How a single cell, the fertilized egg, transforms into a complex multicellular organism is one of the most fascinating questions in biology (see Development). Two processes that are critical in this transformation are the generation of distinct cell types and the patterning of these cells into functional tissues and organs. Molecular genetic experiments in the fruit fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans first revealed that similar transmembrane receptors (called the Notch receptor in Drosophila) play critical roles in numerous cell-cell interactions that are involved in the establishment and patterning of a wide array of cell types. Remarkably, homologous Notch receptors in echinoderms, amphibians, fish, birds, and mammals have subsequently been found to mediate a similar broad range of cellular interactions. This suggests that the Notch receptor is a fundamental intercellular signaling molecule used in the patterning and generation of different cell types in all metazoans. Work from a number of organisms has demonstrated that the Notch receptor is a central component of a conserved intercellular signaling pathway that has at least three additional core members. These are the transmembrane ligands Delta and Serrate and the transcription factor CSL. Consistent with the diverse interactions that the Notch pathway mediates during development, the regulation of the receptor and ligands varies a great deal in different developmental contexts. One of the most intriguing questions about the Notch signaling pathway is how this single pathway regulates the generation of such a broad range of cell types. Gaining insight into this aspect of Notch signaling has been challenging, but answers to this question promise to shed light on the fundamental question of how cells are patterned and how they assume different fates during development.
1. Components of the Notch Signaling Pathway
Notch homologous proteins are large membrane proteins that span the membrane once. In the extracellular domain, all Notch proteins contain multiple, tandemly arranged EGF motifs and three Notch/Lin-12 repeats. In the intracellular domain, Notch proteins contain six ankyrin repeats, a motif involved in direct protein-protein interactions, as well as a PEST sequence that may regulate the stability of the protein (Fig. 1). Only a single Notch protein is encoded in the Drosophila and sea urchin genomes (1). In C. elegans, there are two Notch-like proteins, called GLP-1 and LIN-12. In vertebrates, four Notch homologous proteins have been identified.
Figure 1. Schematic diagram of the primary structures of Drosophila Notch receptor and its ligands, Delta and Serrate.
Delta and Serrate, the two identified ligands for Notch, are also single-spanning transmembrane proteins. Much as Notch does, all ligands for Notch contain multiple EGF-like repeats in the extracellular domain, as well as a unique cysteine-rich motif called the DSL domain (Fig. 1). In Drosophila, there appears to be only one Delta and one Serrate protein although multiple homologues of each have been identified in vertebrates (eg, 2). In C. elegans, there are two identified ligands, LAG-2 and APX-1, which are smaller than, but similar in structure to, Delta and Serrate. Remarkably, of the 36 EGF-like repeats in Drosophila Notch, only two (repeats 11 and 12) are necessary for binding to either Serrate or Delta. This has raised the possibility that additional ligands may exist that bind to the other EGF-like repeats in the extracellular domain; to date, however, no other ligands have been identified.
An additional shared component in the Notch pathway has been demonstrated by studies in Drosophila, C. elegans, and vertebrates. These are the homologous CSL proteins, which encode nuclear DNA-binding proteins (3, 4). CSL proteins appear to act both as repressors and activators of transcription, providing a link for the Notch pathway to alterations in gene expression (5-7).
2. Activation of the Notch Receptor
Interestingly, almost all identified Notch proteins contain nuclear localization signals in their intracellular domain (see Nuclear import /export). In addition, the intracellular domains of most Notch proteins, when expressed alone in cells and untethered to the membrane, enter the nucleus and cause phenotypes in cells similar to the one caused by overactivation of the Notch pathway. Furthermore, the intracellular domains of some Notch proteins bind physically to CSL proteins and activate transcription of target genes. These findings have led to the proposal that the interaction of Notch with its ligands results in the proteolytic release and translocation of the intracellular domain into the nucleus (Fig. 2 and Ref. 8). The amount of intracellular domain that enters the nucleus to signal in vivo, however, appears to be extremely low and requires very sensitive assays to detect (9, 10). One common transcriptional target in both vertebrates and Drosophila for the Notch-CSL protein complex is the Enhancer of Split complex (7, 11). This complex encodes seven proteins related to basic helix-loop-helix proteins, as well as a protein that shares homology with GTP-binding proteins. Another target of Notch signaling in Drosophila is the vestigial gene, which is activated by Notch signaling at the dorsal-ventral boundary in the wing to stimulate cell proliferation. Other targets of Notch-mediated signaling are likely to exist but have yet to be identified (eg, 12). Intriguingly, during some cell-cell interactions, Notch proteins do not require CSL proteins to signal (7, 12, 13). But how Notch proteins signal independently of CSL proteins is unknown at this time.
