Contenidos

Vertebrates may look bilaterally symmetrical from the outside, but many of their internal organs—the heart, the stomach, the liver, and so on—are highly asym- metric. This asymmetry is quite reproducible: 99.98% of people have their heart on the left. We have seen how a vertebrate embryo develops its internal and external tissue layers and its anteroposterior and dorsoventral axes. But how does the left–right asymmetry arise?

Genetic studies in mammals show that this problem can be broken down into two distinct questions—one concerning the creation of asymmetry and the other concerning its orientation. Several mutations are known, in humans and in mice, that cause a randomization of the left–right axis: 50% of the mutant individuals have their internal organs arranged in the normal way, while the other 50% have an inverted anatomy, with the heart on the right. In these indi- viduals, it seems, the mechanism that makes the left and right sides different has functioned correctly, but the mechanism that decides between the two possible orientations of the left–right axis is defective.

A key to the basis of these phenomena comes from the discovery of molec- ular asymmetries that precede the first gross anatomical asymmetries. The ear- liest signs are seen in patterns of gene expression in the neighborhood of the node—the homolog in mouse and chick of the frog Organizer. In particular, the gene Nodal, coding for a member of the TGFb superfamily, is expressed asym- metrically in this region (not only in the mouse, but also in chick, frog and zebrafish) (Figure 22–87). Asymmetry of Nodal expression in the immediate neighborhood of the node is relayed outward to create a broad stripe of Nodal

Figure 22–86 Effect of mutations in the Kit gene. Both the baby and the mouse

are heterozygous for a loss-of-function mutation that leaves them with only half the normal quantity of Kit gene product. In both cases pigmentation is defective because pigment cells depend on the Kit product as a receptor for a survival factor. (Courtesy of R.A. Fleischman, from Proc. Natl Acad. Sci. U.S.A. 88:10885–10889, 1991. With permission from National Academy of Sciences.) LEFT RIGHT node (A) (B) asymmetric Nodal expression developing notochord beating cilia drive asymmetric flow of extracellular fluid primitive streak 100 mm LEFT RIGHT Nodal Pitx2 Lefty heart develops on left side

Figure 22–87Helical beating of cilia at the node, and the origins of left-right asymmetry. (A) The beating of the cilia

drives a fluid flow toward one side of the node, and this leads to asymmetric gene expression in the neighborhood of the node. According to one theory, the flow exerts this effect by carrying extracellular signal proteins to one side. Another theory notes that cilia can also function as mechanosensors, and proposes that a subset of cilia at the node respond to deflection due to the fluid flow by opening Ca2+_{channels so as to create an} increased Ca2+_{concentration in the cells} on one side. (B) The resulting asymmetric expression pattern of Nodal, coding for a signal protein belonging to the TGFb superfamily, in the neighborhood of the node (lower two blue spots) in a mouse embryo at 8 days of gestation, as shown by in situ hybridization. At this stage, the asymmetry has already been relayed outward to the lateral plate mesoderm, where Nodal is expressed on the left side (large elongated blue patch) but not the right. (B, courtesy of Elizabeth Robertson.)

expression in the mesoderm along the left side—and only the left side—of the embryo’s body. The mechanism that relays the asymmetry from the node and localizes Nodal expression is not understood and may vary from one class of ver- tebrates to another. In all species, however, it seems to depend on feedback loops involving Nodal together with a second set of genes, the Lefty genes. These, like Nodal itself, are directly regulated by the Nodal signaling pathway and their products, the Lefty proteins, are related to Nodal; but Lefty proteins diffuse more widely and act oppositely, as Nodal antagonists. Mice with a knockout mutation in the Lefty1 gene frequently have the right side converted into a mirror image of the left, so that left–right asymmetry is lost.

Another gene that is directly regulated by the Nodal pathway, Pitx2, coding for a gene regulatory protein, links the outcome of the Nodal/Lefty interactions to subsequent anatomical development. Nodal drives Pitx2 expression on the left side of the body and thereby confers asymmetry on the heart and other internal organs.

This leaves us with the puzzle of how the initial asymmetry of Nodal expres- sion originates. Whatever the mechanism, the outcome of events at the node in a normal animal must be biased so that left-specific genes are regularly expressed on the left side: there has to be a link between the mechanism that creates asymmetry and the mechanism that orients it. A clue to the orienting mechanism first came to light in a Swedish infertility clinic. A small subset of infertile men were found to have sperm that were immotile because of a defect in the dynein molecules needed for beating of cilia and flagella. These men also suffered from chronic bronchitis and sinusitis because the cilia in their respira- tory tract were defective. And strikingly, 50% of them had their internal organs left–right inverted, with the heart on the right. The findings originally seemed completely mysterious; but similar effects are seen in mammals with other mutations resulting in defective cilia. This suggests that ciliary beating somehow controls which way the left–right axis is oriented.

