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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

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Molecular Biology of the Cell. 4th edition.

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Caenorhabditis Elegans: Development from the Perspective of the Individual Cell

The nematode worm Caenorhabditis elegans is a small, relatively simple, and precisely structured organism. The anatomy of its development has been described in extraordinary detail, and one can map out the exact lineage of every cell in the body. Its complete genome sequence is also known, and large numbers of mutant phenotypes have been analyzed to determine gene functions. If there is any multicellular animal whose development we should be able to understand in terms of genetic control, this is it.

There are, it is true, some factors that make such an understanding difficult. Anatomically simple though it is, it has more genes (about 18,000) and more cells (about 1000) than one can easily keep track of without a computer. It is not the most directly instructive model organism if one wants to understand how animals such as ourselves form organs such as eyes or limbs, which the worm lacks, and it is hard to do transplantation experiments. Moreover, DNA sequence comparisons indicate that, while the lineages leading to nematodes, insects, and vertebrates diverged from one another at about the same time, the rate of evolutionary change in the nematode lineage has been substantially greater: both its genes and its body structure are more divergent from our own than are those of Drosophila.

In spite of all this, the worm presents us with an excellent introductory example: it poses the basic general questions of animal development in a relatively simple form, and it lets us answer them in terms of the behavior of individual, identified cells.

Caenorhabditis elegans Is Anatomically Simple

As an adult, C. elegans consists of only about 1000 somatic cells and 1000–2000 germ cells (exactly 959 somatic cell nuclei plus about 2000 germ cells are counted in one sex; exactly 1031 somatic cell nuclei plus about 1000 germ cells in the other) (Figure 21-16). The anatomy has been reconstructed, cell by cell, by electron microscopy of serial sections. The body plan of the worm is simple: it has a roughly bilaterally symmetrical, elongate body composed of the same basic tissues as in other animals (nerve, muscle, gut, skin), organized with mouth and brain at the anterior end and anus at the posterior. The outer body wall is composed of two layers: the protective epidermis, or “skin,” and the underlying muscular layer. A tube of endodermal cells forms the intestine. A second tube, located between the intestine and the body wall, constitutes the gonad; its wall is composed of somatic cells, with the germ cells inside it.

Figure 21-16. Caenorhabditis elegans.

Figure 21-16

Caenorhabditis elegans. A side view of an adult hermaphrodite is shown. (From J.E. Sulston and H.R. Horvitz, Dev. Biol. 56:110–156, 1977. © Academic Press.)

C. elegans has two sexes—a hermaphrodite and a male. The hermaphrodite can be viewed most simply as a female that produces a limited number of sperm: she can reproduce either by self-fertilization, using her own sperm, or by cross-fertilization after transfer of male sperm by mating. Self-fertilization allows a single heterozygous worm to produce homozygous progeny. This is an important feature that helps to make C. elegans an exceptionally convenient organism for genetic studies.

Cell Fates in the Developing Nematode Are Almost Perfectly Predictable

C. elegans begins life as a single cell, the fertilized egg, which gives rise, through repeated cell divisions, to 558 cells that form a small worm inside the egg shell. After hatching, further divisions result in the growth and sexual maturation of the worm as it passes through four successive larval stages separated by molts. After the final molt to the adult stage, the hermaphrodite worm begins to produce its own eggs. The entire developmental sequence, from egg to egg, takes only about three days.

The lineage of all of the cells from the single-cell egg to the multicellular adult was mapped out by direct observation of the developing animal. In the nematode, a given precursor cell follows the same pattern of cell divisions in every individual, and with very few exceptions the fate of each descendant cell can be predicted from its position in the lineage tree (Figure 21-17).

Figure 21-17. The lineage tree for the cells that form the gut (the intestine) of C. elegans.

Figure 21-17

The lineage tree for the cells that form the gut (the intestine) of C. elegans. Note that although the intestinal cells form a single clone (as do the germ-line cells), the cells of most other tissues do not. Nerve cells (not shown in the drawing of the (more...)

