Germ cell determination in nematodes

Theodor Boveri (Figure 4.2; 1862–1915) was the first person to observe an organism's chromosomes throughout its development. In so doing, he discovered a fascinating feature in the development of the roundworm Parascaris aequorum (formerly Ascaris megalocephala). This nematode has only two chromosomes per haploid cell, allowing for detailed observations of the individual chromosomes. The cleavage plane of the first embryonic division is unusual in that it is equatorial, separating the animal half from the vegetal half of the zygote (Figure 19.1A). More bizarre, however, is the behavior of the chromosomes in the subsequent division of these first two blastomeres. The ends of the chromosomes in the animal-derived blastomere fragment into dozens of pieces just before this cell divides. This phenomenon is called chromosome diminution, because only a portion of the original chromosome survives. Numerous genes are lost in these cells when the chromosomes fragment, and these genes are not included in the newly formed nuclei (Tobler et al. 1972; Müller et al. 1996). Meanwhile, in the vegetal blastomere, the chromosomes remain normal. During second cleavage, the animal cell splits meridionally while the vegetal cell again divides equatorially. Both vegetally derived cells have normal chromosomes. However, the chromosomes of the more animally located of these two vegetal blastomeres fragment before the third cleavage. Thus, at the 4-cell stage, only one cell—the most vegetal—contains a full set of genes. At successive cleavages, nuclei with diminished chromosomes are given off from this vegetalmost line, until the 16-cell stage, when there are only two cells with undiminished chromosomes. One of these two blastomeres gives rise to the germ cells; the other eventually undergoes chromosome diminution and forms more somatic cells. The chromosomes are kept intact only in those cells destined to form the germ line. If this were not the case, the genetic information would degenerate from one generation to the next. The cells that have undergone chromosome diminution generate the somatic cells.

Figure 19.1

Distribution of germ plasm during cleavage of (A) normal and (B) centrifuged zygotes of Parascaris. (A) The germ plasm is normally conserved in the most vegetal blastomere, as shown by the lack of chromosomal diminution in that particular cell. Thus, (more...)

Boveri has been called the last of the great “observers” of embryology and the first of the great experimenters. Not content with observing the retention of the full chromosome complement by the germ cell precursors, he set out to test whether a specific region of cytoplasm protects the nuclei within it from diminution. If so, any nucleus happening to reside in this region should be protected. Boveri (1910) tested this hypothesis by centrifuging Parascaris eggs shortly before their first cleavage. This treatment shifted the orientation of the mitotic spindle. When the spindle forms perpendicular to its normal orientation, both resulting blastomeres should contain some of the vegetal cytoplasm (see Figure 19.1B). Indeed, Boveri found that after the first division, neither nucleus underwent chromosomal diminution. However, the next division was equatorial along the animal-vegetal axis. Here the resulting animal blastomeres both underwent diminution, whereas the two vegetal cells did not. Boveri concluded that the vegetal cytoplasm contains a factor (or factors) that protects nuclei from chromosomal diminution and determines germ cells.

In the nematode C. elegans, the germ line precursor cell is the P4 blastomere. The P-granules enter this cell, and they appear critical for instructing it to become the germ line precursor (see Figure 8.45). The components of the P-granules remain largely uncharacterized, but they appear to contain several transcriptional inhibitors and RNA-binding proteins, including homologues of the Drosophila Vasa and Nanos proteins, whose function we will see below (Kawasaki et al. 1998; Seydoux and Strome 1999; Subramanian and Seydoux 1999).

Germ cell determination in insects

In Drosophila, PGCs form as a group of cells (pole cells) at the posterior pole of the cellularizing blastoderm. These nuclei migrate into the posterior region at the ninth nuclear division, and they become surrounded by the pole plasm, a complex collection of mitochondria, fibrils, and polar granules (Figure 19.2; Mahowald 1971a,Mahowald 1971b; Schubiger and Wood 1977). If the pole cell nuclei are prevented from reaching the pole plasm, no germ cells will be made (Mahowald et al. 1979).

Figure 19.2

The pole plasm of Drosophila. (A) Electron micrograph of polar granules from particulate fraction of Drosophila pole cells. (B) Scanning electron micrograph of a Drosophila embryo just prior to completion of cleavage. The pole cells can be seen at the (more...)

Nature has provided confirmation of the importance of both pole plasm and its polar granules. One of the components of the pole plasm is the mRNA of the germ cell-less (gcl) gene. This gene was discovered by Jongens and his colleagues (1992) when they mutated Drosophila and screened for those females who did not have “grandoffspring.” They assumed that if a female did not place functional germ plasm in her eggs, she could still have offspring, but those offspring would be sterile (since they would lack germ cells). The wild-type gcl gene is transcribed in the nurse cells of the fly's ovary, and its mRNA is transported into the egg. Once inside the egg, it is transported to the posteriormost portion and resides within what will become the pole plasm (Figure 19.3A, Figure 19.3B). This message gets translated into protein during the early stages of cleavage (Figure 19.3C,Figure 19.3D). The gcl-encoded protein appears to enter the nucleus, and it is essential for pole cell production. Flies with mutations of this gene lack germ cells.