Time-lapse videomicroscopy in the living mouse embryo reveals that the cells at the node, on its internal face, have cilia that beat in a helical fashion: like a screw-thread, they have a definite handedness, and at the node they are set in a little hollow that is shaped so that their beating drives a current of fluid towards the left side (see Figure 22–87A). According to one theory, signal proteins carried in this current toward the left side provide the bias that orients the left–right axis of the mouse body. Another theory proposes that cilia in this system, as in cer- tain other contexts, act not only as drivers of fluid flow but also as mechanical sensors, responding to deflection by generating an asymmetric current of Ca2+ ions across the node to influence adjacent tissue.

The handedness of the ciliary beating reflects the handedness—the left–right asymmetry—of the organic molecules of which all living things are made. It seems that this, therefore, is the ultimate director of the left–right asym- metry of our anatomy.

Summary

Animal development involves dramatic cell movements. Thus, in gastrulation, cells from the exterior of the early embryo tuck into the interior to form a gut cavity and create the three germ layers—endoderm, mesoderm, and ectoderm—from which higher animals are constructed. In vertebrates, the movements of gastrulation are organized by signals from the Organizer (the dorsal lip of the amphibian blastopore, corresponding to the node in a chick or mouse embryo). These signals specify the dorsoventral axis of the body and govern convergent extension, in which the sheet of cells moving into the interior of the body lengthens along the head-to-tail axis while narrowing at right angles to this axis. The active repacking movements of individual cells that drive convergent extension are coordinated through the Frizzled/Dishev- elled planar-polarity signaling pathway—a branch of the Wnt signaling pathway that regulates the actin cytoskeleton.

Subsequent development involves many further cell movements. Part of the ecto- derm thickens, rolls up, and pinches off to form the neural tube and neural crest. In the

midline, a rod of specialized cells called the notochord elongates to form the central axis of the embryo. The long slabs of mesoderm on either side of the notochord become segmented into somites. Migrant cells, such as those of the neural crest, break loose from their original neighbors and travel through the embryo to colonize new sites. Pri- mordial germ cells and many other migrants are guided by chemotaxis dependent on the receptor CXCR4 and its ligand SDF1. Specific cell adhesion molecules, such as cad- herins and integrins, help to guide the migrations and control the selective cohesion of cells in new arrangements.

Ultimately, the pattern of cell movements is directed by the pattern of gene expres- sion, which determines cell surface properties and motility. Thus, the formation of somites depends on a periodic pattern of gene expression, which is laid down by a bio- chemical oscillator—the segmentation clock—in the mesoderm and dictates the way the mass of cells will break up into separate blocks. Similarly, the left–right anatomi- cal asymmetry of the vertebrate body is foreshadowed by left–right asymmetry in the pattern of gene expression in the early embryo. This asymmetry, in mammals at least, is thought to be directed ultimately by the handedness of ciliary beating in the neigh- borhood of the node.

THE MOUSE

The mouse embryo—tiny and inaccessible in its mother’s womb—presents a hard challenge to developmental biologists. It has, however, two immediate attractions. First, the mouse is a mammal, and mammals are the animals that we, as humans, care about most. Second, among mammals, it is one of the most convenient for genetic studies, because it is small and breeds rapidly. These two factors have spurred an enormous research effort, resulting in the development of some remarkably powerful experimental tools. In this way, the mouse has become the main model organism for experimentation in mammalian genetics and the most intensively studied surrogate for humans. It is separated from humans by only about 100 million years of evolution. Its genome is the same as ours in size, and there is very nearly a one-to-one correspondence between mouse and human genes. Our proteins are typically 80–90% identical in amino acid sequence, and large blocks of close nucleotide sequence similarity are also evident when the regulatory DNA sequences are compared.

Through ingenuity and perseverance, developmental biologists have now found ways to gain access to the early mouse embryo without killing it and to generate mice to order with mutations in any chosen gene. Almost any genetic modification that can be made in a worm, a fly, or a zebrafish can now also be made in the mouse, and in some cases made better. The costs of research in the mouse are far greater, but so are the incentives. As a result, the mouse has become a rich source of information about all aspects of the molecular genetics of development—a key model system not only for mammals, but also for other animals. It has provided, for example, much of what we know about Hox genes, left–right asymmetry, cell death controls, the role of Notch signaling, and a host of other topics.

We have already drawn repeatedly upon data from the mouse. We shall make use of it even more in the next chapter, where we discuss adult tissues and the developmental processes that occur in them. In this section, we examine the special features of mouse development that have been exploited to make the genetic manipulations possible. By way of example, we shall also outline how the mouse has been used to illuminate one further important developmental process—the creation of organs such as lungs and glands by interactions between embryonic connective tissue and epithelium.

Mammalian Development Begins With a Specialized Preamble