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p24 proteins and quality control of Notch family protein in C. elegans

Genetic screens in C. elegans and Drosophila have identified factors that influence lin-12/Notch activity. Many genes have been identified in sensitized genetic backgrounds, by suppressing or enhancing mutations in lin-12. Most of these genes have been named sel genes, for suppressor/ enhancer of lin-12. The suppressor/enhancer approach mitigates potential difficulties arising from possible functional redundancy of mechanisms that control receptor activity as well as gene redundancy in each step. Several sel genes that have been characterized are involved in basic cell biological processes. Two sel genes, sel-12 (presenilin) and sup-17 (ADAM10/Kuzbanian), appear to affect processing of LIN-12 and GLP-1. Two other sel genes, sel-1 and sel-10, are likely to affect LIN-12 and GLP-1 turnover. Therefore, the sel genes and their interactions with lin-12 and glp-1 may illuminate connections between basic cell biological processes and signaling (Wen, 1999 and references).

Mutations in the Caenorhabditis elegans sel-9 gene elevate the activity of lin-12 and glp-1, which encode members of the LIN-12/NOTCH family of receptors. Sequence analysis indicates SEL-9 is one of several C. elegans p24 proteins. Allele-specific genetic interactions suggest that reducing sel-9 activity increases the activity of mutations altering the extracellular domains of LIN-12 or GLP-1. Reducing sel-9 activity restores the trafficking to the plasma membrane of a mutant GLP-1 protein that would otherwise accumulate within the cell. These results suggest a role for SEL-9 and other p24 proteins in the negative regulation of transport of LIN-12 and GLP-1 to the cell surface, and favor a role for p24 proteins in a quality control mechanism for endoplasmic reticulum-Golgi transport (Wen, 1999).

Multiple members of the p24 family are found in all eukaryotes, from yeast to mammals. Members of the p24 family are type I membrane proteins with a signal peptide at the amino terminus, a lumenal (extracytosolic) domain, a single transmembrane domain, and a short cytoplasmic tail. p24 proteins have a predicted lumenal coiled-coil domain, conserved amino acids in the transmembrane domain and cytoplasmic tail, and similar overall size and organization. They may be grouped into at least three subfamilies based on primary sequence. One subfamily comprises yeast Emp24p and mammalian p24A; SEL-9 appears to be a member of this subfamily. Another subfamily comprises yeast Erv25p and mammalian Tmp21, and the third subfamily comprises mammalian gp25L proteins (Wen, 1999 and references).

In eukaryotic cells, secretory protein trafficking is mediated by transport vesicles, which bud from a donor membrane of one compartment and fuse with a recipient membrane of a different compartment. Distinct vesicle coat protein complexes mediate different budding/fusion events. Anterograde transport from ER to Golgi is mediated by COPII-coated vesicles. Bidirectional transport between the ER and Golgi, and intra-Golgi transport, is mediated by COPI-coated vesicles. Endocytic trafficking is mediated by clathrin-coated vesicles. A key feature of vesicle-mediated trafficking is the net transfer of cargo from one compartment to another, while components of the donor compartment are selectively excluded from vesicles and/or recycled. Furthermore, there appears to be a quality control mechanism, so that cargo proteins of misfolded or mutant proteins are not transferred. Little is known about how selective packaging or quality control occurs. Signals on cargo and the coat proteins appear to influence assembly of the COPII coat complex. However, other factors appear to influence selectivity: for example, null mutations that bypass the anterograde secretion block associated with the absence of Sec13p (one component of the COPII coat complex) also cause leakage of ER resident proteins and mutant invertase (Wen, 1999 and references).