This degree of stereotyped precision is not seen in the development of larger animals. At first sight, it might seem to suggest that each cell lineage in the nematode embryo is rigidly and independently programmed to follow a set pattern of cell division and cell specialization, making the worm a woefully unrepresentative model organism for development. We shall see that this is far from true: as in other animals, development depends on cell-cell interactions as well as on processes internal to the individual cells. The outcome in the nematode is almost perfectly predictable simply because the pattern of cell-cell interactions is highly reproducible and is accurately correlated with the sequence of cell divisions.

Products of Maternal-Effect Genes Organize the Asymmetric Division of the Egg

In the developing worm, as in other animals, most cells do not become restricted to a single pathway of differentiation until quite late in development, and cells of a particular type, such as muscle, usually derive from several spatially dispersed precursors that also give rise to other types of cells. The exceptions, in the worm, are the gut and the gonad, each of which forms from a single dedicated founder cell, born at the 8-cell stage of development for the gut-cell lineage and at the 16-cell stage for the germ-cell lineage, or germ line. Other cells in the embryo, meanwhile, are also becoming different from one another, even though they are not yet committed with regard to their terminal mode of differentiation. How do these early differences between cells arise?

The worm is typical of most animals in the early specification of the cells that will eventually give rise to the germ cells (eggs or sperm). The worm's germ line is produced by a strict series of asymmetric cell divisions of the fertilized egg. The asymmetry originates with a cue from the egg's environment: the sperm entry point defines the future posterior pole of the elongated egg. The proteins in the egg then interact with one another and organize themselves in relation to this point so as to create a more elaborate and extreme asymmetry in the interior of the cell. The proteins involved are mainly translated from the accumulated mRNA products of the genes of the mother. Because this RNA is made before the egg is laid, it is only the mother's genotype that dictates what happens in the first steps of development. Genes acting in this way are called maternal-effect genes.

A subset of maternal-effect genes are specifically required to organize the asymmetric pattern of the nematode egg. These are called par (partitioning-defective) genes, and at least six have been identified, through genetic screens for mutants where this pattern is disrupted. The par genes have homologs in insects and vertebrates, where they are also involved in organizing cell asymmetry, coordinating the polarization of the cytoskeleton with the distribution of other cell components

In the nematode egg, the Par proteins (the products of the par genes) serve to bring a set of ribonucleoprotein particles called P granules to the posterior pole, so that the posterior daughter cell inherits P granules and the anterior daughter cell does not. Throughout the next few cell divisions, the Par proteins operate in a similar way, orienting the mitotic spindle and segregating the P granules to one daughter cell at each mitosis, until, at the 16-cell stage, there is just one cell that contains the P granules (Figure 21-18). This one cell gives rise to the germ line.

Figure 21-18. Asymmetric divisions segregating P granules into the founder cell of the C. elegans germ line.

Figure 21-18

Asymmetric divisions segregating P granules into the founder cell of the C. elegans germ line. The micrographs in the upper row show the pattern of cell divisions, with cell nuclei stained blue with a DNA-specific fluorescent dye; below are the same cells (more...)

The specification of the germ-cell precursors as distinct from somatic-cell precursors is a key event in the development of practically every type of animal, and the process has common features even in phyla with very different body plans. Thus in Drosophila, particles similar to P granules are also segregated into one end of the egg, and become incorporated into the germ-line precursor cells to determine their fate. Similar phenomena occur in fish and frogs. In all these species, one can recognise at least some of the same proteins in the germ-cell-determining material, including homologs of an RNA-binding protein called Vasa. How Vasa and its associated proteins and RNA molecules act to define the germ line is still unknown.

Progressively More Complex Patterns Are Created by Cell-Cell Interactions

The egg, in C. elegans as in other animals, is an unusually big cell, with room for complex internal patterning. In addition to the P granules, other factors become distributed in an orderly way along its anteroposterior axis under the control of the Par proteins, and thus are allocated to different cells as the egg goes through its first few cell-division cycles. These divisions occur without growth (since feeding cannot begin until a mouth and a gut have formed) and therefore subdivide the egg into progressively smaller cells. Several of the localized factors are gene regulatory proteins, which act directly in the cell that inherits them to either drive or block the expression of specific genes, adding to the differences between that cell and its neighbors and committing it to a specialized fate.

