Spiral cleavage

Developmental biologists are acutely interested in asymmetries in development: they are visible cues to some underlying regional differences. For instance, we'd like to know the molecules and interactions involved in taking a seemingly featureless sphere, the egg, and specifying one side to go on to form a head, and the the opposite side to form a tail. We'd like to understand why our back (or dorsal) side looks different from our belly (or ventral) side. One particularly intriguing distinction, though, is the left-right axis. For the most part, left and right are nearly identical, mirror-images of one another, but there are also key asymmetries. Your heart, for instance, is larger on the left side than the right, your liver lies mostly on the right side of your abdomen while the stomach arcs to the left, and these arrangements are essential for normal function. Left-right asymmetries are more subtle than anterior-posterior or dorsal-ventral differences, and that makes them especially fascinating.

One way to study left-right asymmetries is to pick an animal in which they are more pronounced. Snails are the classic example, since they have a coiled shell which can have either a right-handed or left-handed twist, so you can spot the handedness of the asymmetry with a glance at the adult. Snails have become the canonical example of left-right asymmetries for another reason, too: the asymmetry is established by the third division (the eight-cell stage) of the developing mollusc embryo.

Many molluscs and annelids exhibit an early pattern of highly determinate cleavages. That is, the zygote goes through an amazingly stereotyped pattern of divisions that produces a characteristic arrangement of cells—so stereotyped, that investigators can give each cell individual names, and find that same cell in each and every individual of that species (and even in different, related species). Here, for instance, is Conklin's summary diagram of early development in the mollusc, Crepidula^*.

The top row is the two-cell stage, and you can see the nuclei in the named cells (AB and CD) maneuvering and going into mitosis. The middle row shows the similar events in the four-cell stage, with the four cells named A, B, C, and D, and then the bottom left picture is the 8-cell stage, the bottom middle is the 29-cell stage (I don't know about you, but just the concept that there is a 29-cell stage tickles),and the last image is the snail gastrula. Everything in this sequence is reproducible and predictable. For instance, the cells labeled "M" in the last picture are the mesodermal precursors that will go on to form muscle and connective tissue, and we know that those cells always arise from the 4d cell at the 29-cell stage. Isn't that amazing? It's like clockwork development, fixed and unchanging.

But for now, let's just focus on the third division, when four cells become eight (figure E to figure F). The molluscan pattern of early divisions is called spiral cleavage after this one step. The A, B, C, and D cells are called macromeres because they are very large cells, and their daughter cells from this division are called micromeres (1a, 1b, 1c, 1d) because, obviously, they are much smaller. In addition, when they bud off the macromeres, the micromeres don't lie directly above them, but are rotated clockwise just a bit, so the cell bodies lie in the furrows between the macromeres. That third division with a twist is what gives this the name 'spiral cleavage'. A division without the twist, such as we have, is called 'radial cleavage'.

If you have a little trouble visualizing that, try watching this short time-lapse movie of the third cleavage (QuickTime, 1.8MB) from Shibazaki et al..

The little twist doesn't seem like much, but since the highly ordered cascade of divisions that follow are dependent on the relationships between all eight of these cells, changing it changes the asymmetries we can see in the 29-cell stage and the gastrula. Those asymmetries are perpetuated right into the adult. Here's another figure from the Shibazake et al. paper showing what the divisions lead to.

The Third Cleavage Patterns of the Dextral and Sinistral L. stagnalis Embryos and Their Adult Snails. (A) The 4-cell to the 8-cell stage of the sinistral and dextral L. stagnalis embryos observed by light microscopy. A quartet of micromeres is displaced anticlockwise in the levotropic cleavage (blue arrow, left) and clockwise in the dextrotropic cleavage (blue arrow, right) when viewed from the animal pole. (B) These 8-cell stage embryos eventually become the sinistral (left) and the dextral (right) adult snails. Scale bars equal 20 µm in(A) and 10 mm in (B).