One gene identified as a bypass suppressor of delta sec13 was EMP24, a defining member of the p24 subfamily to which SEL-9 belongs. p24 proteins are transmembrane protein components of COPI- and COPII-coated vesicles, and interact with coat proteins via their transmembrane/carboxy-terminal domains. Strains lacking the p24 proteins Emp24p or Erv25p have similar secretion defects: there is reduced ER to Golgi transport of a subset of secretory proteins and leakage of ER resident proteins. Two different roles for p24 proteins have been proposed. One possibility is that p24 proteins are receptors/adaptors for lumenal cargo. Alternatively, it has been proposed that the proteins encoded by EMP24 and other delta sec13 bypass suppressors are part of a quality control mechanism that prevents the premature formation of vesicles that have not properly segregated cargo from ER-resident proteins. The genetic interactions between sel-9 and lin-12 or glp-1 are consistent with an Emp24p-like role for SEL-9 in the transport of LIN-12 and GLP-1. SEL-9 and F47G9.1 may act during the sorting process to keep misfolded or mutant LIN-12 and GLP-1 proteins from transport vesicles as a general block to the progress of vesicles containing aberrant proteins. The genetic data provided in this paper are more consistent with a role for p24 proteins in a quality control mechanism as opposed to a role in cargo reception. In functional interactions, sel-9 behaves as a negative regulator. If sel-9 were principally to function as a LIN-12/GLP-1 cargo receptor, it might have been expected to behave as a positive factor: loss or reduction of a cargo receptor should reduce the amount of LIN-12 or GLP-1 at the cell surface. Instead, loss or reduction of sel-9 activity increases the amount of lin-12 or glp-1 activity, enhances the weak gain-of-function activity associated with overexpression of an essentially wild-type LIN-12 protein, and demonstrably increases the amount of a mutant GLP-1 protein at the cell surface. The results presented here are therefore more consistent with a major role for p24 proteins in quality control as opposed to cargo reception (Wen, 1999 and references).

All of the mutations that were affected by reducing sel-9 activity alter the extracellular domain of LIN-12 or GLP-1. These mutations may lead to general structural defects in the extracellular domain, since the mutations affect different subregions and have different effects (some elevate and some reduce activity). SEL-9(+) may directly or indirectly recognize the abnormal extracellular domains of the mutant LIN-12 or GLP-1 proteins and block their transport, thus effectively functioning to negatively regulate the amount of LIN-12/GLP-1 in the plasma membrane. In sel-9 mutants, however, abnormal LIN-12/GLP-1 proteins may instead be transported to the cell surface, where they may be able to function. This inference is supported by an examination of the cell biology underlying these genetic interactions. When sel-9 activity is normal, the mutant GLP-1(q415) protein appears to be retained within the cell, and the hermaphrodites display a glp-1 mutant phenotype. In contrast, when sel-9 is mutant, the GLP-1(q415) mutant protein is found in the plasma membrane, and the hermaphrodites display a wild-type phenotype. It is postulated that the effect of sel-9 on mutant LIN-12 or GLP-1 reflects a role for SEL-9(+) in the transport of LIN-12(+) and GLP-1(+). SEL-9(+) may inhibit the transport of aberrant LIN-12(+) and GLP-1(+) proteins, which may occur at some frequency due to misfolding or misprocessing. The finding that sel-9 mutations enhance the weak gain-of-function defect associated with overexpression of a tagged LIN-12 protein with a wild-type extracellular domain is consistent with this postulated role (Wen, 1999 and references).

One issue that deserves comment is the lack of a severe phenotype associated with reduced sel-9 activity. In yeast, delta emp24 causes only a moderate reduction of secretion of a select group of proteins and does not cause a marked visible phenotype. This lack of a visible phenotype might be attributable to functional redundancy among the multiple p24 proteins in yeast; however, particularly if p24 proteins depend on each other for stability, then perhaps elimination of all p24 protein activity might not result in a deleterious phenotype. In C. elegans, there also appear to be multiple p24 proteins. The sel-9 alleles isolated so far appear to reduce sel-9 activity, but the sel-9 null phenotype is not known with certainty: none of the existing mutations cause early stop codons or deletions of the coding region. Like Emp24p, SEL-9 may be involved in the transport of a select group of proteins, including LIN-12 and GLP-1. If sel-9 activity were essential for all secretory protein transport, one might reasonably have expected to see some evidence for a phenotype caused by RNAi. The definitive answer to the question of the phenotype caused by a lack of p24 proteins will be most readily addressed in yeast, where it will be feasible to construct strains lacking multiple genes for p24 proteins (Wen, 1999 and references).

