Adp

receptors receptors

EphA EphB

receptors receptors

▲ FIGURE 15-34 General structure of Eph receptors and their ligands. The cytosolic domain of Eph receptors has tyrosine kinase activity. Within the Eph receptor family, the receptors exhibit some 30-70 percent homology in their extracellular domains and 65-90 percent homology in their kinase domains. Their ligands, the ephrins, either are linked to the membrane through a hydrophobic GPI anchor (class A) or are single-pass transmembrane proteins (class B). The core domains of various ephrin ligands show 30-70 percent homology. Ephrin-B ligands and their receptors can mediate reciprocal signaling. [Adapted from V. Dodelet and E. Pasquale, 2000, Oncogene 19:5614; see J. G. Flanagan and P Vanderhaegen, 1998, Ann. Rev. Neurosci. 21:309.]

which can act as a receptor and ligand. To illustrate this phenomenon, we consider the role of the ephrins, a family of cell-surface ligands, and the Eph receptors in the development of mammalian blood vessels.

The Eph receptors, a novel type of receptor tyrosine ki-nase, have two classes of ligands (Figure 15-34). Ephrin-A ligands are tethered to the plasma membrane by a glyco-sylphosphatidylinositol (GPI) anchor. These ephrin ligands play a crucial role in forming connections between neurons in the developing nervous system. Ephrin-B ligands are single-pass transmembrane proteins. The results of biochemical experiments showed that ephrin-B ligands stimulate tyrosine phosphorylation of EphB receptors and of their own cytosolic domain. These observations led to the intriguing notion that ephrin-B ligand/EphB receptor complexes promote bidirectional reciprocal interactions. Strong support for this hypothesis has come from the study of blood vessel formation.

Blood vessels, arteries and veins, form a complex network of branched structures in the adult. An early network of vessels is remodeled during angiogenesis as larger branches assemble from smaller ones and vessels become surrounded by support cells. Knockout mice lacking ephrin-B2 exhibit striking defects in angiogenesis. This finding led scientists to explore the pattern of expression of ephrin-B2 and its receptor, EphB4, in the developing embryo (Figure 15-35). In normal embryos, ephrin-B2 is expressed only in arteries; EphB4, only on veins. Although ephrin-B2 is expressed only on arterial capillaries, venous capillaries also fail to undergo angiogenesis in ephrin-b2 knockouts. These data suggest that interaction between an arterial cell producing ephrin-B2 and a venous cell producing EphB4 causes the induction of both cells (see Figure 15-35c). In other words, ephrin-B2 and EphB4 each functions as both a ligand and a receptor to control the development of both veins and arteries.

Umbilical a. Allantoic stalk

Dorsal aorta Atrium

Ventricle

Dorsal aorta Atrium

Ventricle

Juvenile vascular system

Mature vascular system

Primary plexus

Juvenile vascular system

Arterial endothel cell

Venous endothelial cell

▲ FIGURE 15-35 Reciprocal induction mediated by ephrin-B2 and its receptor, EphB4, in angiogenesis in the yolk sac.

(a) Ephrin-B2 (red) is expressed on arteries and EphB4 (blue) on veins in the early mouse embryo. (b) The early vascular network is remodeled during angiogenesis. In ephrin-B2 knockout mice, angiogenesis is blocked at the primary plexus stage. The absence of ephrin-B2 thus interrupts the development of both arteries and veins. (c) Formation of intercalating arteries and veins results from interactions between developing arterial and venous endothelial cells mediated by ephrin-B2 (arterial) and EphB4 (venous). These reciprocal interactions induce the development of both cell types. [Adapted from H. U. Wang et al., 1998, Cell 93:741.]

Mature vascular system

Venous endothelial cell

Primary plexus

Arterial endothel cell

▲ FIGURE 15-35 Reciprocal induction mediated by ephrin-B2 and its receptor, EphB4, in angiogenesis in the yolk sac.

(a) Ephrin-B2 (red) is expressed on arteries and EphB4 (blue) on veins in the early mouse embryo. (b) The early vascular network is remodeled during angiogenesis. In ephrin-B2 knockout mice, angiogenesis is blocked at the primary plexus stage. The absence of ephrin-B2 thus interrupts the development of both arteries and veins. (c) Formation of intercalating arteries and veins results from interactions between developing arterial and venous endothelial cells mediated by ephrin-B2 (arterial) and EphB4 (venous). These reciprocal interactions induce the development of both cell types. [Adapted from H. U. Wang et al., 1998, Cell 93:741.]

