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The fates of cells distributed along the anterior/posterior axis are specified early in fly development. At the same time, cells are responding to the dorsal/ventral control system. Each cell is thus uniquely specified along both axes, in a two-dimensional grid. If each of the five gap genes were expressed in its own section of the embryo, at just one concentration, only five cell types could be formed. The actual situation permits far greater diversity among the cells. As noted earlier, the amount of each gap protein varies from low to high to low along the anterior/posterior axis, and the expression domains of different gap genes overlap. This complexity creates combinations of transcription factors that lead to the creation of many more than five cell types. Remarkably, the next step in the Drosophila development process generates a repeating pattern of cell types from the rather chaotic nonrepeating pattern of gap-gene expression domains.

Combinations of Gap Proteins Direct Transcription of Pair-Rule Genes in Stripes

Our vertebrae and the body segments of an insect are both examples of a commonly employed tactic in animal structure and music: repeats with variations. The first sign of such repeats in fly embryos is a pattern of repeating stripes of transcription of eight genes collectively called pair-rule genes. The body of a larval fly consists of 14 segments, and each pair-rule gene is transcribed in half of its primordia, or seven stripes, separated by "interstripes" where that pair-rule gene is not transcribed (Figure 15-22c). Mutant embryos that lack the function of a pair-rule gene have their body segments fused together in pair-wise fashion—hence the name of this class of genes. The expression stripes for each pair-rule gene partly overlap with those of other pair-rule genes; so each gene must be responding in a unique way to gap-gene and other earlier regulators.

The transcription of three "primary" pair-rule genes is controlled by transcription factors encoded by gap and maternal genes. Because gap and maternal genes are expressed in broad, nonrepeating bands, the question arises: How can such a nonrepeating pattern of gene activities confer a repeating pattern such as the striped expression of pair-rule genes? To answer this question, we consider the transcription of the even-skipped (eve) gene in stripe 2, which is controlled by the maternally derived Bicoid protein and the gap proteins Hunchback, Krüppel, and Giant. All four of these transcription factors bind to a clustered set of regulatory sites, or enhancer, located upstream of the eve promoter (Figure 15-23a). Hunchback and Bicoid activate the transcription of eve in a broad spatial domain, whereas Krüppel and Giant repress eve transcription, thus creating sharp posterior and anterior boundaries. The combined effects of these proteins, each of which has a unique concentration gradient along the anteroposterior axis, initially demarcates the boundaries of stripe 2 expression (Figure 15-23b).

The initial pattern of pair-rule stripes, which is not very sharp or precise, is sharpened by autoregulation. The Eve

(a) eve gene transcription regulation Giant ( \ ) Krüppel ( \ )

(b) eve stripe 2 regulation

Hunchback ( f ) Giant ( \) eve stripe 2 Krüppel ( ^ )

(b) eve stripe 2 regulation

Hunchback ( f ) Giant ( \) eve stripe 2 Krüppel ( ^ )

Parasegments

Parasegments

▲ FIGURE 15-23 Expression of the Even-skipped (Eve) stripe 2 in the Drosophila embryo. (a) Diagram of an 815-bp enhancer controlling transcription of the pair-rule gene eve. This regulatory region contains binding sites for Bicoid and Hunchback proteins, which activate the transcription of eve, and for Giant and Krüppel proteins, which repress its transcription. Enhancer is shown with all binding sites occupied, but in an embryo occupation of sites will vary with position along the anterior/ posterior axis. (b) Concentration gradients and of the four proteins that regulate eve stripe 2. The coordinated effect of the two repressors (i) and two activators (T) determine the precise boundaries of the second anterior eve stripe. Only in the orange region is the combination of regulators correct for the eve gene to be transcribed in response to the stripe 2 control element. Further anterior Giant turns it off; further posterior Krüppel turns it off and the level of Bicoid activator is too low. Expression of other stripes is regulated independently by other combinations of transcription factors that bind to enhancers not depicted in part (a). [See S. Small et al., 1991, Genes & Devel. 8:827.]

protein, for instance, binds to its own gene and increases transcription in the stripes, a positive autoregulatory loop. This enhancement does not occur at the edges of stripes where the Eve protein concentration is low; so the boundary between stripe and interstripe is fine-tuned.

Each primary pair-rule gene is regulated by multiple enhancers that are organized in modules. Each stripe is formed in response to a different combination of transcriptional regulators acting on a specific module, so the nonrepeating distributions of regulators can create repeating patterns of pair-rule gene repression and activation. If even one enhancer is bound by an activating combination of transcriptional regulators, the presence of other enhancers in an inactive, "off" state (not bound to a regulator) will not prevent transcription. For instance, in Eve stripe 2, the right combination and amounts of Hunchback and Bicoid create an "on" state that activates transcription even though other enhancers are present in the inactive state. In each stripe, at least one enhancer is bound by an activating combination of regulators. Note that this system of gene control is flexible and could be used to produce nonrepeating patterns of transcription if that were useful to an animal.

Similar responses to gap and maternal proteins govern the striped patterns of transcription of the two other primary pair-rule genes, runt and hairy. Because the enhancers of runt and hairy respond to different combinations of regulators, the eve, runt, and hairy expression stripes partly overlap one another, with each stripe for any one gene offset from a stripe

Time after fertilization

(2000 nuclei)

bicoid mRNA

bicoid mRNA

(2000 nuclei)

Antennapedia

Engrailed

Antennapedia

Engrailed

Protein expressed

Fushi tarazu

Even skipped

Paired

Engrailed

Wingless

Protein expressed

Fushi tarazu

Even skipped

Paired

Engrailed

Wingless

4th parasegment

•A.

5th parasegment

6th parasegment

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