Cleavage in Snail Eggs
Spiral holoblastic cleavage is characteristic of several animal groups, including annelid worms, some flatworms, and most molluscs. It differs from radial cleavage in numerous ways. First, the cleavage planes are not parallel or perpendicular to the animal-vegetal axis of the egg; rather, cleavage is at oblique angles, forming a “spiral” arrangement of daughter blastomeres. Second, the cells touch one another at more places than do those of radially cleaving embryos. In fact, they assume the most thermodynamically stable packing orientation, much like that of adjacent soap bubbles. Third, spirally cleaving embryos usually undergo fewer divisions before they begin gastrulation, making it possible to follow the fate of each cell of the blastula. When the fates of the individual blastomeres from annelid, flatworm, and mollusc embryos were compared, many of the same cells were seen in the same places, and their general fates were identical (Wilson 1898). Blastulae produced by radial cleavage have no blastocoel and are called stereoblastulae.
Figures 8.26 and 8.27 depict the cleavage of mollusc embryos. The first two cleavages are nearly meridional, producing four large macromeres (labeled A, B, C, and D). In many species, the blastomeres are different sizes (D being the largest), a characteristic that allows them to be individually identified. In each successive cleavage, each macromere buds off a small micromere at its animal pole. Each successive quartet of micromeres is displaced to the right or to the left of its sister macromere, creating the characteristic spiral pattern. Looking down on the embryo from the animal pole, the upper ends of the mitotic spindles appear to alternate clockwise and counterclockwise. This causes alternate micromeres to form obliquely to the left and to the right of their macromere. At the third cleavage, the A macromere gives rise to two daughter cells, macromere 1A and micromere 1a. The B, C, and D cells behave similarly, producing the first quartet of micromeres. In most species, the micromeres are to the right of their macromeres (looking down on the animal pole). At the fourth cleavage, macromere 1A divides to form macromere 2A and micromere 2a; and micromere 1a divides to form two more micromeres, 1a1 and 1a2. Further cleavage yields blastomeres 3A and 3a from macromere 2A, and micromere 1a2 divides to produce cells 1a21 and 1a22.
Figure 8.26
Spiral cleavage of the mollusc Trochus viewed (A) from the animal pole and (B) from one side. In (B), the cells derived from the A blastomere are shown in color. The mitotic spindles, sketched in the early stages, divide the cells unequally and at an (more...)
Figure 8.27
Spiral cleavage of the snail Ilyanassa. The D blastomere is larger than the others, allowing the identification of each cell. Cleavage is dextral. (A) 8-cell stage. PB is a polar body. (B) Mid-fourth cleavage (12-cell embryo). The macromeres have already (more...)
The orientation of the cleavage plane to the left or to the right is controlled by cytoplasmic factors within the oocyte. This was discovered by analyzing mutations of snail coiling. Some snails have their coils opening to the right of their shells (dextral coiling), whereas other snails have their coils opening to the left (sinistral coiling). Usually, the direction of coiling is the same for all members of a given species. Occasionally, though, mutants are found. For instance, in species in which the coils open on the right, some individuals will be found with coils that open on the left. Crampton (1894) analyzed the embryos of such aberrant snails and found that their early cleavage differed from the norm. The orientation of the cells after the second cleavage was different in the sinistrally coiling snails owing to a different orientation of the mitotic apparatus (Figure 8.28). All subsequent divisions in left-coiling embryos are mirror images of those in dextrally coiling embryos. In Figure 8.28, one can see that the position of the 4d blastomere (which is extremely important, as its progeny will form the mesodermal organs) is different in the two types of spiraling embryos. Eventually, two snails are formed, with their bodies on different sides of the coil opening.
Figure 8.28
Looking down on the animal pole of left-coiling and right-coiling snails. The origin of sinistral and dextral coiling can be traced to the orientation of the mitotic spindle at the second cleavage. The left-coiling (A) and right-coiling (B) snails develop (more...)
The direction of snail shell coiling is controlled by a single pair of genes (Sturtevant 1923; Boycott et al. 1930). In the snail Limnaea peregra, most individuals are dextrally coiled. Rare mutants exhibiting sinistral coiling were found and mated with wild-type snails. These matings showed that there is a right-coiling allele D, which is dominant to the left-coiling allele d. However, the direction of cleavage is determined not by the genotype of the developing snail, but by the genotype of the snail's mother. A dd female snail can produce only sinistrally coiling offspring, even if the offspring's genotype is Dd. A Dd individual will coil either left or right, depending on the genotype of its mother. Such matings produce a chart like this:
The genetic factors involved in snail coiling are brought to the embryo by the oocyte cytoplasm. It is the genotype of the ovary in which the oocyte develops that determines which orientation cleavage will take. When Freeman and Lundelius (1982) injected a small amount of cytoplasm from dextrally coiling snails into the eggs of dd mothers, the resulting embryos coiled to the right. Cytoplasm from sinistrally coiling snails did not affect the right-coiling embryos. These findings confirmed that the wild-type mothers were placing a factor into their eggs that was absent or defective in the dd mothers.
