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diffusion, and in the mainly cellular tissue of the wing disk, Dynamin here acts to move Dpp from cell to cell to help it diffuse from its source.

Figure 9-3C described the workings of a simple concentration gradient mechanism, for DV patterning in the fly. This establishes the dorsal dominance of Dpp signal that, among other things, inhibits neural development, which takes place more ventrally. This is all part of a broader DV gradient patterning mechanism, that results in regionalized DV areas that develop into the major tissue fates in the fly body plan. This process is worth describing briefly to show the nested or layered

Dorsal inhibits Tolloid, Dpp, and Zerknüllt

Cross Sect

Dorsal protein gradien

Zerknüllt

Cross Sect

Dorsal protein gradien

Lateral ectoderm

Dorsal ectoderm

Mesoderm

Mesoderm

/^Neurogenic ectoderm Mesectoderm

Lateral ectoderm

Dorsal ectoderm

Dorsal activates Twist, Snail, Rhomboid

Figure 9-4. Gradient mechanism for dorsoventral body plan patterning in Drosophila.The center figure shows a cross section of the early embryo, identifying locations of gene expression as well as (right side) the tissue fates of the various regions. Above the figure is shown schematically that Dorsal protein inhibits genes that must be expressed for dorsal tissue fates; below the figure is a schema of gene induction by Dorsal, of Twist and Snail expression to induce mesodermal ventral fates, and inhibiting Rhomboid whose expression (ventrolater-ally) is associated with neural fates. For details, see text and (Gilbert 2003).

nature of patterning and the numerous ways it can be brought about (Figure 9-4, and for details see Gerhart and Kirschner 1997; Gilbert 2003).

Early DV polarity involves a cascade of effects beginning with a protein, called Gurken (that is, coded by the Gurken gene) whose gradient is provided by the oocyte nucleus, which by the process of oocyte formation is located dorsally within an ovarian follicle that also contains within it 15 other cells, called nurse cells that supply mRNA and other materials to the oocyte (for details see Gerhart and Kirschner 1997; Gilbert 2003; Lawrence 1992). Gurken protein inhibits ventralizing signal from forming in the dorsal site of its (Gurken's) production. Another protein, Dorsal (i.e., a TF encoded by the Dorsal gene), in part provided by the follicle to the oocyte, is initially distributed throughout the egg. However, Dorsal's effect is to inhibit dorsal structures, so that it must form higher concentrations in the ventral side (the gene is so named because in its absence the entire embryo takes on dorsal fates). A series of events involving 11 known genes causes the translocation of Dorsal protein to the ventral nuclei in the syncytium.

As indicated in Figure 9-4, dorsal tissues form at the lowest concentration of Dorsal. Dorsal is complexed with a gene product, Cactus (not shown in the figure), and is activated only when released, which occurs via the Nudel protein whose concentration is highest in the ventral part of the egg, because Gurken protein, which is dorsally concentrated, represses Nudel transcription.Thus, a DV gradient of active Dorsal TF is established; via a series of threshold-level effects such as those shown in Figure 9-3A (where the cartoon at the bottom suggests AP patterning), Dorsal acts as a selector for differential gene expression initiating the major embryonic tissues. Ventrally, where the Dorsal concentration is greatest, it activates genes including Twist and Snail that form mesoderm and undergo gastrulation when external tissue moves inside the embryo to form the gut and other internal structures. Just lateral to that, the lower Dorsal concentration enables the gene Rhomboid to be expressed, and so on. On the dorsal side of the embryo, where Dorsal concentration is least, dorsalizing genes including Tolloid, Zerknüllt, and the familiar Dpp are activated. We saw in Figure 9-3C how Sog and other proteins, whose expression is induced as a result of the Dorsal gradient, then interact to establish the Dpp gradient, that in turn inhibits neural development, that occurs in cells in the region shown in Figure 9-4.

The Dorsal gradient mechanism is interesting in that it is a TF not a signaling factor, showing that it is the gradient, and not the type of gene that establishes it, that is important in the logic of patterning. In this case also, depending on the circumstances, the Dorsal protein appears to activate some genes and inhibit others. The same TF can have both kinds of effects, depending on the REs to which it binds near a target gene—for example, what else must bind there, or must be prevented from binding there.

Development Is Plastic ... and Other Caveats

The patterning results just discussed are among the many phenomena that have been carefully studied experimentally. Many if not most of the genes involved were first found in mutant flies, and even named for those mutant effects, and their expression was then documented and manipulated to identify their effects. But traits are often studied first in terms of expression patterns of known genes, and it is important to be careful about interpreting expression patterns because they can be misleading: expression of a gene in a tissue or developmental stage does not automatically mean critical function at that stage. For example, the importance of EMI in pharyngeal development in vertebrates seemed somewhat perplexing because pharyngeal region patterning seemed to have arisen in evolution before neural crest. Experimental ablation of NC cells in the chick to prevent their migration did not disrupt early pharyngeal arch patterning (Veitch et al. 1999).Thus, although NC may be involved, normal development does not entirely depend on it. In a similar way, it is a common experience that experimental inactivation of developmental genes in mice does not affect a structure in which the gene is expressed and/or in the way the expression pattern might suggest. Unfortunately, it is not practicable to do experimental manipulations of all gene expression patterns, especially in large or long-lived species.

The Bicoid and Dorsal stories are classics in the discovery of morphogenic gradients. However, organisms are robust and the actual effects are subtle. Bicoid gradients can be manipulated experimentally in Drosophila, and the result shows that to a considerable extent the embryo somehow compensates for experimentally induced aberrant dose levels and develops more or less normally (Driever and Nusslein-Volhard 1988; Namba et al. 1997). This is a point to stress something we have noted earlier, the role of chance even in the biochemical concentrations of cellular constituents. Organisms must be resistant to such changes. Bicoid concentrations have been found to vary considerably among embryos (Houchmandzadeh et al. 2002), probably buffered by scale-information built into the Hunchback concentration.

This variation may be a reflection not just of chance variation in transcription rates but of variability even within species in the structure and sequence details of enhancers more generally (Wray et al. 2003). We referred in earlier chapters to the fluidity and variability in enhancer binding location, number, and sequences.

But there is more. This early patterning might seem to be quite a fundamental aspect of development, one hard to change, but a fruit fly does not all insects make. Fruit flies are "long-germband" insects, in which the blastoderm (the part from which the embryo develops) takes up most of the egg and the body segments are specified simultaneously by processes such as those we have described. By contrast, "short-germband" insects develop more structures outside of the egg cell itself, sequentially during development. Recent work on the short-germband beetle Tribolium (another popular model arthropod species) shows that two other genes, Orthodenticle (Otd) and Hunchback, play the Bicoid role in establishing AP patterning (Schroder 2003). Whatever the common ancestor, there has been a substitution of mechanism for a conserved basic polarity-establishing phenomenon, a kind of phenogenetic equivalence and perhaps a manifestation of phenogenetic drift.

Genes from all four Hox clusters (denoted HoxA, HoxB, HoxC, and HoxD) are expressed in mouse embryonic development and are thought to be required for pattern formation. Many examples of effect, including homeotic shift (of identity of segments) have been observed in natural or experimental mutation of these genes. But the picture is not always so straightforward. As one of several known examples, mice experimentally lacking their HoxC cluster genes establish normal patterning (they have subsequent other problems, so the genes, which are used in many tissues, do have important function) (Suemori and Noguchi 2000).

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