Slg. 12 on
FIGURE 4.10 Early development of trachea in Drosophila. (A) Expression of trachealess (trh) gene, which encodes a bHLH-PAS transcription factor, and acts as a determinant of tracheal identity. The locus contains a [3-gal expression enhancer trap (/-eve-1) and expression of the trh gene is conveniently monitored by use of an a(3-gal antibody. (AI) trh(l-eve-l) expression in ten tracheal placodes; normal pattern, stage 11. (A2) trh autoregulates: expression is extended anteriorly to generate an ectopic tracheal placode, (tpo), in embryos expressing trh under heat shock control (hs-trh). (A3) trh cross-regulation by drifter (dfr) gene product. Extensive ectopic trh (l-eve-l) expression is seen in an embryo expressing both dfr and trh under heat shock control (arrows). In (A2) ectopic expression driven by hs-trh occurs in tpo because dfr is already being expressed in that location. (B) Expression of dfr gene, which encodes a POU domain transcription factor. (Bl) Expression of a dfr.lacz construct (visualized
FIGURE 4.10 Early development of trachea in Drosophila. (A) Expression of trachealess (trh) gene, which encodes a bHLH-PAS transcription factor, and acts as a determinant of tracheal identity. The locus contains a [3-gal expression enhancer trap (/-eve-1) and expression of the trh gene is conveniently monitored by use of an a(3-gal antibody. (AI) trh(l-eve-l) expression in ten tracheal placodes; normal pattern, stage 11. (A2) trh autoregulates: expression is extended anteriorly to generate an ectopic tracheal placode, (tpo), in embryos expressing trh under heat shock control (hs-trh). (A3) trh cross-regulation by drifter (dfr) gene product. Extensive ectopic trh (l-eve-l) expression is seen in an embryo expressing both dfr and trh under heat shock control (arrows). In (A2) ectopic expression driven by hs-trh occurs in tpo because dfr is already being expressed in that location. (B) Expression of dfr gene, which encodes a POU domain transcription factor. (Bl) Expression of a dfr.lacz construct (visualized as in A i -A3 i.e., by immunocytological detection of the Lacz gene product) at stage 12, as tracheal cells (t) begin migration; oen, oenocytes, where this gene is also expressed. (B2) Expression of dfr.lacz in trh~ embryo at stage 10: the initial activation of dfr in tracheal placodes (tp) does not depend on trh expression. (B3) Same as (B2) but at stage 12; expression of dfr.lacz has now disappeared from the tracheal placodes, in trunk remaining only in oenocytes. [(A, B) From Zelzer and Shilo (2000a) Mech. Dev. 91,163-173; copyright Elsevier Science.] (C) Regulatory interactions of the trh and dfr specification genes. At stage 9 the A/P and D/V patterning systems of the embryo set up the ten bilateral trh and dfr expression domains. By stage 12 the initial spatial inputs have been supplanted by an auto- and cross-regulatory system that maintains trh and dfr expression in an enforcing activation loop, as shown in (A) and (B). Downstream interactions of trh (red) and dfr (purple) are shown: target genes require activity of either trh, dfr, or both. These gene products interact physically (Zelzer and Shilo, 2000a), and the bHLH-PAS product of trh also requires a second factor, encoded by the arnt gene (Ohshiro and Saigo, 1997). Target genes which encode components of signal transduction systems are shown in green (for evidence and references, Boube et al2000): btl, breathless, an FGF receptor; rho, rhomboid, a transmembrane facilitator of EGF signaling; dof, which encodes a component of the EGF signal transduction system; tkv, thickveins, which encodes a Dpp receptor. Two genes encoding transcription factors are targets of Trh, shown in blue: tracheal defective (tdf), which encodes a bZip factor; and hindsight, encoding a Zn finger factor required for further tracheal morphogenesis (Wilket al., 2000). The interactions with the btl c/s-regulatory system are known to be direct (Anderson et al., 1996; Ohshiro and Saigo, 1997); the other interactions are inferred from genetic evidence, i.e., observations on expression in trh~ and dfr embryos. However, while necessary, trh and dfr gene products are not sufficient to induce expression of any but the btl gene when those factors are expressed ectopically. (D) Expression of gene encoding the FGF ligand Branchless (Bnl; dark spots) in five domains surrounding a trh(l-eve-l) tracheal placode (red). The cells closest to the bnl expressing patches migrate in their direction, as indicated in the cartoon below. Control of migration and branching of tracheal tubules is mediated by subsequently