WEBSITE
8.3 Alfred Sturtevant and the genetics of snail coiling. By a masterful thought experiment, Sturtevant demonstrated the power of applying genetics to embryology. To do this, he brought Mendelian genetics into the study of the oocyte. http://www.devbio.com/chap08/link0803.shtml
The polar lobe: cell determination and axis formation
Molluscs provide some of the most impressive examples of mosaic development, in which the blastomeres are specified autonomously, and of cytoplasmic localization, wherein morphogenetic determinants are placed in a specific region of the oocyte (see Chapter 3). Mosaic development is widespread throughout the animal kingdom, especially in protostomal organisms such as annelids, nematodes, and molluscs, all of which initiate gastrulation at the future anterior end after only a few cell divisions. Moreover, the cytoplasmic factors responsible for specification are actively moved to one pole of the cell so that a blastomere containing these factors can restrict their transmission to only one of its two daughter cells. The fate of the two daughter cells is thus changed by which one of them gets the morphogenetic determinant.
E. B. Wilson demonstrated that mosaic development characterizes the early snail embryo (see Figure 3.7). He also was able to demonstrate that such development is predicated on the segregation of specific morphogenetic determinants into specific blastomeres. Certain spirally cleaving embryos (mostly in the mollusc and annelid phyla) extrude a bulb of cytoplasm immediately before first cleavage (Figure 8.31). This protrusion is called the polar lobe. In certain species of snails, the region uniting the polar lobe to the rest of the egg becomes a fine tube. The first cleavage splits the zygote asymmetrically, so that the polar lobe is connected only to the CD blastomere. In several species, nearly one-third of the total cytoplasmic volume is present in this anucleate lobe, giving it the appearance of another cell. This three-lobed structure is often referred to as the trefoil-stage embryo (Figure 8.32). The CD blastomere then absorbs the polar lobe material, but extrudes it again prior to second cleavage. After this division, the polar lobe is attached only to the D blastomere, which absorbs its material. Thereafter, no polar lobe is formed.
Figure 8.31
Cleavage in the mollusc Dentalium. Extrusion and reincorporation of the polar lobe occur twice. (After Wilson 1904.)
Figure 8.32
Polar lobes of molluscs. (A) Scanning electron micrograph of the extending polar lobe in the uncleaved egg of Buccinum undatum. The surface ridges are confined to the polar lobe region. (B) Section through first-cleavage, or trefoil-stage, embryo of (more...)
Wilson (1904) showed that if one removes the polar lobe at the trefoil stage, the remaining cells divide normally. However, instead of producing a normal trochophore (snail) larva, they produce an incomplete larva, wholly lacking its mesodermal organs—muscles, mouth, shell gland,* and foot. Moreover, Wilson demonstrated that the same type of abnormal embryo can be produced by removing the D blastomere from the 4-cell embryo. Wilson concluded that the polar lobe cytoplasm contains the mesodermal determinants, and that these determinants give the D blastomere its mesoderm-forming capacity. Wilson also showed that the localization of the mesodermal determinants is established shortly after fertilization, thereby demonstrating that a specific cytoplasmic region of the egg, destined for inclusion in the D blastomere, contains whatever factors are necessary for the special cleavage rhythms of the D blastomere and for the differentiation of the mesoderm.
The morphogenetic determinants sequestered within the polar lobe are probably located in the cytoskeleton or cortex, not in the diffusible cytoplasm of the embryo. Evidence for this localization came from the studies of A. C. Clement (1968). When he separated the animal hemisphere of the egg of the snail Ilyanassa obsoleta from the vegetal hemisphere, the animal hemisphere formed ectodermal organs that resembled those formed by lobeless embryos. Clement then took embryos that had begun resorbing their second polar lobe and placed them into gelatin slabs. He centrifuged the embedded embryos, forcing the fluid, yolky cytoplasm from the vegetal part of the cell into the animal hemisphere. By centrifuging these embryos in a second, viscous medium, he caused the separation of the animal and vegetal hemispheres. The animal halves from such centrifuged embryos did not develop any more mesodermal or endodermal structures than those of uncentrifuged eggs. Thus, the determinants of the polar lobe were not transferred to the animal hemisphere in the fluid contents of the vegetal hemisphere. Van den Biggelaar (1977) obtained similar results when he removed the cytoplasm from the polar lobe with a micropipette. Cytoplasm from other regions of the cell flowed into the polar lobe, replacing the portion that he had removed. The subsequent development of these embryos was normal. In addition, when he added the soluble polar lobe cytoplasm to the B blastomere, duplications of structures were not seen (Verdonk and Cather 1983). Therefore, the diffusible part of the cytoplasm does not contain the morphogenetic determinants. They probably reside in the nonfluid cortical cytoplasm or on the cytoskeleton.
