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Signals

Cells change their properties rapidly in response to signals, both during and after development. The segregation and progressive restriction of cell potential during development are changes that take place as an organism grows and generates vast numbers of new cells that must be organized into new tissues and shapes. The cells in some adult tissues (e.g., blood, gut epithelia, and skin) also continue to proliferate and differentiate. These cells build on a substantial preexisting framework and have less "original" construction of tissues to do. Both dividing and nondividing cells in adult tissues remain highly responsive to hormones and other signaling molecules and to environmental changes.

Discussion in this chapter and in Chapters 13 and 14 calls attention to the enormous scientific effort that has been made in identifying the components of signaling pathways, how they transduce signals, and the resulting cellular responses. Many current projects are aimed at learning how multiple signaling pathways are mustered to control normal tissue growth and function in embryonic and adult tissues. In many circumstances, the appropriate response depends on the ability of receiving cells to integrate multiple signals and to con-

trol the availability of active signals. The examples of signal integration and modulation described in this section illustrate some general mechanisms used in a wide variety of contexts.

Competence Depends on Properties of Cells That Enable Them to Respond to Inductive Signals

Early embryologists noted that cells differ in their ability to respond to various inductive signals. The ability to respond to a particular signaling molecule, referred to as competence, depends on several properties of the receiving cell: the presence of receptors specific for the signal, the ability of these receptors to activate specific intracellular pathways, the presence of transcription factors that stimulate the expression of the genes required to implement the appropriate response, and a chromatin structure that makes these genes accessible for transcription.

In some cases, the reception of one signal may make cells competent to receive another. After a part of the liver is damaged or removed surgically, increased amounts of two signals, tumor necrosis factor (TNF) and interleukin-6 (IL-6), are produced as part of the response to liver damage. These signals cause hepatocytes to enter a "primed" state in which the cells increase their production of certain transcription factors (e.g., NF-kB, Stat3, AP1, and CEBP) but do not divide. Primed cells are competent to respond to a combination

Liver regeneration

Liver regeneration

Primed hepatocytes^

"Priming" with TNF and IL-6 allows a response to growth factors. TNF effect can be ■ apoptotic or proliferative, depending on glutathione content and reactive oxygen species.

Growing hepatocytes

Cyclin D

▲ FIGURE 15-39 Priming of resting hepatocytes for later responses to signaling molecules that induce growth. Injury to the liver or removal of part of the liver leads to priming of cells in response to interleukin-6 (IL-6) and tumor necrosis factor (TNF) signals. Primed cells increase their production of the indicated transcription factors but do not divide. Subsequent increase in the blood level of hepatocyte growth factor (HGF) induces primed cells to produce cyclin D, which is required for cell division (Chapter 21). HGF acts in conjunction with epidermal growth factor (EGF) and transforming growth factor a (TGFa).

of three signals that together induce the synthesis of cyclin D and mitosis (Figure 15-39).

Liver regeneration is an important survival tactic after a part of the liver is damaged or poisoned, but unregulated liver growth would lead to an unduly large organ or possibly cancer. In the primed state, hepatocytes can measure their own physiological state, their location within a tissue, their proximity to other cells, the need for healing, and the spatial organization of cells within a structure. On the basis of this assessment, the cells can respond most appropriately to subsequent signals.

Some Signals Can Induce Diverse Cellular Responses

Several classes of cell-surface receptors discussed in Chapters 13 and 14 are linked to more than one intracellular signal-transduction pathway (see Table 14-1). Multiple intra-cellular signaling possibilities are most evident with G protein-coupled receptors, cytokine receptors, and receptor tyrosine kinases. This phenomenon raises a general question: What governs how a cell responds to a signal that can be transduced by multiple pathways? Conversely, if the signaling pathway is the same in many cell types, why does one cell respond by dividing, another by differentiating, and still another by dying? For instance, signaling through the RTK-Ras-MAP kinase pathway (see Figure 14-16) is used repeatedly in the course of development, yet the outcome in regard to cell-fate specification varies in different tissues. If there is no specificity beyond the ligand and receptor, an activated Ras might substitute for any signal. In fact, activated Ras can do so in many cell types. In one DNA microarray study of fibroblasts, for instance, the same set of genes was transcrip-tionally induced by platelet-derived growth factor (PDGF) and by fibroblast growth factor (FGF), suggesting that exposure to either signaling molecule had similar effects. The PDGF receptor and the FGF receptor are both receptor ty-rosine kinases, and the binding of ligand to either receptor can activate Ras.

Several mechanisms for producing diverse cellular responses to a particular signaling molecule seem possible in principle: (1) the strength or duration of the signal governs the nature of the response; (2) the pathway downstream of the receptor is not really the same in different cell types, for example because different complements of transcription factors are present in the receiving cells; and (3) converging inputs from other pathways modify the response to the signal.

Differences in Signal Strength or Duration Evidence supporting the use of the first mechanism comes from studies with PC12 cells, a cultured cell line capable of differentiating into adipocytes or neurons. Nerve growth factor (NGF) promotes the formation of neurons, whereas epidermal growth factor (EGF) promotes the formation of adipocytes.

Strengthening the EGF signal by prolonging exposure to it causes neuronal differentiation. Although both NGF and EGF are RTK ligands, NGF is a much stronger activator of the Ras-MAP kinase transduction pathway than is EGF. The EGF receptor can apparently activate this pathway only after prolonged stimulation.

