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Pipe + Windbeutel + Nudel

Eggshell (ventral)

▲ FIGURE 15-17 Dorsoventral axis determination in Drosophila. This process relies on two signal systems, one in follicle cells and the other in the oocyte or embryo or both, plus a proteolytic cascade within the perivitelline space. (a, b) Movement of the oocyte nucleus creates an initial asymmetry ( 1). The dorsal location of the oocyte nucleus ultimately results in the production of Pipe protein only in ventral follicle cells ( 2|- 5). (c) Subsequent events in the perivitelline space along the ventral surface generate a gradient of active Spätzle ( 6- 9). By this time, the egg has been fertilized and the embryo is a syncytium with many nuclei; the region around only one nucleus is shown. Activation of Toll on the embryo's surface by Spätzle causes Dorsal protein to enter the nucleus (10 and 11) where it activates transcription of specific target genes, depending on its concentration. (d) The concentration of Spätzle—hence Toll activation and nuclear localization of Dorsal— is greatest along the ventral midline, conferring ventral fates (e.g., muscle) on cells in this region. More laterally, less Dorsal enters the nuclei, and in consequence the cell fates are different (e.g., neural). Dorsal cell fates arise where no Dorsal enters the nucleus. Mutants lacking Toll receptor, or Dorsal, form only dorsal cell types. [Adapted from Gilbert and Hashimoto, 1999, Trends Cell Biol. 9:102.]

underway. Spätzle binds to its transmembrane receptor, called Toll, on the ventral embryo surface. Thus the signaling has come full circle: Gurken ligand produced dorsally in oocyte ^ activation of its EGF-like receptor on dorsal follicle cells, and then back through the protease cascade on the ventral side ^ Spätzle ligand ^ activation of its receptor, Toll, on the ventral side of the embryo. The net effect is to coordinate the eggshell structures produced by the follicle cells with the embryo structures produced inside.

Within the embryo, association of the cytosolic domain of activated Toll with two proteins (Tube and Pelle) leads to phosphorylation of Cactus protein. In the absence of Toll signaling, Cactus binds to a transcription factor called Dorsal and traps it, but phosphorylated Cactus is rapidly degraded by the proteasome. The newly freed Dorsal is able to enter the nuclei of the embryo's cells and activate the transcription of different target genes, depending on its concentration (see Figure 15-17c). Spätzle and Dorsal thus function as graded regulators, inducing ventral fates where their concentration is highest and other fates laterally as their concentration diminishes (Figure 15-17d). Dorsal function reaches its peak after cellularization has taken place.

The central features of the Toll-Dorsal pathway in flies, which are analogous to those of the mammalian NF-kB pathway discussed in Chapter 14, exist in mammals and probably in all animals. Dorsal is similar to the NF-kB transcription factor; Cactus, to its inhibitor, I-kB; and the Toll receptor, to the receptor for interleukin 1, which acts through Tube and Pelle equivalents to cause the phosphorylation of I-kB and the release of NF-kB (see Figure 14-28). NF-kB is a critical regulator of genes required for immune responses in mammals and insects and appears to function in mammalian development as well. It nicely exemplifies the utilization of one signal-transduction pathway to accomplish multiple tasks, such as patterning in development plus the immune response to infection or injury. This phenomenon appears to be fairly common and partly explains the small number of signaling pathways that have evolved over biological time despite the increasing complexity of organisms.

Nuclear Dorsal and Decapentaplegic, a Secreted Signal, Specify Ventral and Dorsal Cell Fates

The remarkable series of steps depicted in Figure 15-17 results in a gradient in the nuclear localization of the transcription factor Dorsal. The concentration of nuclear Dorsal decreases gradually from highest in cells at the ventral midline to lower values in lateral cells and eventually to none in dorsal cells. Mutants lacking dorsal function cannot make cells with ventral character; so the entire embryo develops dorsal structures. (Note that fly genes are named according to their mutant phe-notypes; thus the dorsal gene controls ventral fates.) Once inside the nucleus, Dorsal controls the transcription of specific target genes by binding to distinct high- and low-affinity regulatory sites and by interacting in a combinatorial fashion with other transcription factors. Dorsal represses the transcription of decapentaplegic (dpp), tolloid, short gastrulation, and zerknüllt and activates the transcription of twist, snail, single-minded, and rhomboid. Each of these genes contains a unique combination of cis-acting regulatory sequences to which Dorsal and other transcription factors bind.

Figure 15-18 illustrates how nuclear Dorsal specifies different target-gene expression patterns, depending on its con-

Dorsal protein

/\ Snail ^

—i Low-affinity dorsal-binding sites

Twist protein

CQ bHLH ^^

^ High-affinity dorsal-—~ binding sites

twist gene snail gene twist protein-coding sequence twist gene protein-coding sequence snail gene rhomboid^Z.

rhomboid gene

twist gene ;

snail gene rhomboid^Z.