Figure 19.3

Localization of Germ cell-less gene products in the posterior of the egg and embryo. The gcl mRNA can be seen in the posterior pole of early-cleavage embryos produced by wild-type females (A), but not in embryos produced by gcl-deficient mutant females (more...)

A second set of pole plasm components are the posterior determinants mentioned in Chapter 9. Oskar appears to be the critical protein of this group, since injection of oskar mRNA into ectopic sites of the embryo will cause the nuclei in those areas to form germ cells. The genes that restrict Oskar to the posterior pole are also necessary for germ cell formation (Ephrussi and Lehmann 1992; Newmark et al. 1997). Moreover, Oskar appears to be the limiting step of germ cell formation, since adding more oskar message to the oocyte causes the formation of more germ cells (Ephrussi and Lehmann 1992). Oskar functions by causing the localization of the proteins and RNAs necessary for germ cell formation. One of these RNAs is the nanos message, whose product is essential for posterior segment formation. Nanos is also essential for germ cell formation. Pole cells lacking Nanos do not migrate into the gonads and fail to become gametes. Nanos appears to be important in preventing mitosis and transcription during germ cell development (Kobayashi et al. 1996; Deshpande et al. 1999). Another one of these RNAs encodes Vasa, an RNA-binding protein. The mRNAs for this protein are seen in the germ plasm of many species.

A third germ plasm component was a big surprise: mitochondrial ribosomal RNA (mtrRNA). Kobayashi and Okada (1989) showed that the injection of mtrRNA into embryos formed from ultraviolet-irradiated eggs restores the ability of these embryos to form pole cells. Moreover, in normal fly eggs, the small and large mitochondrial rRNAs are located outside the mitochondria solely in the pole plasm of cleavage-stage embryos. Here they appear as components of the polar granules (Kobayashi et al. 1993; Amikura et al. 1996; Kashikawa et al. 1999). While mitochondrial RNA is involved in directing the formation of the pole cells, it does not enter them.

A fourth component of Drosophila pole plasm (and one that becomes localized in the polar granules) is a nontranslatable RNA called polar granule component (Pgc). While its exact function remains unknown, the pole cells of transgenic female flies making antisense RNA against Pgc fail to migrate to the gonads (Nakamura et al. 1996).

Germ cell determination in amphibians

Cytoplasmic localization of germ cell determinants has also been observed in vertebrate embryos. Bounoure (1934) showed that the vegetal region of fertilized frog eggs contains material with staining properties similar to those of Drosophila pole plasm (Figure 19.4). He was able to trace this cortical cytoplasm into the few cells in the presumptive endoderm that would normally migrate into the genital ridge. By transplanting genetically marked cells from one embryo into another of a differently marked strain, Blackler (1962) showed that these cells are the primordial germ cell precursors.

Figure 19.4

Germ plasm at the vegetal pole of frog embryos. (A) Germ plasm (dark regions) near the vegetal pole of a newly fertilized zygote. (B) In situ hybridization localizing the mRNA for Xcat2 (the Xenopus homologue of Nanos) in the vegetal cortex of first-cleavage (more...)

The early movements of the germ plasm in amphibians have been analyzed in detail by Savage and Danilchik (1993), who labeled the germ plasm with fluorescent dye. They found that the germ plasm of unfertilized eggs consists of tiny “islands” that appear to be tethered to the yolk mass near the vegetal cortex. These germ plasm islands move with the vegetal yolk mass during the cortical rotation of fertilization. After the rotation, the islands are released from the yolk mass and begin fusing together and migrating to the vegetal pole. Their aggregation depends on microtubules, and the movement of these clusters to the vegetal pole is dependent on a kinesin-like protein that may act as the motor for germ plasm movement (Robb et al. 1996). Later, periodic contractions of the vegetal cell surface also appear to push this germ plasm along the cleavage furrows of the newly formed blastomeres, enabling it to enter the embryo.

When ultraviolet light is applied to the vegetal surface (but nowhere else) of frog embryos, the resulting frogs are normal but lack germ cells in their gonads (Bounoure 1939; Smith 1966). Very few primordial germ cells reach the gonads; those few that do have about one-tenth the volume of normal PGCs and have aberrantly shaped nuclei (Züst and Dixon 1977). Savage and Danilchik (1993) found that UV light prevents vegetal surface contractions and inhibits the migration of germ plasm to the vegetal pole. The Xenopus homologues of nanos and vasa are specifically localized to this region (Forristall et al. 1995; Ikenishi et al. 1996; Zhou and King 1996). So, like the Drosophila pole plasm, the cytoplasm from the vegetal region of the frog zygote contains the determinants for germ cell formation. Moreover, several of the components are the same.

The components of the germ plasm have not all been catalogued. Indeed, in the birds and mammals, such a list has hardly even been started. Moreover, we still do not know the functions of the proteins (such as Vasa and Nanos) and nontranslated RNAs found in the germ plasm. One hypothesis (Nieuwkoop and Sutasurya 1981; Wylie 1999) is that the components of the germ plasm inhibit both transcription and translation, thereby preventing the cells containing it from differentiating into anything else. According this hypothesis, the cells become germ cells because they are forbidden to become any other type of cell.