type seem likely to be responsible for a large fraction of the organization of complex organisms. There has been direct molecular demonstration of reaction-diffusion-like patterning in the use of Fgf and Bmp signaling as activators and inhibitors, respectively (Hogan 1999; Jernvall and Jung 2000; Jernvall and Thesleff 2000; Jung et al. 1998; Jung et al. 1999). Here we add the qualifying terminology, "reaction-diffusion-

like" to indicate that these repetitive patterning systems vary in most of their details, the nature and number of interacting agents, their diffusion mechanisms, and the like.

In this kind of dynamic patterning, the frequency and location of the waves, peaks, or zones of expression are determined not by the specific ingredients of the system but by its dynamic parameters: timing and intensity of expression, number of cells, diffusion rates, receptor/enhancer binding efficiencies, and the like. Combinatorial expression is also involved, because it is the combination of activator(s) and inhibitor(s) and the cascade of gene expression they trigger that specifies the zones of growth and inhibition, and while there may only be quantitative differences between the two, combinatorial differences consequent to the signaling process account for the structural differences.

While nested patterns may involve several patterning signals, there is not a separate gene or even gene combination for each hair, feather, or tooth cusp. Instead, each unit is produced by a reinvocation of the same developmental cascade. The dynamic interaction of SFs determines when, where, and how many such instances will occur. There are genes for the process, but not "for" each individual component. The units and their intervening regions are part of a single trait.

For repetitive organ systems, an initial process called prepatterning occurs, which involves the priming of genetic switches that are "remembered" in the cells (or their descendants); the "enabled" genes are activated only at some subsequent time and tissue context. That is how they "know" to express receptors for the SFs they will have to be able to detect in order to be induced. Traits like dentition or taste buds, mammalian coloration patterns, and perhaps feathers in birds or respiratory spiracles along the sides of insects are examples. EMI may be the triggering phenomenon, when migrating NC cells in the jaws encounter inductive signals from specific locations in overlying prepatterned or prepared epithelium.

Feathers and hair are arrayed along what first appear as linear axes and then become more spatially distributed. Extensive studies have been done on the patterning of chick feathers (Figure 9-7) (Jung, Francis-West et al. 1998). Lines of comparably pluripotent cells expand in the epithelium, ultimately forming stripes, detectable experimentally by the presence of SFs including Shh, Fgf4, and Ptc (from the Patched gene). The stripe then organizes into local "condensations" of cells, or slightly thickened placodes that are the initial indication that a structure will develop. Primordial placodes serve as autonomous local signaling centers or organizers, which interact inductively with underlying NC-derived mesenchyme to differentiate into feathers. Zones of activation that then generate surrounding zones of inhibition are typical of periodic patterning processes. SFs Bmp2 and Bmp4 are produced and diffuse away from the feather placode, where Bmp receptors on surrounding cells receive these ligands, and are inhibited from forming placodes, thus producing inhibition zones around each placode. Bmp antagonists like Noggin or Follistatin in the placode prevent self-inhibition. Much the same pattern is responsible for hair patterning (Jung, Francis-West et al. 1998; Oro and Scott 1998) and as noted earlier teeth as well (Jernvall and Jung 2000; Weiss et al. 1998).

This general and rather simple process is nested in various ways. As the inhibiting signal diminishes with distance from an initiation site, new placodes appear. The original stripe differentiates into periodic feather buds along a line, but the diffusion process then produces new feathers laterally, to produce the two-dimensional array. Different types of feathers arise even among adjacent locations, and in dif-


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Figure 9-7. Feather patterning reaction-diffusion schematic at standard times during chick development. Original signal develops along a line, The signal then develops activation spots where initial feather buds arise. As the process continues, diffusing activator and inhibitor signal leads to the formation of additional rows lateral to the original one. Bottom figure shows the activator-inhibitor gene system with gray-shade that corresponds to the top figure. Redrawn after (Jung, Francis-West et al. 1998).

ferent parts of the bird (see Figure 9-12), much as different color patterns are generated in different parts of a mammal's fur or different types of vertebrae or segments appear along the AP axis of vertebrates and invertebrates, respectively. Hair, fish scales, mouse facial sensory whiskers, and mammary glands are patterned structures that develop similarly.