While the first few differences between cells along the anteroposterior axis of C. elegans result from asymmetric divisions, further patterning, including the pattern of cell types along the other axes, depends on interactions between one cell and another. The cell lineages in the embryo are so reproducible that individual cells can be assigned names and identified in every animal (Figure 21-19); the cells at the four-cell stage, for example, are called ABa and ABp (the two anterior sister cells), and EMS and P2 (the two posterior sister cells). As a result of the asymmetric divisions we have just described, the P2 cell expresses a signal protein on its surface—a nematode homolog of the Notch ligand Delta—while the ABa and ABp cells express the corresponding transmembrane receptor—a homolog of Notch. The elongated shape of the eggshell forces these cells into an arrangement such that the most anterior cell, ABa, and the most posterior cell, P2, are no longer in contact with one another. Thus only the ABp cell and the EMS cell are exposed to the signal from P2. This signal acts on ABp, making it different from ABa and defining the future dorsal-ventral axis of the worm (Figure 21-20).

Figure 21-19. The pattern of cell divisions in the early nematode embryo, indicating the names and fates of the individual cells.

Figure 21-19

The pattern of cell divisions in the early nematode embryo, indicating the names and fates of the individual cells. Cells that are sisters are shown linked by a short black line. (After K. Kemphues, Cell 101:345–348, 2000.)

Figure 21-20. Cell-signaling pathways controlling assignment of different characters to the cells in a four-cell nematode embryo.

Figure 21-20

Cell-signaling pathways controlling assignment of different characters to the cells in a four-cell nematode embryo. The P2 cell uses the Notch signaling pathway to send an inductive signal to the ABp cell, causing this to adopt a specialized character. (more...)

At the same time, P2 also expresses another signaling molecule, a Wnt protein, which acts on a Wnt receptor (a Frizzled protein) in the membrane of the EMS cell. This signal polarizes the EMS cell in relation to its site of contact with P2, controlling the orientation of the mitotic spindle. The EMS cell then divides to give two daughters that become committed to different fates as a result of the Wnt signal from P2. One daughter, the MS cell, will give rise to muscles and various other body parts; the other daughter, the E cell, is the founder cell for the gut, committed to give rise to all the cells of the gut and to no other tissues (see Figure 21-20).

Having described the chain of cause and effect in early nematode development, we now examine some of the methods that have been used to decipher it.

Microsurgery and Genetics Reveal the Logic of Developmental Control; Gene Cloning and Sequencing Reveal Its Molecular Mechanisms

To discover the causal mechanisms, we need to know the developmental potential of the individual cells in the embryo. At what points in their lives do they undergo decisive internal changes that determine them for a particular fate, and at what points do they depend on signals from other cells? In the nematode, laser microbeam microsurgery can be conveniently used to kill one or more of a cell's neighbors and then to observe directly how the cell behaves in the altered circumstances. Alternatively, cells of the early embryo can be pushed around and rearranged inside the eggshell using a fine needle. For example, the relative positions of ABa and ABp can be flipped at the four-cell stage of development. The ABa cell then undergoes what would normally be the fate of the ABp cell, and vice versa, showing that the two cells initially have the same developmental potential and depend on signals from their neighbors to make them different. A third tactic is to remove the eggshell of an early C. elegans embryo by digesting it with enzymes, and then to manipulate the cells in culture. The existence of a polarizing signal from P2 to EMS was demonstrated in this way.

Genetic screens were used to identify the genes involved in the P2-EMS cell interaction. A search was made for mutant strains of worms in which no gut cells were induced (called mom mutants, because they had more mesoderm—mesoderm being the fate of both of the EMS cell daughters when induction fails). Cloning and sequencing the mom genes revealed that one encodes a Wnt signal protein that is expressed in the P2 cell, while another encodes a Frizzled protein (a Wnt receptor) that is expressed in the EMS cell. A second genetic screen was conducted for mutant strains of worms with the opposite phenotype, in which extra gut cells are induced (called pop mutants, because they have plenty of pharynx as a result of the extra gut). One of the pop genes (pop-1) turns out to encode a gene regulatory protein (a LEF-1/TCF homolog) whose activity is down-regulated by Wnt signaling in C. elegans. When Pop-1 activity is absent, both daughters of the EMS cell behave as though they have received the Wnt signal from P2. Similar genetic methods were used to identify the genes whose products mediate the Notch-dependent signaling from P2 to ABa.