The characterization of sel-9 emphasizes a link between the secretory apparatus and cell signaling during development. The characterization of other developmental genes is providing other linkages between secretion and cell signaling processes. For example, the establishment of dorsoventral polarity occurs during oogenesis and involves a signal from the oocyte to the follicle cells and a second signal from follicle cells back to the oocyte. The gene windbeutel, which is required for proper dorsoventral polarity, acts in the follicle cells and encodes an ER protein that has been proposed to chaperone a secreted signal produced in the follicle cells (Konsolaki, 1998). Whether the linkages that have been found between the secretory apparatus and cell signaling processes reflect constitutive secretory functions or serve as points of regulation will be an issue for future study (Wen, 1999 and references).

Notch signaling in C. elegans

Much of the patterning of early C. elegans embryos involves a series of Notch interactions that occur in rapid succession and have distinct outcomes; however, none of the targets for these interactions have been identified. The REF-1 family of bHLH transcription factors is a major target of Notch signaling in all these interactions and most examples of Notch-mediated transcriptional repression can be attributed to REF-1 activities. The REF-1 family is expressed and has similar functions in both Notch-dependent and Notch-independent pathways, and this dual mode of deployment is used repeatedly to pattern the embryo. REF-1 proteins are unusual in that they contain two different bHLH domains and lack the distinguishing characteristics of Hairy/Enhancer of Split (HES) bHLH proteins that are Notch targets in other systems. These results show that the highly divergent REF-1 proteins are nonetheless HES-like bHLH effectors of Notch signaling (Neves, 2005).

Most of the 39 bHLH genes in C. elegans can be grouped within Drosophila and vertebrate families of bHLH genes. However, there are six related orphan genes that are unique to nematodes; these are referred to as the ref-1 family after the first described member, ref-1. The ref-1 genes encode unusual bHLH proteins that each contain two distinct bHLH domains, a configuration thus far described only for a rice protein. The basic regions of the REF-1 proteins have moderate similarity to the basic regions of E(spl) or HERP proteins. Otherwise, the proteins are highly divergent and they lack the Orange domain, the conserved proline/glycine in the basic domain, and the terminal WRPW sequence. Only ref-1 has been studied genetically, and it was found to be required for cell fusion events during larval development that are not known to involve Notch signaling. This report provides evidence that the ref-1 family is a major target of Notch signaling in nematodes and that these genes function in all six embryonic Notch interactions examined. REF-1 and at least one additional family member appear to utilize the corepressor UNC-37/Groucho, thus linking UNC-37 with Notch-mediated repression in C. elegans. These results provide insight into the network of Notch signaling events in the embryo and suggest that the ref-1 and E(spl) genes may be highly diverged relatives of the same ancestral bHLH target of Notch signaling (Neves, 2005).

During organogenesis of the C. elegans digestive system, epithelial cells within a cyst-like primordium develop diverse shapes through largely unknown mechanisms. This study analyzed two adjacent, dorsal epithelial cells, called pm8 and vpi1, that remodel their shapes and apical junctions to become donut-shaped, or toroidal, single-cell tubes. pm8 and vpi1 delaminate from the dorsal cyst epithelium and migrate ventrally, across the midline of the cyst, on a transient tract of laminin. pm8 appears to encircle the midline by wrapping around finger-like projections from neighboring cells. Finally, pm8 and vpi1 self-fuse to become toroids by expressing AFF-1 and EFF-1, two fusogens that are each sufficient to promote crossfusion between other cell types. Notch signaling in pm8 induces AFF-1 expression, while simultaneously repressing EFF-1 expression; vpi1 expresses EFF-1 independent of Notch. Thus, the adjacent pm8 and vpi1 cells express different fusogens, allowing them to self-fuse into separate, single-cell tubes while avoiding crossfusion (Rasmussen, 2008).