Figure 2. Hypothetical model for activation of the Notch signaling pathway. The Notch receptor can bind to either Delta neighboring cell. Ligand binding leads to proteolytic release of the intracellular domain of Notch from the membrane. Al intracellular domain is translocated into the nucleus, where it interacts with a CSL protein. This interaction leads to a com the transcriptional activity of target genes of the Notch pathway.
3. Pleiotropy of Notch-mediated signaling
Notch was originally identified in Drosophila as a "neurogenic" gene involved in the segregation of neuroblasts and epidermal cells in the embryonic neuroectoderm. However, temperature-sensitive mutants in Drosophila Notch and C. elegans Notch homologues, specific mutations in Notch homologues, and expression and misexpression studies have uncovered an amazing diversity of cell-fate decision and patterning processes that Notch receptors mediate in Drosophila, C. elegans, and vertebrate development. For example, in Drosophila, Notch signaling has been implicated in the establishment of distinct cell types in the mesoderm, endoderm, Malpighian tubules, eye, and ovary. In addition, Notch signaling is crucial to the organization of the Drosophila dorsal-ventral wing boundary, which is a patterning process that directs wing growth and wing margin formation (eg, 14). In C. elegans, the two Notch-like proteins GLP-1 and LIN-12 similarly mediate numerous cell-fate decisions, and GLP-1 plays a central role in an important patterning process, the maintenance of mitotic nuclei in the distal end of the C. elegans ovary. In vertebrates, Notch signaling is involved in cell-fate specification during neurogenesis, hematopoiesis, and muscle cell development (3, 15-17). In addition, Notch signaling is necessary for the segmentation of somites, a critical process in patterning the segmental organization of vertebrate embryos (18, 19). Furthermore, studies of human diseases have shown that altered forms of Notch signaling can lead to T-cell lymphoblastic leukemia and to a developmental disorder called Alagille syndrome, which results in developmental abnormalities in the liver, kidney, heart, eye, vertebrae, and facial structure (reviewed in 20).
Intriguingly, particular mutations in the human Notch receptor, Notch3, have also been implicated in the adult onset disease CADASIL, which causes vascular alterations and leads to repeated strokes and dementia (21). The late onset of this disease suggests that Notch signaling may play an unknown function in differentiated cells of adult tissues.
4. Diverse interactions between Notch and its ligands
Reflecting the numerous cell-fate and patterning processes mediated by Notch signaling, the ligands for Notch are used in diverse ways to activate and sometimes even repress the Notch receptor. In some developmental contexts, Notch and its ligands are arranged in what is known as an inductive interaction. In inductive cell-cell interactions, the signaling and receiving cells are not equivalent to one another, and one cell or tissue selectively signals to the other (Fig. 3a). An example of this is germ-line induction in C. elegans: the Notch-like protein GLP-1 is selectively expressed in the receiving germ-line tissue, and the ligand LAG-2 is restricted to the signaling distal-tip cell.
Figure 3. Diverse interactions between the Notch receptor and its ligands. (a) In inductive signaling, the signaling and receiving cells are not equivalent to one another at the onset of the interaction. The signaling cell expresses only the ligand, and the receiving cell expresses only the Notch receptor. In lateral signaling, (b) interacting cells are initially equivalent to one another, and both express ligand and receptor. A stochastic fluctuation in signaling results in one cell receiving above-threshold Notch signaling. This triggers a feedback mechanism that reinforces this signaling difference such that one cell ultimately expresses only ligand and the other only receptor. In mutual signaling, ( c) a group of cells express ligand and receptor, and all signal one another. This type of signaling may be used to coordinate patterning processes or cell-fate decisions within groups of cells. High levels of Notch ligand can inhibit Notch signaling within the same cell in which they are expressed (d). The mechanism for this inhibition is not understood, but it may be mediated by direct interactions between Notch and its ligands within the same cell.
Notch and its ligands also mediate lateral signaling, where the signaling and receiving cells are initially equivalent to one another in developmental potential and signaling ability. In this type of interaction, it appears that random fluctuations in signaling activity are reinforced and strengthened through feedback mechanisms so that one cell becomes the signaler and the other cell the receiver (Fig. 3b). An example of this is the interaction between the anchor cell (AC) precursor and the ventral uterine (VU) precursor cell during C. elegans development. Both cells are initially equivalent in their ability to become either the AC or the VU cell, and both express the Notch-like receptor LIN-12 and the ligand LAG-2. Interactions between these cells result in the activation of a feedback mechanism, such that one cell ultimately expresses only the ligand LAG-2 (signaling cell, which becomes the AC) and the other cell expresses only the Notch-like receptor LIN-12 (receiving cell, which becomes the VU).
