Cleavage and gastrulation

After fertilization, the zygote divides rapidly without growing, partitioning the egg's large cytoplasm into many smaller cells. This is cleavage. It ends in a blastula, a ball or sheet of cells, often surrounding a fluid-filled cavity. Then comes gastrulation, the most dramatic reorganization in an animal's life, in which cells move into the interior to form the three germ layers and the body plan takes shape. As the embryologist Lewis Wolpert put it, "It is not birth, marriage or death, but gastrulation, which is truly the most important time in your life." This lesson compares how different animals cleave and gastrulate and asks what controls these processes.

The cell cycle in cleavage

Cleavage divisions are unusually fast because they skip the growth phases of the cell cycle. In Xenopus the first cleavage occurs about 90 minutes after fertilization and the next eleven follow at intervals of about 30 minutes, alternating S phase and M phase with almost no G1 or G2. A Drosophila embryo is faster still, with nuclear cycles of under 10 minutes. The driving engine is the oscillation of maturation-promoting factor, a complex of cyclin B and the kinase Cdk1, whose activity rises to trigger mitosis and falls as cyclin is destroyed. Because the egg is stocked with maternal cyclin mRNA, histones and nucleotides, the embryo can run many cycles without transcription.

The rapid cycles eventually slow. In Xenopus this happens at the twelfth cleavage, after about 4,000 cells have formed, an event called the mid-blastula transition. Cell cycles lengthen and become asynchronous, G phases appear, cells become motile and zygotic transcription begins on a large scale. A key trigger is the ratio of nuclear DNA to cytoplasm: each cleavage doubles the DNA while the total volume stays the same, and when enough DNA has accumulated to titrate out maternal factors that repress transcription or limit replication, the transition occurs. Embryos made with extra DNA reach the transition earlier. More generally, the handover from maternal to zygotic control, the maternal-to-zygotic transition, involves degradation of maternal mRNAs as well as activation of the zygotic genome; it occurs at the 2-cell stage in mice and over 4 to 8 cell stages in humans.

import math
# Cleavage without growth: cell number doubles each cycle while total volume is fixed.
egg_diameter_um = 1200.0     # Xenopus egg, about 1.2 mm
egg_volume = math.pi / 6 * egg_diameter_um ** 3
first_cycle_min, cycle_min = 90, 30
print("cycle  time(h)   cells    mean cell diameter (um)")
for n in range(0, 14):
    cells = 2 ** n
    t = 0 if n == 0 else (first_cycle_min + (n - 1) * cycle_min) / 60
    d = (6 * egg_volume / cells / math.pi) ** (1 / 3)
    flag = "  <- mid-blastula transition" if n == 12 else ""
    print(f"{n:5d}  {t:6.1f}  {cells:7d}   {d:8.1f}{flag}")
# nuclear-to-cytoplasmic ratio rises in proportion to cell number
print(f"DNA:cytoplasm ratio rises {2 ** 12}-fold between the zygote and cycle 12")

cycle  time(h)   cells    mean cell diameter (um)
    0     0.0        1     1200.0
    1     1.5        2      952.4
    2     2.0        4      756.0
    3     2.5        8      600.0
    4     3.0       16      476.2
    5     3.5       32      378.0
    6     4.0       64      300.0
    7     4.5      128      238.1
    8     5.0      256      189.0
    9     5.5      512      150.0
   10     6.0     1024      119.1
   11     6.5     2048       94.5
   12     7.0     4096       75.0  <- mid-blastula transition
   13     7.5     8192       59.5
DNA:cytoplasm ratio rises 4096-fold between the zygote and cycle 12

The table of output shows why cleavage partitions rather than builds: by the mid-blastula transition, about 7 hours after fertilization, the embryo has thousands of cells, each only about 75 µm across, but the embryo as a whole is no bigger than the egg. The thousandfold increase in the ratio of DNA to cytoplasm is the timer for the transition.

Patterns of cleavage

The geometry of cleavage depends on two factors: the amount and distribution of yolk, which impedes division, and the orientation of the mitotic spindles, which is inherited from the egg's organization. In holoblastic (complete) cleavage the whole egg divides, as in sea urchins, frogs and mammals. In meroblastic (incomplete) cleavage only part of the egg divides, leaving a large undivided yolk mass, as in birds, reptiles, most fish and insects.

Radial cleavage, in sea urchins and with modification in frogs, divides the egg along planes parallel and perpendicular to the animal–vegetal axis, so tiers of cells sit directly above one another. In Xenopus, yolk concentrated in the vegetal hemisphere slows division there, so animal cells (micromeres) are smaller than vegetal cells (macromeres). Spiral cleavage, characteristic of molluscs, annelids and flatworms, the spiralians, tilts the spindles at an angle so that each tier is offset from the one below; it is usually accompanied by highly autonomous specification and a nearly invariant cell lineage. The direction of the spiral, left or right, is set by a maternal gene and determines the coiling of snail shells: Lymnaea snails with a mutation in the maternal formin gene Lsdia1 produce embryos with mirror-image cleavage and left-coiling shells.