The Conserved Notch Signaling Pathway Mediates Lateral Inhibition

Now we shift our attention to lateral inhibition, which causes adjacent developmentally equivalent or near-equivalent cells to assume different fates. Genetic analyses in Drosophila and C. elegans revealed the role of the highly conserved Notch/Delta pathway in lateral inhibition. The Drosophila proteins Notch and Delta are the prototype receptor and ligand, respectively, in this signaling pathway. Both proteins are large transmembrane proteins whose extracellular domains contain multiple EGF-like repeats and binding sites for the other protein. Although Delta is cleaved to make an apparently soluble version of its extracellular domain, findings from studies with genetically mosaic Drosophila have shown that the Delta signal reaches only adjacent cells.

Interaction between Delta and Notch triggers the prote-olytic cleavage of Notch, releasing its cytosolic segment,

Intrinsically biased

Extrinsically biased

Intrinsically biased

Extrinsically biased

Cluster

Field

Cluster

▲ FIGURE 15-36 Amplification of an initial bias to create different cell types by Notch-mediated lateral inhibition.

(a) A difference between two initially equivalent cells may arise randomly (left ). Alternatively, interacting cells may have an intrinsic bias (center ) or an extrinsic bias (right ). For instance, cells that have received different proteins in an asymmetric cell division will be intrinsically biased; those that have received different signals (orange) will be extrinsically biased. Regardless of how the small initial bias arises, Notch becomes predominant in one of the two cells, promoting its own expression and repressing production of its ligand Delta in that cell. In the other cell, Delta predominates. The outcome is reinforcement of the small initial difference. (b) Notch-mediated lateral inhibition may create a sharp boundary in an initial field of cells, such as along the edge of the developing Drosophila wing, or distinguish a central cell from a surrounding cluster of cells, as in neural precursor establishment. [Adapted from S. Artavanis-Tsakonas et al., 1999, Science 284:770.]

which translocates to the nucleus and regulates the transcription of specific target genes (see Figure 14-29). In particular, Notch signaling activates the transcription of Notch itself and represses the transcription of Delta, thereby intensifying the difference between the interacting cells (Figure 15-36a). Notch-mediated signaling can give rise to a sharp boundary between two cell populations or can single out one cell from a cluster of cells (Figure 15-36b). Notch signaling controls cell fates in most tissues and has consequences for differentiation, proliferation, the creation of cell asymmetry, and apoptosis. In the immune system, for instance, Notch signaling helps prevent the formation of T cells that attack an individual's own proteins. Here, we describe two examples of Notch signaling in cell-fate determination.

Determination of AC and VU Cell Fates in C. elegans Two equivalent cells, designated Zl.ppp and Z4.aaa, in roundworms can give rise to an anchor (AC) cell or a ventral uterine (VU) precursor cell. The results of laser ablation studies showed that, if either the Zl.ppp or Z4.aaa cell is removed, the remaining cell always becomes AC. In worms lacking functional LIN-12, the C. elegans homolog of Notch, both cells become AC. Conversely, constitutive activation of LIN-12 in both Z1.ppp and Z4.aaa results in both cells becoming VU. Thus LIN-12 activity levels specify AC and VU cell fates.

Both Z1.ppp and Z4.aaa produce the receptor, LIN-12, and its ligand, the Delta homolog LAG-2, at similar levels (Figure 15-37). As development proceeds, one cell begins to express more receptor through random fluctuations in proReceptor (LIN-12) Ligand (LAG-2)

▲ FIGURE 15-37 Determination of different cell fates by lateral inhibition in C. elegans development. LIN-12, a Notch homolog, and LAG-2, a Delta homolog, regulate interactions between two equivalent cells, designated Z1.ppp and Z4.aaa. Either cell can assume a ventral uterine (VU) or anchor (AC) fate. See text for discussion. [Adapted from I. Greenwald, 1998, Genes & Dev. 12:1751.]

▲ FIGURE 15-37 Determination of different cell fates by lateral inhibition in C. elegans development. LIN-12, a Notch homolog, and LAG-2, a Delta homolog, regulate interactions between two equivalent cells, designated Z1.ppp and Z4.aaa. Either cell can assume a ventral uterine (VU) or anchor (AC) fate. See text for discussion. [Adapted from I. Greenwald, 1998, Genes & Dev. 12:1751.]

tein levels or differences in the ambient level of signaling through the pathway. The cell receiving a slightly higher signal begins to increase its expression of the receptor and decrease its expression of the ligand. In the neighboring cell, now exposed to a reduced level of ligand, expression of the receptor falls and that of the ligand increases. In this way, the initial asymmetry resulting from a random event is amplified, finally leading to the commitment of one cell as a pre-VU cell and its partner as a pre-AC cell. When formed, the AC cell begins sending out a LIN-3 signal that functions in vulva development. Notch-mediated lateral inhibition also operates in that process when the P6.p cell inhibits the neighboring P5.p and P7.p cells (see Figure 15-12b).

