FIGURE 2.1 cis-Regulatory control of evenskipped stripe 2 in Drosophila. (A)

Diagram of "minimal stripe element" (—1070 to —1550 of the upstream sequence). When introduced into the germ line this DNA fragment suffices to produce accurate

FIGURE 2.1 cis-Regulatory control of evenskipped stripe 2 in Drosophila. (A)

Diagram of "minimal stripe element" (—1070 to —1550 of the upstream sequence). When introduced into the germ line this DNA fragment suffices to produce accurate design: it creates a novel spatial regulatory domain, which did not previously exist, in a field of pluripotential nuclei (cf. Fig. 2.1C-F).

Spatial repression plays an essentially similar role in regulation of the cyllla gene of Strongylocentrotus purpuratus, as shown in Fig. 2.2. cyllla encodes a terminal product of the regulatory apparatus, viz a cytoskeletal actin, one of five in this genome. The cyllla gene is regulated in a way that distinguishes it from any other of the actin genes: it is expressed exclusively in the aboral ectoderm and its precursor cells from mid-cleavage on. The aboral ectoderm ultimately forms the single cell-thick squamous epithelial wall of the late embryo and larva, which is bounded by the neurogenic ciliated band and the oral ectoderm surrounding the mouth. The regulatory problem for the cyllla gene is to ensure its expression only in aboral ectoderm, and to prevent expression in oral ectoderm progenitors, in skeletogenic cells, and in all endomesodermal cells (see Fig. 2.2B, C). This problem is compounded by the constraint that oral/aboral (O/A) polarity cannot be set up in the sea urchin egg prior to fertilization. Any point on the equatorial circumference of the egg may serve as either oral or aboral pole, and in typical sea expression of a lacz reporter in the exact location of endogenous stripe 2 (see Fig. 1.4). Target sites for transcription factors are indicated. Repressors, viz, Kruppel (Kr) and Giant (Gt), are shown as solid rectangles above the line representing the DNA. Activators, viz Bicoid (Bed) and Hunchback (Hb), are shown as solid circles below the line. A, anterior; P, posterior. [(A) From Small et al. (1992) EMBOJ. I1, 4047-4057 and by permission of the European Molecular Biology Organization.] (B) Immunofluorescence display of the location of the Hb activator and the anterior Bed domain, with respect to the stripes of Eve protein, 14th cleavage. Hb is stained in dark green; Bed in blue fading to magenta more posteriorly; and Eve in orange-red; the overlap of Eve and Hb appears yellow. The anterior domain of Hb includes Eve stripes 1, 2, and 3. [(B) From J. Relnitz, personal communication.] (C, D) eve mRNA (black) and kr mRNA (red) as displayed by in situ hybridization. In (C) and (D) the numerals indicate the eve stripes. (C) The Kr domain directly abuts the posterior boundary of eve stripe 2 in a normal embryo. (D) Forced expression of kr in the region overlapping eve stripe 2 (by placing the kr gene under control of the stripe 2 c/'s-regulatory element) almost completely represses eve stripe 2. [(C, D) From Wu et al. (1998) Development 125, 3765-3774 and The Company of Biologists, Ltd.] (E) Gt protein revealed by immu-nostainlng (brown), together with a lacz mRNA stripe generated by the cis-regulatory element shown in (A), as revealed by in situ hybridization (blue-green). The Gt domain abuts eve stripe 2 at its anterior border. (F) Effect on expression of mutating all three of the Gt target sites. c/s-Regulatory element of (A) at top, same color coding of target sites. Open boxes indicate mutated sites. Lacz mRNA is now transcribed in an anteriorly expanded region, since repression mediated by Gt (and its unknown cofac-tor; Wu et al., 1998) can no longer occur. Anterior is to the left in (B-F). [(E, F) From Small et al. (1992) EMBO J. II, 4047-4057 and by permission of the European Molecular Biology Organization.]

FIGURE 2.2 Spatial repression in the regulation of the Strongylocentrotus purpuratus cyllla gene. (A) Target sites and spatial inputs in the cyllla c/s-regulatory system (Kirchhamer and Davidson, 1996; Coffman et al., 1997). Sites for a DNA looping protein (SpGcf; Zeller et al., 1995) are omitted. The proteins which bind the sites shown are: R, SpRuntl; M, SpMybl; Z, SpZ 12-1; P, SpP3a2; CT, SpCtfl; T, SpTefl; >, SpOctl; and the open downward triangles represent an unknown positive regulator. Repressors are shown in red; activators in dark blue. The spatial domains where these regulators are functional are indicated (open boxes). (B) Normal distribution of endogenous cyllla mRNA, displayed by in situ hybridization in a late gastrula-stage embryo. Cyllla transcripts are confined to the aboral ectoderm; oral ectoderm is indicated as the region between arrowheads in panels (B-D, F). (C-F) Whole mount in situ hybridization display of a CAT mRNA reporter, transcribed under control of various cyllla c/s-regulatory elements. [(A, B) From Bogarad et al. (1998) Proc. Natl. Acad. Sci. USA 95, 14827-14832; copyright National Academy of Sciences, USA.] (C) Expression of complete wild-type (control) cyllla»CAT transgene containing the regulatory system shown in (A), plus some distal sites for SpGcfl which have a mild enhancing effect on the level of activity. CAT mRNA is confined to clones of aboral ectoderm cells. When DNA is injected into eggs it is usually incorporated into single blastomeres early in cleavage, resulting in mosaic expression patterns. Incorporation is stable, and completely random with respect to cell lineage (Livant et al., 1991). The total expression pattern is obtained from observations on a population of injected embryos. (D) Effect on expression of mutating one of the P3a2 sites in the otherwise complete construct. Expression now occurs in oral ectoderm as well as in aboral ectoderm at the normal level; about 30% of embryos (i.e., the fraction in which DNA will be incorporated in clones generating oral ectoderm on a random basis) display only ectopic oral ectoderm expression, as in this particular embryo. [(C, D) From Kirchhamer et al. (1996) Development 122, 333-348 and The Company of Biologists, Ltd.] (E) Expression in skeletogenic mesenchyme, as well as in a patch of ectoderm, in an embryo bearing the cyllla»CAT vector in which the two Z sites were mutated. The embryo is viewed from the bottom (i.e., blastopore end). [(E) from Wang et al. (I995) Development 121, 1111-1 122 and The Company of Biologists, Ltd.] (F) Ectopic expression in oral ectoderm and endoderm, plus normal expression in patch of aboral ectoderm, caused by a mutation of the M site which prevents binding of the Myb repressor [(F) From Coffman et al. (1997) Development 124, 4717-4727 and The Company of Biologists, Ltd.] (G) Immunocytological display of Myb protein, in a late pluteus-stage embryo. The bilaterally organized oral ectoderm forms the upper wall of the embryo, and the aboral ectoderm constitutes the remainder of the external wall of the embryo. The Myb repressor is confined to oral ectoderm and gut (the circular midgut is clearly visible in this image), plus in some mesenchyme cells (not clearly visible here). [(G) From J. A. Coffman and E. H. Davidson, unpublished data.]

Embryo Devolopment

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