Although elegant experiments have shown the reaction-diffusion-like nature of this process, knockout and other experimental manipulation of these signals also show that they are not all required for successful patterning. This suggests that other factors are involved (e.g., Chuong et al. 2000). Of course they must be: as we have noted in many places, at the very least the cells must be primed to express the SFs, receptors, and signal processing machinery and to make the appropriate parts of chromosomes available to respond to the signal.

In vertebrates, the oral epithelium expresses the SF Fgf8, but this signal becomes inhibited by zones of Bmp4 expression, and local Fgf8 foci remain that will become tooth-forming placodes along the upper and lower jaws (and/or elsewhere in the oral cavity, depending on species). Other genes, including the SF Shh and the TF Lef1, are also expressed in the initiation sites. The first known indicators are organizer-like zones of SF production that are similar to those just described for feathers in birds (indeed, hair and teeth can be made to grow in similar areas in some experimental manipulations in mice) (Jernvall and Jung 2000; Jernvall and Thesleff 2000; Thesleff et al. 2001).

Specific genes in the underlying mesenchyme (e.g.,TFs Msx1, Msx2, Pax9) whose presence allows the NC-derived cells to respond to these epithelial inductive signals and others (e.g., Bmp2) appear to inhibit laterally adjacent mesenchyme from participating, thus focusing the mesenchymal response into a local responding center under the epithelial initiation site, in which a tooth will form. Subsequently, the local signaling centers within each tooth, the enamel knots (EKs) referred to earlier, appear and secrete similar (though not entirely identical) combinations of SFs. Interestingly, the EKs themselves do not express receptors for the factors they secrete, but the surrounding cells do (Kettunen et al. 2000); this stimulates cell division and downgrowth around the EK, which eventually undergoes apopto-sis, but not before secondary EKs repeat this process to generate other cusps. The dentition has several axes: along the jaw, the breadth and width of individual teeth, and the crown-to-root vertical axis. Each is produced by signaling systems, sometimes involving the same genes. In the end, the pattern can be modeled closely by reaction-diffusion-like models for the array of teeth along jaws (Kulesa et al. 1996), and for periodic cuspal variation within teeth (Salazar-Ciudad and Jernvall 2002).

Feathers and teeth are among the model systems that illustrate what are much more widespread phenomena (there are many similar examples in insects and other animals and the equivalent in plants). In the case of teeth, experimental evidence suggests that the epithelial tissue layer has been prepatterned long before the initiation sites appear (Weiss, Zhao et al. 1998). Taste buds have comparable spatial patterning on the tongue, and direct experimental evidence suggests that these locations are prepatterned as early as gastrulation (Barlow and Northcutt 1998; Northcutt and Barlow 1998).

There are good reasons why this early preparation might be expected, which also illustrate why differentiation is hierarchical: a tissue like hair or feathers cannot form until there is skin to form on. Upper and lower teeth develop in anatomically separate regions of the jaws that suggest that prepatterning may have occurred very early there as well (Weiss, Zhao et al. 1998) and we referred above to evidence that the limb may also be prepatterned before it becomes a physically distinct structure. Another indicator that prepatterning occurs is that closely related animals can achieve similar results even though they are of very different size (such as the stripes on a Bengal tiger and a small tabby house cat, or teeth in an elephant and a mouse). One might say that this solves the allometric problem: the same basic mechanism is likely to be involved across the species size range, and the initial patterning event is likely to occur at a developmental stage when the embryos of all the species are more or less the same size (e.g., Murray 1993). The early stage can be much earlier than the first manifestation of the actual trait in the embryo. Then later, growth differences can generate structures of size appropriate to the species and these can be allometrically stretched or distorted by the growth factor (a subset of SF class gene products) patterns.

In a regionally patterned organ system, it is typical that the simplest and most global aspects of structure arise first and then more complex structures are produced. Axial region-specific expression of Hox genes is induced by gene- and region-specific enhancers (e.g., Shashikant et al. 1998). Once a region is specified, differentiation of hindbrain segments, mouthparts, vertebrae, and limbs occurs (Figure 9-8). Thus the Hox system is involved in the regionalization of major AP zones like thorax and abdomen, but these have multiple nested structures within

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