Fertility depends on germline stem cell proliferation, meiosis and gametogenesis, yet how these key transitions are coordinated is unclear. In C. elegans, this study shows that GLP-1/Notch signaling functions in the germline to modulate oocyte growth when sperm are available for fertilization and the major sperm protein (MSP) hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large when MSP is present and G{alpha}s-adenylate cyclase signaling in the gonadal sheath cells is active. By contrast, gain-of-function glp-1 mutations lead to the production of small oocytes. Surprisingly, proper oocyte growth depends on distal tip cell signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling also affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1 reduction-of-function mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Furthermore, abnormal oocyte growth in glp-1 mutants, but not the inappropriate differentiation of germline stem cells, requires the function of the cell death pathway. The data support a model in which GLP-1 function in MSP-dependent oocyte growth is separable from its role in the proliferation versus meiotic entry decision. Thus, two major germline signaling centers, distal GLP-1 activation and proximal MSP signaling, coordinate several spatially and temporally distinct processes by which germline stem cells differentiate into functional oocytes (Nadarajan, 2009).

Other invertebrate Notch homologs

Early neurogenesis in the spider is characterized by a stereotyped pattern of sequential recruitment of neural cells from the neuroectoderm, comparable with neuroblast formation in Drosophila. However, in contrast to Drosophila, where single cells delaminate from the neuroectoderm, groups of cells adopt the neural fate and invaginate into the spider embryo. This raises the question of whether Delta/Notch signaling is involved in this process, since this system normally leads to a singling out of individual cells through lateral inhibition. Homologs of Delta and Notch have been cloned from the spider Cupiennius salei and their expression and function have been studied. The genes are indeed expressed during the formation of neural cells in the ventral neuroectoderm. Loss of function of either gene leads to an upregulation of the proneural genes and an altered morphology of the neuroectoderm that is comparable with Delta and Notch mutant phenotypes in Drosophila. Thus, although Delta/Notch signaling appears to be used in the same way as in Drosophila, the lateral inhibition process produces clusters of invaginating cells, rather than single cells. Intriguingly, neuroectodermal cells that are not invaginating seem to become neural cells at a later stage, while the epidermal cells are derived from lateral regions that overgrow the neuroectoderm. In this respect, the neuroectodermal region of the spider is more similar to the neural plate of vertebrates, than to the neuroectoderm of Drosophila (Stollewerk, 2002).

Molecular data suggest that myriapods are a basal arthropod group and may even be the sister group of chelicerates. To find morphological indications for this relationship neurogenesis has been analyzed in the myriapod Glomeris marginata (Diplopoda). Groups of neural precursors, rather than single cells as in insects, invaginate from the ventral neuroectoderm in a manner similar to that in the spider: invaginating cell groups arise sequentially and at stereotyped positions in the ventral neuroectoderm of Glomeris, and all cells of the neurogenic region seem to enter the neural pathway. As in the spider, 30-32 invaginating cell groups are arranged in a regular pattern of seven rows consisting of four to five invaginaton sites each. However, analysis of serial transverse sections reveals that up to 11 cells contribute to an individual invagination site, while in the spider only five to nine cells were counted. Furthermore, in contrast to the spider, the ventral neuroectoderm has a multi-layered structure: the apical region covered by a single invagination site seems to be larger and the spacing between the individual invagination sites is narrower than in the spider. The reason for these morphological features is that the invaginating cell groups are located closer together and because of limited space come to lie over and above each other. The invaginating cells of a group do not all occupy a basal position as in the spider, but they also form stacks of cells. Since more cells contribute to an invagination site and the cell processes of the invaginating cells are not as constricted as in the spider, the apical region occupied by an individual invagination site is larger than in the spider (Dove, 2003).

Homologs for achaete-scute, Delta and Notch have been identified in Glomeris. The genes are expressed in a pattern similar to that found in spider homologues and show more sequence similarity to the chelicerates than to the insects (Dove, 2003).