Notch and its ligands can also be expressed and activated in groups of cells. This type of interaction is known as mutual signaling (Fig. 3c). An example of this occurs during the early development of the Drosophila nervous system in the ventral neurogenic ectoderm. All cells in this region express both Notch and the ligand Delta. Usually, only a subset of the cells delaminate from the neurogenic ectoderm and become neuroblasts; in embryos lacking either Notch or Delta, however, most cells in the neurogenic ectoderm delaminate as neuroblasts. It appears that nearly every cell in the neurogenic ectoderm has the potential to become a neuroblast but that neighboring cells interact through Notch-Delta signaling to inhibit this potential, thus restricting the number of cells that develop as neuroblasts.
In some developmental contexts, high levels of Delta or Serrate inhibit Notch signaling within the same cell in which they are expressed (Fig. 3d). An example of this type of interaction is found at the Drosophila dorsal-ventral wing boundary. High levels of Delta or Serrate within cells adjacent to the wing boundary inhibit these cells from receiving Notch signaling (22). This ensures that Notch signaling is activated only in cells at the boundary that do not express ligands for Notch. How ligands for Notch inhibit Notch signaling within the same cell is not fully understood, but evidence suggests that the inhibition could be mediated by direct interactions between the ligand and receptor within the same cell. Thus, the levels of ligand present on a cell may be a critical component in controlling whether neighboring cells can signal one another with the Notch pathway.
5. Additional regulators of the Notch pathway
Several other molecules have been identified as important regulators of the Notch pathway. A protein called Numb was first identified in Drosophila as playing a role in the generation of the different cell types that are required to build neuronal sensory organs located in the epidermis. Protein localization studies found that Numb is distributed asymmetrically during cell divisions in cells that construct the sensory organ. Interestingly, Numb appears to affect the generation of distinct cell types by binding to the intracellular domain of Notch, which inhibits Notch signaling. Therefore, Numb creates a signaling interaction between neighboring sister cells from a cell division very similar to inductive signaling, where one cell is the signaler and the other the receiver (see Fig. 3a).
Genetic studies have indicated that many interactions occur between the Notch pathway and another important cell-cell signaling pathway called the Wnt/Wingless pathway. During some interactions, these pathways appear to act synergistically; in others, however, they act antagonistically to one another. Interestingly, the disheveled gene product, a cytoplasmic protein and member of the Wnt/Wingless signaling pathway, may mediate direct antagonistic interactions between these two pathways. The Disheveled protein can bind to the intracellular domain of Notch and inhibit Notch signaling during the specification of Drosophila wing margin bristles.
Another protein that regulates the Notch pathway is Fringe, which is a secreted protein that appears to function in the extracellular space between cells. In Drosophila, Fringe can modulate interactions between Delta and Serrate and with Notch by blocking the ability of Serrate to activate Notch and potentiating the ability of Delta to activate the receptor (23, 24). Several vertebrate Fringe homologues have been identified as well, and they appear to mediate a similar function (25).
6. How does the Notch pathway control the generation of many cell types?
One of the most puzzling questions about Notch signaling is how this one pathway is involved in the formation of so many distinct cell types and patterning processes during development. Experiments in vertebrates and Drosophila have demonstrated that, during many Notch-mediated cell-cell interactions, Notch signaling appears to block the ability of cells to respond to differentiation signals or regulatory factors, maintaining cells in an undifferentiated state (15, 26). For example, in the Drosophila compound eye, inappropriate activation of Notch signaling in the presumptive R7 cell, at the time this cell is receiving the differentiation signal to become R7, inhibits this cell from differentiating as an R7 cell. Instead, after Notch signaling subsides, this cell differentiates into a cone cell, the default fate for this cell when it does not receive the R7 differentiation signal. Therefore, Notch signaling may be crucial in the establishment of many different cell types in controlling whether and when cells can respond to specific differentiation cues.
Notch signaling, however, does not always inhibit differentiation in cells. During C. elegans development, the Notch pathway mediates the formation of numerous cell types, yet there is currently no evidence that Notch signaling guides the generation of distinct cell types by inhibiting differentiation. Indeed, during the development of the vulva and uterus, Notch signaling appears to act as a differentiation signal in the generation of specific cells that contribute to the formation of both these tissues (27, 28). Furthermore, during Drosophila wing development, activation of Notch leads to the expression of growth and patterning genes (eg, 29), a finding inconsistent with the notion that Notch signaling is keeping these cells in an undifferentiated state. Therefore, although, in many cell-cell interactions, Notch signaling functions to establish distinct cell types by regulating the ability of cells to differentiate, Notch signaling does not always function in this manner. To gain a deeper understanding of how Notch signaling affects the establishment of numerous cell types and patterning process, it will be necessary to identify additional target genes regulated by Notch signaling and elucidate how these genes mediate the differentiation and patterning of distinct cell types.
