Two Very Different Examples of Similar Import

If the same mechanism is found in a Drosophila and in a sea urchin developmental regulatory system, there is a pretty fair case for its generality. The arthropods and the deuterostomes lie distant from one another in the bilaterian cladogram (Fig. 1.6). Yet at the c/s-regulatory level the operating principles in these two systems are indistinguishable. Within the syncytial Drosophila embryo, inputs for the initial tiers of specification functions depend on internuclear diffusion of transcription factors. The maternal bicoid mRNA is localized at the anterior end, and the transcription factor it encodes diffuses inward, forming an anterior (A) to posterior (P) gradient. The transcription factor caudel forms an opposite P to A gradient to repression of translation of the uniformly distributed caudal mRNA by Bicoid. Early zygotic mRNAs encoding transcription factors are generated in relatively broad bands of nuclei that are spaced along the A/P axis ("gap genes;" for reviews, Lawrence, 1992; Rivera-Pomar and Jackie, 1996). The sea urchin embryo, in contrast, is cellular from the start. The inputs for zygotic specification functions are again maternal and zygotic transcription factors, but their activities and/or presentation in the nuclei depend crucially on early intercellular signaling, as well as on localized maternal activation systems (for review, Davidson etal., 1998). But for present concerns these distinctions do not matter: they lie upstream of the cis-regulatory systems that must differentially interpret the regulatory states that they encounter in the various spatial domains of the embryo. This they do by very similar means, as the following examples show.

The output of the evenskipped (eve) stripe 2 cz's-regulatory element was illustrated in Fig. 1.4. It produces a thin circumferential stripe of gene expression a few nuclei wide located at an exact position along the A/P axis. Figure 2.1 A displays the minimal hardwiring required to generate eve stripe 2, i.e., the sequence of transcription factor target sites necessary and sufficient to cause accurate expression of the reporter gene. Because it is well understood, due to an incisive series of studies by M. Levine and colleagues (Small et al, 1991, 1992; Arnosti etal., 1996), this regulatory element provides a classic example of a spatial specification system which sets its boundaries by repression. The stripe 2 module activates transcription in response to the positive Bicoid (Bed) regulator, and a second zygotically transcribed positive regulator, Hunchback (Hb), is also required (the hb gene is activated by Bed as well). The Bed protein is distributed in a broad concentration cline that decreases from anterior to posterior, and the Hb protein is present in about the anterior 40% of the early embryo (Fig. 2. IB). As Fig. 2.1 A shows, there are multiple Bed target sites in the stripe 2 element, some of which bind the factor more tightly than others. However, the multiple Bed activators binding within the element work synergistically with one another, and with the Hb factor; these sites all contribute, but some are more important than others. Their synergism is required to obtain strong activation of the element in spite of low endogenous activator concentrations (Arnosti et al, 1996).

The distribution of the activators to which the eve stripe 2 element responds would suffice for expression extending from far anterior of eve stripe 2 to far posterior of it, i.e., throughout almost the whole anterior half of the embryo. The actual boundaries of eve stripe 2 depend entirely on repression, mediated by the Kriippel (Kr) and Giant (Gt) target sites shown in Fig. 2.1 A. The posterior boundary is produced as a response of the cw-regulatory element to the Kr repressor (Small et al., 1992; Gray and Levine, 1996). If the Kr expression domain is caused to expand in an anterior direction, so that it overlaps rather than abuts eye stripe 2, it wipes out all expression of the eve stripe 2 element (Fig. 2.1C and D). Thus the location of the Kr repressor in the syncytial embryo constitutes an essential spatial input to the system. The anterior boundary is produced by interactions with the Gt repressor, working together with another as yet unknown corepressor which requires Gt binding for function (Small et al., 1992; Arnosti et al, 1996; Wu et al., 1998). The Gt repressor is normally present in the embryo in a band extending up to the anterior border of eve stripe 2, as illustrated in Fig. 2.IE. Figure 2.IF shows that eve stripe 2 expression expands in the anterior direction when the Gt sites of the element shown in Fig. 2.1 A are mutated. These and many other experiments demonstrate the crucial role of spatial repression in placing eve stripe 2 in the appropriate position. Institution of eve stripe 2 is an excellent example of a cis-regulatory specification function, in that expression of this transcription factor in the seven initial stripes is a key initial step in the embryonic pattern formation process that leads to the metamerization of the body plan. The role of the eve czs-regulatory element controlling stripe 2 expression is to "read" and integrate the broad patterns of its activators and repressors into a sharp

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