First, just look at the right side of the figure. This is the normal pattern seen in the snail they studied, Lymnaea. In the transition from 4 to 8 cells, the micromeres make a clockwise or right-handed rotation, and the adult snail subsequently has a right-handed twist to its shell. We can see that this is a consequence of the early rotation because this species also has a rare form with a left-handed spiral to the shell, shown on the left side of the figure…and at its third division, the micromeres rotate counterclockwise! You can watch it: here's another time-lapse movie of levotropic, or left-handed, cleavage (QuickTime, 1.2MB).

This is dazzling stuff, because it is rare to see such an early, simple event so cleanly and comprehensibly mapped onto a much more complex, later event. It gets even better: we can see that it is associated with the chirality of organelles within the cell, and with chemical properties of their constituent molecules. Here are some embryos that have been stained for mitotic spindles and cytoskeleton.

Dynamics of Mitotic Spindles and Microfilaments at the Third Cleavage of the Dextral and Sinistral L. stagnalis Embryos. (A) 3D-reconstruction images of mitotic spindles visualized by indirect immunofluorescence with anti-β-tubulin antibody. Horizontal orientations of the prometaphase spindles are random in both embryos (top). In metaphase-anaphase, spindles in the dextral embryos are oriented helically (bottom right), but those in the sinistral embryos are positioned radially (bottom left). Red arrows indicate the orientation of the spindles by a horizontal projection and arrowheads are nearest to the viewer. The outline of each embryo is indicated by a white line. (B and C) 3D-reconstruction images of embryos in metaphase-anaphase (B) and in telophase (C). Animal-view (top) and the corresponding lateral-view (bottom) images of embryos that are double-stained for filamentous actin (green) and β-tubulin (red). Arrowheads in (B) indicate the direction of the spiral deformation (SD) of the dextral embryos. Scale bar equals 20 µm.

The bright structures in A are the spindles, or small organelles made up mostly of tubulin that organize the mitotic apparatus, and determine the orientation of cell divisions. Daughter cells will bud off of the parent micromere in the direction of the arrows (which also point upwards out of the plane of the screen, something hard to capture in 2D). One of the unexpected observations of the Shibazaki et al. paper is that the sinistral mutant is not simply a mirror image of the dextral form—the spindles completely lack the skewed twist of the normal snail, and the third cleavage is actually more radial than spiral. Similarly, in B, the spindles are stained red and the actin cytoskeleton is stained green, and we again see a difference. The dextral form telegraphs the coming spiral cleavage by bulges in the cell membrane (arrowheads) called spiral distortions. The sinistral form lacks them.

The mutation in the sinistral form destroys the precise spiral asymmetry of the early embryo, but one unexplained phenomenon is that after the third division, the micromeres still migrate towards the furrows between the macromeres…but they consistently twist to the left instead of the right.

One final observation. The authors also examined a different species, Physa acuta, which always has a left handed twist to its shell. As you might expect, the third division is also always made with a left-handed twist…and unlike the sinistral mutant of Lymnaea stagnalis, its spindles are consistently and appropriately skewed in a mirror-image way with respect to the dextral Lymnaea.

The Third Spiral Cleavage and Organismal Handedness Summarized for Gastropod Enantiomorphs within a Species and across Species. The schematic representation was drawn based on our experiments on the recessive sinistral (left) and dominant dextral (middle) L. stagnalis embryos and the dominant sinistral P. acuta embryo (right). Metaphase spindles are shown in red with daughter chromosomes aligned at the equators. Fainter colored spindles in the lateral view of metaphase 4-cell embryos are in blastomeres further removed from the viewer. Spindles are omitted from telophase figures for clarity. Blue broken lines in the telophase embryos represent the positions of the initial contractile rings. Future micromeres at telophase and ones established in the 8-cell embryos are shaded in dark gray. Pink bars in 8-cell embryos link a daughter micromere and a macromere originated from the same blastomere at 4-cell stage.

^*I love many of the names of the marine invertebrates used in developmental studies. Ciona, Cynthia, even Dentalia are like poetry to me. But in my catalog of unfortunately unlovely names, Crepidula fornicata tops the list. It sounds both crudely scatalogical and pornographic. Poor mollusc!

Shibazaki Y, Shimizu M, Kuroda R (2004) Body handedness is directed by genetically determined cytoskeletal dynamic in the early embryo. Current Biology 14:1462-1467.