Differences in Downstream Pathways Signaling through cell type-specific pathways downstream of an RTK has been demonstrated in C. elegans. In worms, EGF signals induce at least five distinct responses, each one in a different type of cell. Four of the five responses are mediated by the common Ras-MAP kinase pathway; the fifth, hermaphrodite ovulation, employs a different downstream pathway in which the second messenger inositol trisphosphate is generated. Binding of IP3 to its receptor (IP3R) in the endoplasmic reticulum membrane leads to the release of stored Ca2+ from the ER (see Figure 13-29). The rise in cytosolic Ca2 + then triggers ovulation. This alternative pathway was discovered with a genetic screen that implicated IP3R, a Ca2 + channel, in EGF signaling—a good example of how a mutation in an unexpected gene can lead to a discovery.

Integration of Signals The third way that the same signaling ligand/receptor pair can produce diverse effects on cells is to integrate more than one signal, as occurs in Drosophila muscle development. Figure 15-40 depicts the convergence of signal inputs that leads to the formation of a single muscle precursor cell, which is defined by its ability to transcribe the even-skipped gene. Early in muscle development, the Drosophila Wnt signal Wingless (Wg) and the TGFp signal Decapentaplegic (Dpp) prime a cell to make it competent to receive a subsequent signal that is transduced through the MAP kinase pathway downstream of Ras. The Wingless signal is produced in circumferential belts, and the Dpp signal is produced in two longitudinal bands at right angles to the Wnt belts.

One group of cells on each side of each body segment receives both the Wingless and the Dpp signals and thus become competent to respond to an unidentified RTK signal that activates Ras. In these cells, signal integration takes place during transcription of the eve gene. The transcription of eve is activated when a short 312-bp eve enhancer (not the same one described in Figure 15-32) is bound by two muscle-specific transcription factors and by three signal-induced transcription factors: TCF factor by Wingless, Mad by Dpp, and Pnt by an RTK acting through Ras. Thus tissue-specific and signal-responsive information is integrated through the action of five regulators on one short piece of DNA in specifying a cell type. As the result of lateral inhibition, eventually only one eve-expressing cell in each initial group of competent cells is left (see Figure 15-40). That single cell will develop into a particular muscle fiber by recruiting other cells and fusing with them.

Equivalence

Equivalence

L'sc cluster L'sc + Eve cluster Eve progenitor

O L'sc O L'sc + Eve O Eve ED Lateral inhibition

L'sc cluster L'sc + Eve cluster Eve progenitor

O L'sc O L'sc + Eve O Eve ED Lateral inhibition

Competence

Competence

Pre-pattern Pre-cluster

Pre-pattern Pre-cluster

▲ FIGURE 15-40 Sequential action of critical signals in Drosophila muscle development. Signal transduction through the RTK pathway is governed by Wnt and TGFp signals. Wingless (Wg) is produced in a stripe of cells running in a belt around part of each body segment of the embryo (purple). Decapentaplegic (Dpp) is produced in a band of dorsal cells running from head to tail on each side of the embryo (blue), a band that is created by the dorsal/ventral signaling system described in Section 15.3. A patch of cells in each body segment will receive both signals; only these cells (green) are competent to respond to the (unidentified) RTK signal that activates intracellular signaling from Ras. All the cells in the patch activate a gene called L'sc, though further signaling restricts L'sc and then eve transcription to a more restricted set of cells called the pre-cluster (orange). Within the pre-cluster, a central cell begins to use Notch signaling to surrounding cells to repress L'sc and eve transcription there until only one cell is left making eve products (red). That single cell will develop into a particular muscle by recruiting other cells and fusing with them; two such cells are created in each body segment by this elaborate process. Both require RTK-mediated signaling; one cell uses the Drosophila EGF receptor (DER) and the EGF-type receptor called Heartless (Htl), and the second cell uses only Htl. [See Halfon et al., 2000, Cell 103:63-74.]

Limb Development Depends on Integration of Multiple Extracellular Signal Gradients

Vertebrate limbs grow from small "buds" composed of an inner mass of mesoderm cells surrounded by a sheath of ectoderm. Secreted signals from both cell layers coordinate limb development and instruct cells about their proper fates within limbs. The first signal, fibroblast growth factor 10 (FGF10) is secreted from the lateral trunk mesoderm and initiates outgrowth of a limb from specific regions of the em-

bryo's flank. Implantation of a bead soaked in FGF10 into places in the flank where a limb does not normally form causes an extra limb to grow; so FGF has remarkable inductive capabilities.

There are three dimensions to a limb: anterior/posterior (thumb to little finger), dorsal/ventral (palm versus back of hand) and proximal/distal (shoulder to fingers). An embryonic cell that knows its position along each of these dimensions is well along toward knowing what to do. A different signaling system operates in each of the three dimensions; so,

► FIGURE 15-41 Integration of three signals in vertebrate limb development along proximal/distal and anterior/ posterior axes. Each limb bud grows out of the flank of the embryo. (a) A fibroblast growth factor (FGF) signal, probably FGF10, comes from the mesoderm in specific regions of the embryo's flank, one region for each limb. FGF10 acts on a local region of surface ectoderm called the apical ectodermal ridge (AER) because it will form a prominent ridge. (b) The ectoderm that receives a FGF10 signal is induced to produce FGF8, another secreted signal. At the posterior end of the limb bud, FGF8 induces transcription of the Sonic hedgehog (Shh) gene. (c) Shh signaling induces transcription of the gene encoding FGF4 in the AER. FGF8 and FGF4 promote continued proliferation of the mesoderm cells, causing outgrowth of the limb bud. Shh also stimulates this outgrowth and confers posterior characteristics on the posterior part of the limb. Development along the dorsal/ventral axis depends on a Wnt signal that is not shown here.

Fgf8 induced by FGF10X

Anterior _

Posterior -

Proliferation maintained by FGF8

Lateral plate mesoderm shh induced by FGF8

Surface ectoderm

Proliferation maintained by FGF8

Proliferation maintained by FGF8 + FGF4

shh maintained by FGF8 + FGF4

Proliferation maintained by FGF8 + FGF4

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