The data support the hypothesis that myriapods are closer to chelicerates than to insects. The spider and the millipede share several features that cannot be found in equivalent form in the insects: in both the spider and the millipede, about 30 groups of neural precursors invaginate from the neuroectoderm in a strikingly similar pattern. Furthermore, in contrast to the insects, there is no decision between epidermal and neural fate in the ventral neuroectoderm of both species analysed. It is concluded that the myriapod pattern of neural precursor formation is compatible with the possibility of a chelicerate-myriapod sister group relationship (Dove, 2003).

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin (phylum Echinodermata) embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. In the early sea urchin blastula embryo, LvNotch, the Notch homolog, is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells that surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm (dorsal) and presumptive endoderm (ventral), with an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. These experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. The specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains (apical LVNotch is coincident with the presumptive secondary mesenchyme cell/endoderm boundary) provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function (Sherwood, 1997).

The molecular mechanisms guiding the positioning of the ectoderm-endoderm boundary along the animal-vegetal axis of the sea urchin embryo remain largely unknown. A role is reported for the sea urchin homolog of the Notch receptor, LvNotch, in mediating the position of this boundary. Overexpression of an activated form of LvNotch throughout the embryo shifts the ectoderm-endoderm boundary more animally along the animal-vegetal axis, whereas expression of a dominant negative form shifts the border vegetally. Mosaic experiments that target activated and dominant negative forms of LvNotch into individual blastomeres of the early embryo, combined with lineage analyses, further reveal that LvNotch signaling mediates the position of this boundary by distinct mechanisms within the animal versus vegetal portions of the embryo. In the animal region of the embryo, LvNotch signaling acts cell autonomously to promote endoderm formation more animally, while in the vegetal portion, LvNotch signaling also promotes the ectoderm-endoderm boundary more animally, but through a cell non-autonomous mechanism. It is further demonstrated that vegetal LvNotch signaling controls the localization of nuclear ß-catenin at the ectoderm-endoderm boundary. Based on these results, it is proposed that LvNotch signaling promotes the position of the ectoderm-endoderm boundary more animally via two mechanisms: (1) a cell-autonomous function within the animal region of the embryo, and (2) a cell non-autonomous role in the vegetal region that regulates a signal(s) mediating ectoderm-endoderm position, possibly through the control of nuclear ß-catenin at the boundary (Sherwood, 2001).

In the sea urchin embryo, the micromeres act as a vegetal signaling center. These cells have been shown to induce endoderm; however, their role in mesoderm development has been less clear. The micromeres play an important role in the induction of secondary mesenchyme cells (SMCs), possibly by activating the Notch signaling pathway. After removing the micromeres, a significant delay was observed in the formation of all mesodermal cell types examined. In addition, there was a marked reduction in the numbers of pigment cells, blastocoelar cells and cells expressing the SMC1 antigen, a marker for prospective SMCs. The development of skeletogenic cells and muscle cells, however, was not severely affected. Transplantation of micromeres to animal cells results in the induction of SMC1-positive cells, pigment cells, blastocoelar cells and muscle cells. The numbers of these cell types are less than those found in sham transplantation control embryos, suggesting that animal cells are less responsive to the micromere-derived signal than vegetal cells. Notch signaling plays a role in the development of SMCs. The micromere-derived signal is necessary for the downregulation of the Notch protein, which is correlated with its activation, in prospective SMCs. It is proposed that the micromeres induce adjacent cells to form SMCs, possibly by presenting a ligand for the Notch receptor (Sweet, 1999).

The current form of a provisional DNA sequence-based regulatory gene network is presented that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see End-mes: Gene Network Update for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the ß-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin, and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated (Davidson, 2002).

Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg2 endomesodermal domain, the blastula-stage separation of the central veg2 mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg2 endodermal domain, the stabilization of specification state within these domains, and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model that treats regulatory events within the relevant embryonic nuclei at particular stages (Davidson, 2002).