Neuronal Development in Drosophila and Vertebrates

Loss-of-function mutations in the Notch or Delta genes produce a wide spectrum of phenotypes in Drosophila. One consequence of such mutations in either gene is an increase in the number of neuroblasts in the central nervous system. In Drosophila embryogenesis, a sheet of ectoderm cells becomes divided into two populations of cells: those that move inside the embryo eventually develop into neuroblasts; those that remain external form the epidermis and cuticle. As some of the cells enlarge and then loosen from the ectodermal sheet to become neuroblasts, they signal to surrounding cells to prevent their neighbors from becoming neuroblasts—a case of lateral inhibition. Notch signaling is used for this in hibition; in embryos lacking the Notch receptor or its ligand, all the ectoderm precursor cells become neural.

The role of Notch signaling in specifying neural cell fates has been studied extensively in the developing Drosophila peripheral nervous system. In flies, various sensory organs arise from proneural cell clusters, which produce bHLH transcription factors, such as Achaete and Scute, that promote neural cell fates. In normal development, one cell within a proneural cluster is somehow anointed to become a sensory organ precursor (SOP). In the other cells of a cluster, Notch signaling leads to the repression of proneural genes, and so the neural fate is inhibited; these nonselected cells give rise to epidermis (Figure 15-38). Temperature-sensitive mutations that cause functional loss of either Notch or Delta lead to the development of additional SOPs from a proneural cluster. In contrast, in developing flies that produce a constitutively active form of Notch (i.e., active in the absence of a ligand), all the cells in a proneural cluster develop into epidermal cells.

To assess the role of the Notch pathway during primary neurogenesis in Xenopus, scientists injected mRNA encoding different forms of Notch and Delta into embryos. Injection of mRNA encoding the constitutively active cytosolic segment of Notch inhibited the formation of neurons. In contrast, injection of mRNA encoding an altered form of Delta that prevents Notch activation led to the formation of too many neurons. These findings indicate that in vertebrates, as in Drosophila, Notch signaling controls neural precursor cell fates.

Induction of proneural cluster Determination

Differentiation

IK Lower level of Emc

▲ FIGURE 15-38 Role of Notch-mediated lateral inhibition in formation of sensory organ precursors (SOPs) in Drosophila. (a) Extracellular signaling molecules and transcription factors, encoded by early-patterning genes, control the precise spatiotemporal pattern of proneural bHLH proteins such as Achaete and Scute (yellow). Most cells within the field express Emc (orange), a related protein that antagonizes Achaete and Scute. A small group of cells, a proneural cluster, produce proneural bHLH proteins. The region of a proneural cluster from which an SOP will form expresses lower levels of Emc, giving these cells a bias toward SOP formation. Interactions between these cells, leading to accumulation of E(spl) repressor proteins in neighboring cells (blue), then restrict SOP formation to a single cell (green). (b) Initially, achaete (ac) and other proneural genes are transcribed in all the cells within a proneural cluster, as are

Differentiation

Cell fate: Epidermal cells

Notch and Delta. Achaete and other proneural bHLH proteins promote expression of Delta. When one cell at random begins to produce slightly more Achaete (left), its production of Delta increases, leading to stronger Notch signaling in all its neighboring cells (right). In the receiving cells, the Notch signaling pathway activates a transcription factor designated Su(H), which in turn stimulates expression of E(spl) genes. The E(spl) proteins specifically repress transcription of ac and other proneural genes. The resulting decrease in Achaete leads to a decrease in Delta, thus amplifying the initial random difference among the cells. As a consequence of these interactions and others, one cell of a proneural cluster is selected as a SOP; all the others lose their neural potential and develop into epidermal cells.

Cell fate: Epidermal cells

Notch and Delta. Achaete and other proneural bHLH proteins promote expression of Delta. When one cell at random begins to produce slightly more Achaete (left), its production of Delta increases, leading to stronger Notch signaling in all its neighboring cells (right). In the receiving cells, the Notch signaling pathway activates a transcription factor designated Su(H), which in turn stimulates expression of E(spl) genes. The E(spl) proteins specifically repress transcription of ac and other proneural genes. The resulting decrease in Achaete leads to a decrease in Delta, thus amplifying the initial random difference among the cells. As a consequence of these interactions and others, one cell of a proneural cluster is selected as a SOP; all the others lose their neural potential and develop into epidermal cells.

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