the cup, normally give epiblast cells move through the primitive streak and into rise only to neurectoderm and . Using the extraembryonic region, no primordial germ cells are 6.0 and 6.5 day transgenically marked donor embryos seen in the cluster location or anywhere else, nor does an and unmarked host embryos, they carried out an in vitro allantois develop. This is the first report of a total inhi- transplantation experiment, grafting small groups of bition of primordial germ cell formation. marked cells both homotypically (proximal to proximal, Bmp4 is expressed before gastrulation in the inverted distal to distal) and heterotypically (proximal to distal, cup of extraembryonic ectoderm that abuts onto the distal to proximal). They cultured the grafted embryos proximal rim of the epiblast cup. In chimeras made be- for two days, then processed them for ␤-galactosidase to tween ES cells (which contribute to the epiblast but not identify the donor cells, and alkaline phosphatase activ- to the extraembryonic ectoderm) and homozygous ity to identify primordial germ cells. The results were Bmp4-null embryos (which therefore form the entire ex- clear-cut. Cells placed in the proximal rim of the epiblast traembryonic ectoderm), neither germ cells nor allantois cup were capable of giving rise to germ cells, whether developed. The authors conclude from these they had been taken from the proximal or from the distal studies that some signal arising from the expression of part of the donor epiblast; conversely, germ cells never Bmp4 in the extraembryonic ectoderm is an essential appeared in the bottom of the cup, even when donor cells requirement for the subsequent establishment of the from the proximal region had been transplanted there. germ cell lineage and for the formation of the allantois. The cell types that differentiated depended on where the Whether or not the secreted molecule BMP4 itself con- grafts were placed, not where they had been taken from. stitutes the signal remains to be investigated; but the absolute requirement for Bmp4 to be expressed is un- questionable. Signals—but from where? Mice heterozygous for the Bmp4-null mutation are fer- So germ cell status is not ‘achieved’ in the mouse em- tile, but Lawson et al. (1999) report that the number of bryo by virtue of some C. elegans-like segregation of cy- primordial germ cells in heterozygous embryos lags be- toplasmic determinants, but rather is ‘thrust upon’ cer- hind that in their wild-type littermates: the germ cell tain cells by means of local signals. But when and where population in the heterozygotes increases at the same do these signals occur, and what do they consist of? Sus- rate as in the controls, but for any given number picion must fall first on the extraembryonic cluster lo- it is reduced ∼50%. A similar though smaller reduction cation, where the movement of the germ cell progenitors in germ cell number was reported for We heterozygotes is halted and germ cell specification takes place. This by Buehr et al. (1993). Lawson et al. (1999) interpret their would time the signal for ∼7 days after fertilization. The findings as indicating a smaller founding population of requirement for progenitors to have come from the germ cells in the heterozygotes, rather than a delay in proximal region of the epiblast cup could then arise germ cell allocation or proliferation, or an early loss of merely because these are the cells that move through the germ cells. A smaller founding population could indicate primitive streak and into the extraembryonic region dur- a weaker signal in the heterozygotes, suggesting that ing gastrulation. germ cell allocation is dosage dependent. The allantois, The first indication that an earlier signal might be in- however, is of normal size in the heterozygotes. volved came from the observation (Y. Masui and T. Yoshimizu, pers. comm.) that proximal epiblast tissue One signal or two? taken at 6.5 days, disaggregated, and then cultured for several days, could give rise to cells showing high alka- Given that a Bmp4-dependent signal from the extraem- line phosphatase activity, resembling primordial germ bryonic ectoderm to the proximal epiblast cells is nec- cells. These cells never could have been exposed to any essary for germ cell development, is it sufficient? Law- cluster-location signals. Cultures derived from other son et al. (1999) discuss both one and two signal models. parts of the epiblast never yielded any alkaline phospha- On a one-signal model, one would have to assume that tase-positive cells. When proximal epiblast was taken only a minority of the proximal epiblast cells receiving from earlier stages, the putative germ cells only appeared the Bmp4-dependent signal, perhaps those above a cer- if some extraembryonic ectoderm was included in the tain threshold level, became programmed to give rise to cultures. allantois and germ cells, whereas the majority ignored So would an earlier signal suffice? Or are there two the signal and continued to migrate as extraembryonic signals? Or more than two? Our understanding of the mesoderm. Of the responding subset, ∼45, perhaps those situation has been greatly enhanced by the important receiving the highest levels of signal, would settle down paper in this issue by Lawson et al. (1999). in the cluster region as the germ cell founder population, Bone Morphogenetic Protein 4 (BMP4) is a member of whereas an unknown number would settle nearby and the TGF␤ superfamily of intercellular signaling proteins. subsequently give rise to the allantois. The cells would Its role in development extends far beyond the morpho- halt in the cluster region not because of the properties of genesis of bone. Most mouse embryos homozygous for a the region, but because it represented the limit of the null mutation in the Bmp4 gene die in the early stages of time or distance or number of cell divisions for which gastrulation; but Lawson et al. (1999) found that some they (or their pioneers) had been programmed to travel. survive long enough to show that, although the proximal Given that only a small fraction of the descendants of