^ rhomboid^

rhomboid gene

▲ FIGURE 15-18 Activation of different target genes by Dorsal subsequent to Toll signaling. Activation of Toll by Spätzle leads to a graded nuclear localization of Dorsal protein (see Figure 15-17d). The resulting Dorsal concentration gradient (ventrally high and dorsally low) can lead to different patterns of gene expression. Shown here are three target genes that have either high-affinity (dark blue) or low-affinity (light blue) Dorsal-binding sites. (a) In ventral regions where the concentration of Dorsal (purple) is high, it can bind to low-affinity sites in twist and snail, activating the transcription of these genes. Twist protein (orange) also activates the transcription of snail, which encodes a repressor (yellow) that prevents the transcription of rhomboid in this region. (b) In lateral regions, the Dorsal concentration is not high enough for the binding of Dorsal to the low-affinity sites regulating twist and snail. The binding of Dorsal to rhomboid is facilitated by the presence of high-affinity sites and the synergistic binding of bHLH heterodimeric activators (green) to neighboring sites. The sharp boundary in the expression of Rhomboid causes formation of distinct cell types in ventral vs. lateral regions. [See A. M. Huang et al., 1997, Genes & Dev. 11:1963.]

centration. For instance, the twist gene, which contains three low-affinity Dorsal-binding sites, is expressed most ventrally where the Dorsal concentration is highest. As the Dorsal concentration decreases, it falls beneath the threshold necessary to activate the transcription of twist. The rhomboid gene, which is expressed only in lateral regions, is controlled through a complex cis-acting regulatory region that contains three high-affinity Dorsal-binding sites. Two of these sites are adjacent to regulatory sequences that bind proteins containing the basic helix-loop-helix (bHLH) motif, which is present in numerous transcription factors. As Twist contains a bHLH motif, it appears that it acts cooperatively with Dorsal to induce transcription of the rhomboid gene in lateral cells. The rhomboid control region also contains four binding sites for Snail, a transcriptional repressor. The production of Snail is induced only at high concentrations of Dorsal because the snail gene contains only low-affinity Dorsal-binding sites within its control region. Because Snail is localized ventrally, its repressor activity defines a sharp ventral-lateral boundary in the transcription of rhomboid. This example nicely illustrates how two transcriptional regulators can collaborate to create a sharp boundary between cell types, something to be discussed further in Sections 15.4 and 15.5.

The dorsal/ventral patterning produced by Dorsal is extended by Decapentaplegic (Dpp). This secreted signaling protein belongs to the TGF ß family, which is found in all animals (Chapter 14). Because transcription of the dpp gene is repressed by Dorsal, Dpp is produced only in the dorsalmost cells of the early fly embryo, which lack Dorsal in their nuclei. A combination of genetic and molecular genetic evidence suggests that Dpp acts as a morphogen to induce the establishment of different ectoderm cell types in the dorsal region of the embryo. For instance, complete removal of Dpp function leads to a loss of all dorsal structures and their conversion into more-ventral ones. Embryos carrying only one wild-type dpp allele show an increase in the number of cells assuming a ventral fate, whereas embryos with three copies of dpp form more dorsal cells.

Thus two graded secreted signals, Spätzle and Dpp, play critical roles in determining the dorsal/ventral axis in Drosophila and in inducing further patterning within the dorsal and ventral regions. Spätzle, acting through nuclear-localized Dorsal, a transcription factor, induces ventral fates and controls the production of Dpp, which induces dorsal fates. Unlike Dorsal, which functions only in early development, the Dpp signal is used repeatedly in later development, participating in many processes such as appendage development, gut formation, and eye development. The Spätzle signal also has other functions, which are discussed later.

The frog TGFß family members called BMP2 and BMP4 have inductive effects similar to those of Dpp protein and indeed are the vertebrate proteins most closely related in sequence to Dpp. Most or all components of the TGFß signaling pathway, including Smad transcription factors, appear to be present and participating in development in all animals

(see Figure 14-2). As discussed in Section 15.5, the vertebrate proteins also control patterning along the dorsal/ventral axis, although the axis is flipped in vertebrates compared with invertebrates. Loss of TGFp signaling, owing to mutations in TGFp receptors or Smad proteins, contributes to the onset of cancer (Chapter 23).

Transcriptional Control by Maternally Derived Bicoid Protein Specifies the Embryo's Anterior

We turn now to determination of the anterior/posterior axis in the early fly embryo while it is still a syncytium. As in determination of the dorsal/ventral axis, specification of anterior/posterior cell fate begins during oogenesis. The initial asymmetry also involves so-called maternal mRNAs, which are produced by nurse cells and transported into the oocyte. In this case they become localized in discrete spatial domains (see Figure 15-14). For example, bicoid mRNA is trapped at the most anterior region, or anterior pole, of the early fly embryo (Figure 15-19). The anterior localization of

▲ EXPERIMENTAL FIGURE 15-19 Maternally derived bicoid mRNA is localized to the anterior region of early Drosophila embryos. All embryos shown are positioned with anterior to the left and dorsal at the top. In this experiment, in situ hybridization with a radioactively labeled RNA probe specific for bicoid mRNA was performed on whole-embryo sections 2.5-3.5 hours after fertilization. This time period covers the transition from the syncytial blastoderm to the beginning of gastrulation. After excess probe was removed, probe hybridized to maternal bicoid mRNA (dark silver grains) was detected by autoradiography. Bicoid protein is a transcription factor that acts alone and with other regulators to control the expression of certain genes in the embryo's anterior region. [From P W. Ingham, 1988, Nature 335:25; photographs courtesy of P W. Ingham.]

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