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Drosophila. This remarkable protein functions both as a transcriptional activator and as a membrane-cytoskeleton linker protein (see Figure 6-7). In the absence of a Wnt signal, p-catenin is phosphorylated by a complex containing GSK3, a protein kinase; the adenomatosis polyposis coli (APC) protein, an important human tumor suppressor; and Axin, a scaffolding protein. Phosphorylated p-catenin is ubiquiti-nated and then degraded in proteasomes. In the presence of Wnt, p-catenin is stabilized and translocates to the nucleus. There, it is believed to associate with the TCF transcription factor to activate expression of particular target genes (e.g., wg, cyclin D1, myc, and metalloprotease genes), depending on cell type. Recent evidence suggests that p-catenin acts by a different mechanism in which it controls the export of TCF from the nucleus and perhaps its activation in the cytosol.

Findings from genetic studies have shown that Wnt-induced stabilization of p-catenin depends on Dishevelled (Dsh) protein. In the presence of Wnt, Dsh and the Lrp membrane protein appear to interact with components of the phos-phorylation complex, thereby inhibiting the phosphorylation and subsequent degradation of p-catenin (see Figure 15-32b). The importance of p-catenin stability and location means that Wnt signals affect a critical balance between the three pools of p-catenin in the cytoskeleton, cytosol, and nucleus.

Wnt signals help control numerous critical developmental events, such as gastrulation, brain development, limb patterning, and organogenesis. The regulated movement of Wnts through tissue is critical to establishing properly placed boundaries between different cell types. As is discussed in Chapter 23, disturbances in signal transduction through the Wnt pathway and many other developmentally important signaling pathways are associated with various human cancers.

Gradients of Hedgehog and Transforming Growth Factor P Specify Cell Types in the Neural Tube

As we have seen in Drosophila, many developmental signals act in a graded fashion, inducing different cell fates depending on their concentration. The same phenomenon exists in vertebrates, for example, in the development of the mammalian central nervous system from the neural tube, which forms early in embryogenesis. The neural tube is a simple rolled-up sheet of cells, initially one cell thick. Cells in the ventral part will form motor neurons; lateral cells will form a variety of interneurons. The different cell types can be distinguished prior to morphological differentiation by the proteins that they produce.

Graded concentrations of Sonic hedgehog (Shh), a vertebrate equivalent of Drosophila Hedgehog, determine the fates of at least four cell types in the chick ventral neural tube. These cells are found at different positions along the dorsoventral axis in the following order from ventral to dorsal: floor-plate cells, motor neurons, V2 interneurons, and V1 interneurons. During development, Shh is initially expressed at high levels in the notochord, a mesoderm structure in direct contact with the ventralmost region of the neural tube (Figure 15-33). On induction, floor-plate cells also produce Shh, forming a Shh-signaling center in the ventral-most region of the neural tube. Antibodies to Shh protein block the formation of the different ventral neural-tube cells in the chick, and these cell types fail to form in mice homozygous for mutations in the Sonic hedgehog (Shh) gene.

To determine whether Shh-triggered induction of ventral neural-tube cells is through a graded or a relay mechanism, scientists added different concentrations of Shh to chick neural-tube explants. In the absence of Shh, no ventral cells formed. In the presence of very high concentrations of Shh, floor-plate cells formed; whereas, at a slightly lower concentration, motor neurons formed. When the level of Shh was decreased another twofold, only V2 neurons formed. And, finally, only V1 neurons developed when the Shh concentration was decreased another twofold. These data strongly suggest that in the developing neural tube different cell types are formed in response to a ventral ^ dorsal gradient of Shh. The accumulating evidence for gradients does not rule out additional relay signals that may yet be discovered.

Cell fates in the dorsal region of the neural tube are determined by BMP proteins (e.g., BMP4 and BMP7), which belong to the TGFp family. Recall that Dpp protein, a Drosophila TGFp signal, is critical in determining dorsal cell fates in early fly embryos. Indeed, TGFp signaling appears to be an evolutionarily ancient regulator of dorsoventral patterning. In vertebrate embryos, BMP proteins secreted from ectoderm cells overlying the dorsal side of the neural tube promote the formation of dorsal cells such as sensory neurons (see Figure 15-33). Thus cells in the neural tube sense multiple signals that originate at opposite positions on the dorsoventral axis, and measure the signals from both origins to decide on a course of differentiation.

Dorsal

Dorsal

Ventral

▲ FIGURE 15-33 Graded induction of different cell types in the neural tube by Sonic hedgehog (Shh) and BMP signaling.

Shh produced in the notochord induces floor-plate development. The floor plate, in turn, produces Shh, which forms a ventral ^ dorsal gradient that induces additional cell fates. In the dorsal region, BMP proteins secreted from the overlying ectoderm cells act in a similar fashion to create dorsal cell fates. [See T M. Jessell, 2000, Nature Rev. Genet. 1:20.]

Ventral

▲ FIGURE 15-33 Graded induction of different cell types in the neural tube by Sonic hedgehog (Shh) and BMP signaling.

Shh produced in the notochord induces floor-plate development. The floor plate, in turn, produces Shh, which forms a ventral ^ dorsal gradient that induces additional cell fates. In the dorsal region, BMP proteins secreted from the overlying ectoderm cells act in a similar fashion to create dorsal cell fates. [See T M. Jessell, 2000, Nature Rev. Genet. 1:20.]

Cell-Surface Proteoglycans Influence Signaling by Some Pathways

How do signals move through or around cells embedded in tissues? The full answer is not known, but the distance that a signal can move has important implications for the size and shape of organs. A signal that causes neurons to form, for example, will create more neurons if its range of movement increases. The binding of signaling proteins to cell-surface proteoglycans not only affects the range of signal action but also facilitates signaling in some cases. A proteoglycan consists of a core protein to which is bound glycosaminoglycan chains such as heparin sulfate and chondroitin sulfate (see Figure 6-22). Proteoglycans are important components of the extracellular matrix. Some are embedded in the plasma membrane by a hydrophobic transmembrane domain or tethered to the membrane by a lipid anchor.

Evidence for the participation of proteoglycans in signaling comes from Drosophila sugarless (sgl) mutants, which lack a key enzyme needed to synthesize heparin (and chon-droitin) sulfate. These mutants exhibit the phenotypes associated with defects in Wingless signaling and have greatly depressed levels of extracellular Wingless protein, a Wnt signal. Mutations in dally and dally-like, both of which encode core proteins of cell-surface proteoglycans, also are associated with defective Wingless signaling.

The Wnt pathway is not the only signaling pathway affected in sugarless and other Drosophila mutants with defective proteoglycan synthesis. For instance, such mutants have phenotypes (e.g., absence of a heart or trachea) that are associated with loss-of-function of Heartless and Breathless, which are receptor tyrosine kinases that bind FGF-like signaling proteins. These mutants also appear to have defective TGFp signaling in metamorphosis, though not in embryos, suggesting specific actions of the proteoglycans. In Chapter 14, we saw that the type III TGFp receptor is a cell-surface proteoglycan. Although not absolutely required for TGFp signaling, the type III receptor binds and concentrates TGFp near the surface of a cell in which it is produced, thereby facilitating signaling from the type I and type II receptors (see Figure 14-2).

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