Continuing in this way, it is possible to build up a detailed picture of the decisive events in nematode development, and of the genetically specified machinery that controls them.

Cells Change Over Time in Their Responsiveness to Developmental Signals

The complexity of the adult nematode body is achieved through repeated use of a handful of patterning mechanisms, including those we have just seen in action in the early embryo. For example, asymmetric cell divisions dependent on the Pop-1 gene regulatory proteins occur throughout C. elegans development, creating differences between anterior and posterior sister cells.

As emphasized earlier, while the same few types of signals act repeatedly at different times and places, the effects they have are different because the cells are programmed to respond differently according to their age and their past history. We have seen, for example, that at the four-cell stage of development, one cell, ABp, changes its developmental potential because of a signal received via the Notch pathway. At the 12-cell stage of development, the granddaughters of the ABp cell and the granddaughters of the ABa cell both encounter another Notch signal, this time from a daughter of the EMS cell. The ABa granddaughter changes its internal state in response to this signal and begins to form the pharynx. The ABp granddaughter does no such thing—the earlier exposure to a Notch signal has made it unresponsive. Thus, at different times in their history, both ABa lineage cells and ABp lineage cells respond to Notch, but the outcomes are different. Somehow a Notch signal at the 12-cell stage induces pharynx, but a Notch signal at the 4-cell stage has other effects—which include the prevention of pharynx induction by Notch at a later stage.

Heterochronic Genes Control the Timing of Development

A cell does not have to receive an external cue in order to change: one set of regulatory molecules inside the cell can provoke production of another, and the cell can thus step through a series of different states autonomously. These states differ not only in their responsiveness to external signals, but also in other aspects of their internal chemistry, including proteins that stop or start the cell-division cycle. In this way, the internal mechanisms of the cell, together with the past and present signals received, dictate both the sequence of biochemical changes in the cell and the timing of its cell divisions.

The specific molecular details of the mechanisms governing the temporal program of development are still mysterious. Remarkably little is known, even in the nematode embryo with its rigidly predictable pattern of cell divisions, about how the sequence of cell divisions is controlled. However, for the later stages, when the larva feeds and grows and moults to become an adult, it has been possible to identify some of the genes that control the timing of cellular events. Mutations in these genes cause heterochronic phenotypes: the cells in a larva of one stage behave as though they belonged to a larva of a different stage, or cells in the adult carry on dividing as though they belonged to a larva (Figure 21-21).

Figure 21-21. Heterochronic mutations in the lin-14 gene of C. elegans.

Figure 21-21

Heterochronic mutations in the lin-14 gene of C. elegans. The effects on only one of the many affected lineages are shown. The loss-of-function (recessive) mutation in lin-14 causes premature occurrence of the pattern of cell division and differentiation (more...)

Through genetic analyses, one can determine that the products of the heterochronic genes act in series, forming regulatory cascades. Curiously, two genes at the top of their respective cascades, called lin-4 and let-7, do not code for proteins but for short untranslated RNA molecules (21 or 22 nucleotides long). These act by binding to complementary sequences in the non-coding regions of mRNA molecules transcribed from other heterochronic genes, thereby controlling their rate of translation or possibly their degradation (perhaps by a mechanism similar to that of RNAi—see p. 451). Increasing levels of lin-4 RNA govern the progression from larval stage-1 cell behavior to larval stage-3 cell behavior, increasing levels of let-7 RNA govern the progression from late larva to adult.

RNA molecules that are identical or almost identical to the let-7 RNA are found in many other species, including Drosophila, zebrafish, and human. Moreover, these RNAs appear to act in a similar way to regulate the level of their target mRNA molecules, and the targets themselves are homologous to the targets of let-7 RNA in the nematode. The evidence therefore suggests that this system has a universal role in governing the timing of a switch from an early to a late style of cell behavior.