expressed genes within the tubule cells (see review of Metzger and Krasnow, 1999). [(D) From Sutherland et al. (1996) Cell 87, 1091-1101; and Metzger and Krasnow (1999) Science 284, 1635-1639; copyright American Association for the Advancement of Science.] (E) Signaling domains which determine tracheal cell fates. (El) Expression of dpp (purple) dorsally and ventrally, by in situ hybridization, about stage I I just prior to migration. Domains of dpp expression border the tracheal placodes, which are revealed by expression of trh(I -eve-1) (Lacz product as above, red). [(EI) From Vincent et al. (1997) Development 124, 2741-2750 and The Company of Biologists Ltd.] (E2) Expression of sal gene within a subset of tracheal cells destined to form specific components of the tracheal system; sal expression (violet) superimposed on trh(l-eve-l) expression (red) at stage II; (E3) sal expression in extending tracheal system (brown) superimposed on trh(l-eve-l) expression (violet), which continues to mark all tracheal cells. The sal expressing cells constitute the dorsal trunk (DT); (sal expression transiently marks the dorsal branches; DBI, dorsal branch I). [(E2, E3) From Kuhnlein
express the Dpp and EGF signal response systems, respectively, and use them to carry out different aspects of migratory tracheal morphogenesis.
Our second example is drawn from the chick limb, the morphogenesis of which depends on a series of regional signaling interactions (reviewed by Johnson and Tabin, 1997; Dudley and Tabin, 2000). The specific aspect shown in Fig. 4.11 concerns dorsoventral specification in the wing bud. The evidence comes from a series of forced ectopic expression experiments using retroviral vectors. The ectodermal signaling ligand, Wnt7a, causes expression of a mesenchymal regulatory gene of the lim class, Imxl, which defines the subpart of the limb bud that will form dorsal structures. Spatial control, i.e., the function of excluding Imxl expression from the ventral side, is the responsibility of the engrailedl (enl) gene, which encodes a transcription factor that is expressed only in cells of ventral ectodermal origin (Altabef et al., 2000). The enl gene performs this function by preventing expression of the wnt7a gene on the ventral side. The normal disposition of these domains of gene expression are shown in the diagram of Fig. 4.11A (Johnson and Tabin, 1997), and some of the experimental results supporting the relations shown there are illustrated in Fig. 4.11B1-B10. The outcome is that if the Imxl transcriptional domain is permitted to extend to the ventral side of the limb bud, by ectopic expression of wnt7a, the limb develops as if it had two dorsal sides (Fig. 4.11B7-B10). The Imxl expression domain is a defining transcriptional and Schuh (1996) Development 122, 2215-2223 and The Company of Biologist Ltd.] (F) Subdivision of tracheal placode into dorsal and ventral "Dpp response" domains (red) and central "EGFR" domain (yellow). (Fl) sal and kni domains. The sal gene product serves as a determinant of anteroposterior tracheal migration and morphogenesis, controlling the activity of certain signaling pathways. In the central domain where sal is expressed, rho expression results in processing of the EGF ligand precursor (Spitz, Spi), activating the EGFR, and maintaining sal expression. These cells produce the dorsal trunk, as seen in (E3). In the regions subject to the Dpp signal, kni and knrl expression are required to mediate Dpp signal effects following reception of the signal by the Tkv receptor. These cells migrate dorsoventrally and ectopic expression of these genes in tracheal cells causes them to migrate in dorsoventral directions, i.e., toward the Dpp stripes. The sal gene is directly repressed by Kni (Chen et al„ 1998). (F2) Migration begins, driven by the Bnl/Btl (FGF) system controlled by the trh and dfr genes [(C, D)]. Migration tips will form at leading cells marked by stars. The red and yellow colors mark the Dpp response domain cells and the EGF signaling domains, as in (Fl). (F3) Resulting migration patterns. The differences in migratory behavior are apparently mediated by different downstream genes that control diverse signal systems, cell adhesion molecules, special guidance cues, etc. (see reviews of Zelzer and Shilo, 2000b; Metzger and Krasnow, 1999). [(Fl) From Zelzer and Shilo (2000b) BioEssays 22, 219-226, copyright Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.]; [(F2, F3) From Wappner et al. (1997) Development 124, 4707—4716 and The Company of Biologists Ltd.]
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