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Signaling for germ cells any germ cell progenitor in the epiblast at 6.0 days ac- space to target the first cells coming through the poste- tually contributes to the germ cell population (Lawson rior primitive streak, leaving later immigrants to become and Hage 1994), it would be necessary to postulate that extraembryonic mesoderm. It would therefore be quite the signal continued to act on the migrating cells until distinct from the earlier broad Bmp4-dependent signal. shortly before germ cell allocation. Epiblast-derived extraembryonic mesoderm has been More attractive seems a two-signal model (Fig. 1), with shown by the chimera experiments of Lawson et al. a Bmp4-dependent signal from the extraembryonic ecto- (1999) to be incapable on its own of inducing germ cell derm sensitizing the proximal epiblast cells, and a fur- formation; the postulated second signal, however, would ther signal or signals of unknown origin capturing the be acting on cells already sensitized by exposure to the allantois progenitors and the germ cell founder popula- first signal. Again it would need to be highly localized, in tion from the sensitized cells moving past towards an contrast to Bmp4, which Lawson et al. (1999) report to be extraembryonic mesoderm fate. Lawson et al. (1999) pos- widely expressed in the extraembryonic mesoderm. The tulate that a single population of epiblast cells is allo- third possibility is the primitive visceral endoderm, the cated to both the allantois and the germ cell lineage. To thin bag of tissue that surrounds both the epiblast and explain the normal-sized allantois in Bmp4-null hetero- the extraembryonic ectoderm. The extraembryonic germ zygous embryos, they suggest that when the founder cell cluster lies closely apposed to this endodermal layer. population is reduced in number, the allantoic rudiment Anterior patterning in the mouse has been shown to de- would retain its normal complement of cells since an pend on signals coming from the embryonic visceral en- allantois is required for further development, while the doderm (Thomas and Beddington 1996) but nothing is less essential germ cell population could be correspond- known of gene expression in the extraembryonic visceral ingly decreased. It may be, however, that the rapidly pro- endoderm posterior to the primitive streak. liferating allantois is capable of a considerable degree of compensatory growth (Snow et al. 1981), unlike the Are mammals so strange? more slowly dividing germ cell population (Lawson and Hage 1994). In that case one could consider separate sig- The establishment of a germ cell lineage must be about nals for allantois and germ cells. the most fundamental issue ever to have faced the Meta- From what tissues could such a signal or signals ema- zoa throughout their long evolutionary history. Once nate? We know little of the siting and make up of the evolution has devised a system based on unequal distri- allantoic primordium: more detailed investigation of bution of germ cell determinants within the fertilized this essential organ is badly needed. What of the germ egg and early embryo, a system that appears to work well cell cluster? One side is bounded by primitive extraem- for C. elegans and Drosophila, and which has extended bryonic visceral endoderm; the other side is bounded by into vertebrate evolution, at least in zebrafish and frogs, extraembryonic mesoderm at the time when the primor- is it not strange that mammals should do things so dif- dial germ cells can first be identified (Ginsburg et al. ferently? 1990), but the first epiblast cells to move into the extra- Mammals may not be alone. Although frogs and toads embryonic region would be in contact with extraembry- (Anura) have visible ‘germ plasm’ present from the oo- onic ectoderm. There are thus three possibilities. Any cyte stage onwards, their cousins the newts (Urodela) effective signal emanating from extraembryonic ecto- lack any obvious germ plasm in the early embryo. Germ derm would need to be strictly regulated in time and cells are first seen in the ventrolateral region during gas-

Figure 1. A two-signal model for germ cell develop- ment in the mouse. (Left) At 6.0 days, the signal (solid arrows) coming from the extraembryonic ecto- derm (blue) predisposes the proximal epiblast cells towards a germ-line fate. This whole layer of primor- dial germ cell precursors (᭺) moves (dashed arrow) toward the primitive streak and up into the extraem- bryonic region. (Right)At7.0days,someofthemi- grating cells are trapped by a signal or signals (solid arrows) in the cluster region and give rise to the de- finitive germ cell lineage. (Red-orange) epiblast; (yel- low) visceral endoderm; (gold) migrating extraembry- onic mesoderm; (white) exocoel.

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McLaren trulation, and were reported by Nieuwkoop and Sata- Hogan. 1999. Bmp4 is required for the generation of primor- surya (1979) to be induced in the mesoderm. Evidently dial germ cells in the mouse embryo. Genes & Dev. (this Urodeles warrant further investigation, using modern issue). methods. In , the evidence remains inconclusive Nieuwkoop, P.D. and L.A. Satasurya. 1979. Primordial germ (Karagenc et al. 1996). cells in the chordates. Cambridge University Press, Cam- bridge, UK. But if mammals do turn out to be unique, is that so Snow, M.H.L., P.P.L Tam, and A. McLaren. 1981. On the con- surprising? The first and most crucial challenge in mam- trol and regulation of size and morphogenesis in mammalian malian development is to achieve complete and stable embryos. In Levels of Genetic Control in Development, pp. implantation in the uterus of the mother. To that end, 201–217, Alan R. Liss, New York, NY. the first cell lineages to differentiate in the mammalian Tam, P.P. and S.X. Zhou. 1996. The allocation of epiblast cells embryo are trophectoderm and primitive endoderm, to the ectodermal and germ-line lineages is influenced by the both of which contribute exclusively to extraembryonic position of the cells in the gastrulating mouse embryo. Dev. structures (placenta and other extraembryonic mem- Biol. 178: 124–132. branes). Until the basic life-support systems to nourish Thomas, P. and R. Beddington. 1996. Anterior primitive endo- and protect the future fetus have been laid down, no derm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr. Biol. 6: 1487–1496. further differentiation takes place. In a sense the dra- matic increase in proliferation rate of the epiblast that heralds the completion of implantation and the onset of gastrulation is analogous to early embryonic develop- ment in other animals. In Drosophila, the unequal distribution of determi- nants within the oocyte is induced by signals emanating from the surrounding somatic tissues, which make no material contribution to later development. In mam- mals, the two extraembryonic lineages (trophectoderm and primitive endoderm) make no material contribution to fetal development, but they may turn out to be the source for many of the signals that regulate that devel- opment. The distinction could be regarded as essentially one of timing.

Acknowledgments I am grateful to the Wellcome Trust for financial support.

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Signaling for germ cells

Anne McLaren

Genes Dev. 1999, 13:

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