Cells Do Not Count Cell Divisions in Timing Their Internal Programs

Since the steps of cell specialization have to be coordinated with cell divisions, it is often suggested that the cell division cycle might serve as a clock to control the tempo of other events in development. In this view, changes of internal state would be locked to passage through each division cycle: the cell would click to the next state as it went through mitosis, so to speak. There is a substantial amount of evidence that this idea is wrong. Cells in developing embryos, whether they be worms, flies, or vertebrates, usually carry on with their standard timetable of determination and differentiation even when progress through the cell-division cycle is artificially blocked. Necessarily, there are some abnormalities, if only because a single undivided cell cannot differentiate in two ways at once. But in most cases that have been studied, it seems clear that the cell changes its state with time regardless of cell division, and that this changing state controls both the decision to divide and the decision as to when and how to specialize.

Selected Cells Die by Apoptosis as Part of the Program of Development

The control of cell numbers in development depends on cell death as well as cell division. A C. elegans hermaphrodite generates 1030 somatic cell nuclei in the course of its development, but 131 of the cells die. These programmed cell deaths occur in an absolutely predictable pattern. In C. elegans, they can be chronicled in detail, because one can trace the fate of each individual cell and see who dies, watching as each suicide victim undergoes apoptosis and is rapidly engulfed and digested by neighboring cells (Figure 21-22). In other organisms, where close observation is harder, such deaths easily go unnoticed; but cell death by apoptosis is probably the fate of a substantial fraction of the cells produced in most animals, playing an essential part in generating an individual with the right cell types in the right numbers and places, as discussed in Chapter 17.

Figure 21-22. Apoptotic cell death in C. elegans.

Figure 21-22

Apoptotic cell death in C. elegans. Death depends on expression of the ced-3 and ced-4 genes in the absence of ced-9 expression—all in the dying cell itself. The subsequent engulfment and disposal of the remains depend on expression of other genes (more...)

Genetic screens in C. elegans have been crucial in identifying the genes that bring about apoptosis and in highlighting its importance in development. Three genes, called ced-3, ced-4, and egl-1, (ced stands for cell death abnormal), are found to be required for the 131 normal cell deaths to occur. If these genes are inactivated by mutation, cells that are normally fated to die survive instead, differentiating as recognizable cell types such as neurons. Conversely, over-expression or misplaced expression of the same genes causes many cells to die that would normally survive, and the same effect results from mutations that inactivate another gene, ced-9, which normally represses the death program.

All these genes code for conserved components of the cell-death machinery. As described in Chapter 17, ced-3 codes for a caspase homolog, while ced-4, ced-9 and egl-1 are respectively homologs of Apaf-1, Bcl-2, and Bad. Without the insights that came from detailed analysis of the development of the transparent, genetically tractable nematode worm, it would have been very much harder to discover these genes and understand the cell-death process in vertebrates.

Summary

The development of the small, relatively simple, transparent nematode worm Caenorhabditis elegans is extraordinarily reproducible and has been chronicled in detail, so that a cell at any given position in the body has the same lineage in every individual, and this lineage is fully known. Also, the genome has been completely sequenced. Thus powerful genetic and microsurgical approaches can be combined to decipher developmental mechanisms. As in other organisms, development depends on an interplay of cell-cell interactions and cell-autonomous processes. Development begins with an asymmetric division of the fertilized egg, dividing it into two smaller cells containing different cell-fate determinants. The daughters of these cells interact via the Notch and Wnt cell signaling pathways to create a more diverse array of cell states. Meanwhile, through further asymmetric divisions one cell inherits materials from the egg that determine it at an early stage as progenitor of the germ line.

Genetic screens identify the sets of genes responsible for these and later steps in development, including, for example, cell-death genes that control the apoptosis of a specific subset of cells as part of the normal developmental program. Heterochronic genes that govern the timing of developmental events have also been found, although in general our understanding of temporal control of development is still very poor. There is good evidence, however, that the tempo of development is not set by the counting of cell divisions.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
Bookshelf ID: NBK26861