Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press 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 embryo, 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, germ cell determinants present in the egg are asym- (i.e., proximal), immediately adjacent to the inverted ex- metrically segregated at each subsequent cell division traembryonic ectoderm 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 mammals, it seems that days they were found nearer to the side of the cup where cells have germ cell status thrust upon them. the primitive streak 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 gastrulation, ∼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 mesoderm 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 blastocyst 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 endoderm. Lawson and to zero by a recent experiment, simple in design but very Hage removed the embryos 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 GENES & DEVELOPMENT 13:373–376 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org 373 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press 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 surface ectoderm. 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 chimera 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 somite 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.