Clement (1962) also analyzed the further development of the D blastomere in order to observe the further appropriation of these determinants. The development of the D blastomere is illustrated in Figure 8.27. This macromere, having received the contents of the polar lobe, is larger than the other three. When one removes the D blastomere or its first or second macromere derivatives (1D or 2D), one obtains an incomplete larva, lacking heart, intestine, velum (the ciliated border of the larva), shell gland, eyes, and foot. When one removes the 3D blastomere (after the division of the 2D cell to form the 3D and 3d blastomeres), one obtains an almost normal embryo, having eyes, foot, velum, and some shell gland, but no heart or intestine (Figure 8.33). Therefore, some of the morphogenetic determinants originally present in the D blastomere must have been apportioned to the 3d cell. After the 4d cell is given off (by the division of the 3D blastomere), removal of the D derivative (the 4D cell) produces no qualitative difference in development. In fact, all the essential determinants for heart and intestine formation are now in the 4d blastomere, and removal of that cell results in a heartless and gutless larva (Clement 1986). The 4d blastomere is responsible for forming (at its next division) the two mesentoblasts, the cells that give rise to both the mesodermal (heart) and endodermal (intestine) organs.
Figure 8.33
Importance of the polar lobe in the development of Ilyanassa. (A) Normal larva. (B) Abnormal larva, typical of those produced when the polar lobe of the D blastomere is removed. (E, eye; F, foot; S, shell; ST, statocyst, a balancing organ; V, velum; VC, (more...)
The material in the polar lobe is also responsible for organizing the dorsal-ventral (back-belly) polarity of the embryo. When polar lobe material is forced to pass into the AB blastomere as well as into the CD blastomere, twin larvae are formed that are joined at their ventral surfaces (Figure 8.34; Guerrier et al. 1978; Henry and Martindale 1987).
Figure 8.34
Formation of twin embryos by the suppression of polar lobe formation in Dentalium. (A) Normal embryo at sixth-cleavage stage. (B) Twin embryos formed when low concentrations of cytochalasin are used to inhibit polar lobe formation and the polar lobe material (more...)
Thus, experiments have demonstrated that the nondiffusible polar lobe cytoplasm is extremely important in normal mollusc development for a number of reasons:
It contains the determinants for the proper cleavage rhythm and cleavage orientation of the D blastomere.
It contains certain determinants (those entering the 4d blastomere and hence leading to the mesentoblasts) for mesodermal and intestinal differentiation.
It is responsible for permitting the inductive interactions (through the material entering the 3d blastomere) leading to the formation of the shell gland and eye.
It contains determinants needed for specifying the dorsal-ventral axis of the embryo.
Although the polar lobe is clearly important for normal snail development, we still do not know the mechanisms of its effects. There appear to be no major differences in mRNA or protein synthesis between lobed and lobeless embryos (Brandhorst and Newrock 1981; Collier 1983, 1984). One possible clue has been provided by Atkinson (1987), who has observed differentiated cells of the velum, digestive system, and shell gland within lobeless embryos. Thus, lobeless embryos can produce these cells, but they appear unable to organize them into functional tissues and organs. Tissues of the digestive tract can be found, but they are not connected; muscle cells are scattered around the lobeless larva, but are not organized into a functional muscle tissue. Thus, the developmental functions of the polar lobe may be very complex.
Fate map of Ilyanassa obsoleta
As detailed above, fate maps of the snail Ilyanassa have been made means of ablation experiments. More recently, Joanne Render (1997) has constructed a more detailed fate map by injecting specific micromeres with beads containing the fluorescent dye Lucifer Yellow. The fluorescence is maintained over the period of embryogenesis and can be seen in the larval tissue derived from the injected cells. Figure 8.35 shows the new fate map of Ilyanassa obsoleta. The second quartet micromeres (2a-d) generally contribute to the shell-forming mantle, the velum, the mouth, and the heart. The third quartet micromeres (3a-d) generate large regions of the foot, velum, esophagus, and heart. The 4d cell, the mesentoblast, contributes to the larval kidney, heart, retractor muscles, and intestine.
Figure 8.35
Fate map of the snail Ilyanassa obsoleta. Beads containing Lucifer Yellow were injected into individual blastomeres at the 32-cell stage. When the embryos developed into larvae , their descendants could be identified by their fluorescence. (After Render (more...)
WEBSITE
8.4 Modifications of cell fate in spiralian eggs. Within the gastropods, differences in the timing of cell fate result in significantly different body plans. Furthermore, the leeches and nemerteans have modified the spiralian cleavage pattern to produce new types of body plans. http://www.devbio.com/chap08/link0804.shtml