Mammalian cleavage is rotational and slow. The first division is meridional, and in the second one cell divides meridionally while the other divides equatorially. Divisions take 12 to 24 hours and are asynchronous. At the 8-cell stage mouse blastomeres undergo compaction, flattening against each other and forming tight junctions, and the outer cells become polarized. Cells on the outside develop into the trophectoderm, which forms the embryonic part of the placenta, while the inside cells form the inner cell mass, from which the embryo proper arises. The trophectoderm pumps fluid into the interior to make a cavity, producing the blastocyst. This first fate decision depends on position and polarity: the Hippo signalling pathway is inactive in polarized outer cells, allowing the coactivator Yap to enter the nucleus and, with the transcription factor Tead4, activate trophectoderm genes such as Cdx2. Inner cells, lacking polarity, keep Hippo signalling active and express pluripotency factors such as Oct4. The inner cell mass then segregates into epiblast, which forms the embryo, and primitive endoderm, which forms the yolk sac.

Discoidal meroblastic cleavage in birds and fish confines division to a small disc of cytoplasm, the blastodisc, on top of the yolk. In the chick, by the time the egg is laid, cleavage has produced a blastoderm of some 20,000 cells. Insects show superficial cleavage: nuclei divide without cell membranes in the centre of the egg, migrate to the periphery to form a syncytial blastoderm, and only then are separated by membranes, forming the cellular blastoderm. This syncytial stage is what allows transcription factor gradients to pattern the Drosophila embryo, as lesson 4 explains.

Gastrulation: forming the germ layers

Gastrulation converts the blastula into an embryo with three germ layers. Ectoderm forms the outer layer and gives rise to the epidermis and the nervous system. Endoderm forms the innermost layer and gives rise to the lining of the gut and its derivatives. Mesoderm forms between them and gives rise to muscle, skeleton, connective tissue, the heart and blood, the kidneys and the gonads. Animals with three germ layers are called triploblasts; cnidarians such as sea anemones have only two and are diploblasts. The germ layers are not absolute commitments, and the neural crest, an ectodermal population that forms bone and cartilage in the head, is sometimes called a fourth germ layer.

Cell movements of gastrulation

Gastrulation uses a small repertoire of cell behaviours, combined differently in different species. Invagination is the infolding of a sheet of cells, like poking a finger into a balloon. Involution is the inturning of an expanding outer layer so that it spreads over the inner surface. Ingression is the migration of individual cells from the surface into the interior, often by epithelial–mesenchymal transition. Delamination is the splitting of one sheet into two. Epiboly is the spreading of an epithelial sheet to cover deeper layers. Convergent extension is the narrowing and lengthening of a tissue as cells intercalate between their neighbours, driven in vertebrates by the planar cell polarity pathway.

Gastrulation in four embryos

In the sea urchin, about 32 small micromere descendants at the vegetal pole undergo epithelial–mesenchymal transition and ingress into the blastocoel as primary mesenchyme cells, which will build the larval skeleton. The vegetal plate then invaginates to form the archenteron, the primitive gut, which elongates by convergent extension and is pulled across the blastocoel by filopodia of secondary mesenchyme cells until it touches the opposite wall, where the mouth forms. The blastopore becomes the anus, the defining feature of deuterostomes. In protostomes, such as insects and molluscs, the blastopore typically gives rise to the mouth.

In Xenopus, gastrulation begins on the dorsal side with the formation of bottle cells, which constrict their apical surfaces and initiate invagination at the dorsal lip of the blastopore. Mesoderm then involutes over the lip and migrates along the inner surface of the blastocoel roof, using a fibronectin matrix, while animal cap ectoderm spreads by epiboly to cover the embryo. The blastopore lip extends laterally and ventrally to form a ring around a plug of yolky endoderm. The dorsal lip is the site of the Spemann–Mangold organizer, the subject of lesson 5.

In the chick, gastrulation proceeds through the primitive streak, a thickening that forms in the posterior epiblast and extends forward. Epiblast cells converge on the streak, undergo epithelial–mesenchymal transition and ingress through it. The first cells to ingress displace the hypoblast to form definitive endoderm; later cells spread between the layers as mesoderm. The anterior end of the streak, Hensen's node, is the bird equivalent of the amphibian organizer. As the streak regresses toward the posterior, it lays down the notochord and somites in its wake, so that the embryo develops from head to tail. Mammals, including humans, gastrulate through a primitive streak in the same way; in humans the streak appears around day 14 after fertilization, which is the developmental basis of the 14-day limit on human embryo research discussed in lesson 12.

Zebrafish gastrulation begins as the blastoderm spreads by epiboly over the yolk. At the margin, cells involute to form an inner layer, the hypoblast, and converge toward the dorsal side, where the embryonic shield, the fish organizer, forms. Convergent extension elongates the body axis. Throughout these embryos the same cell behaviours appear in different proportions, and the same signalling pathways, particularly Nodal, Wnt and fibroblast growth factor, specify the mesoderm and endoderm that move.

Mechanics and molecules of cell movement

Gastrulation is a problem in mechanics as well as in gene regulation. Apical constriction, driven by actomyosin contraction under the control of the small GTPase RhoA, changes cell shape from columnar to wedge-like and bends sheets. Changes in cell adhesion, especially of cadherins, allow cells to separate from or remain within epithelia; epithelial–mesenchymal transition involves loss of E-cadherin under the control of transcription factors such as Snail and Twist, the same factors that promote cancer metastasis. Mesodermal cells crawl on extracellular matrix proteins such as fibronectin. Differential adhesion and cortical tension can make tissues sort into layers, as Malcolm Steinberg showed by mixing dissociated cells from different tissues, which sorted out like immiscible liquids.