During the seventh to ninth cleavage interval, a signal is transmitted from the micromeres to the adjacent surrounding cells, i.e., now the inner ring of veg2 lineage blastomeres. The result is the specification of these cells as mesodermal precursors. The signaling ligand is Delta, which activates the Notch (N) receptor. In response, the N receptor is activated specifically in the progenitors of the future veg2 mesoderm (i.e., the mesoderm formed from progeny of the 8 sixth-cleavage veg2 cells). This event is specifically required for veg2 mesodermal specification (Davidson, 2002).

This work suggests that regulatory genes carrying out several different classes of function are likely to be required for endomesoderm specification. These include genes required for micromere functions, genes required for endomesodermal specification that are dependent for activation on the Tcf system, mesodermal genes that are activated downstream of the N system, regulatory genes required for endoderm or for mesoderm cell type specification, and also batteries of downstream genes that encode skeletogenic, mesodermal, and endodermal differentiation products (Davidson, 2002).

Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. This study provides a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examines the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. Endomesoderm segregation was seen to first occur at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. These experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate (Croce, 2010).

The primary structure of HrNotch is described, an ascidian (phylum Urochordata) homolog of the Drosophila neurogenic protein Notch. HrNotch transcripts encode a protein of 2352 amino acids and share the principal features of the Notch gene family: extracellular epidermal growth factor (EGF)-like repeats, three Notch/Lin-12 repeats and six intracellular ankyrin repeats. Yet ascidian Notch contains only 33 EGF repeats in the putative extramembrane domain and specifically lacks the three EGF-like repeats. In situ hybridization shows that maternal HrNotch mRNA is distributed uniformly in the cytoplasm of the unfertilized egg. During cleavage, maternal HrNotch transcripts are ubiquitous in the ectoderm cells of the animal hemisphere, which contain less yolk granules. During gastrulation, maternal transcripts persist in most ectoderm lineage cells. Zygotic expression of HrNotch seems to start at the neural plate stage in both a-line cells (descendants of anterior-animal blastomeres) of the dorsal neuroectoderm and b-line cells (descendants of the posterior-animal blastomeres) that comprise the neural fold. Following this stage, transcripts are most evident in the descendants of these cells, that is, the brain lineage cells, precursors of a larval adhesive organ, and the dorsal part of the nerve cord (roof plate). Brain lineage cells include the precursors of sensory pigment cells that are known to comprise an equivalence group in ascidian embryos. During tail elongation, transcripts disappear. Predominant expression of HrNotch in epidermal and neural cells is a common feature of chordate Notch genes. The timing of HrNotch expression in sensory pigment cell precursors suggests involvement in the determinative events in the sensory pigment cell equivalence group (Hori, 1997).

A key issue for understanding the early development of the chordate body plan is how the endoderm induces notochord formation. In the ascidian Ciona, nuclear accumulation of ß-catenin is the first step in the process of endoderm specification. Nuclear accumulation of ß-catenin directly activates the gene (Cs-FoxD) for a winged helix/forkhead transcription factor and this gene is expressed transiently at the 16- and 32-cell stages in endodermal cells. The function of Cs-FoxD, however, is not associated with differentiation of the endoderm itself but is essential for notochord differentiation or induction. In addition, it is likely that the inductive signal that appears to act downstream of Cs-FoxD does not act over a long range. It has been suggested that FGF or Notch signal transduction pathway mediates ascidian notochord induction. Previous work suggests that Cs-FGF4/6/9 is partially involved in the notochord induction. The present experimental results suggest that the expression and function of Cs-FGF4/6/9 and Cs-FoxD are not interdependent, and that the Notch pathway is involved in B-line notochord induction (B-line cells represent a secondary notochord lineage) downstream of Cs-FoxD (Imai, 2002).

At fourth cleavage of sea urchin embryos four micromeres at the vegetal pole separate from four macromeres just above them in an unequal cleavage. The micromeres have the capacity to induce a second axis if transplanted to the animal pole and the absence of micromeres at the vegetal pole results in the failure of macromere progeny to specify secondary mesenchyme cells (SMCs). This suggests that micromeres have the capacity to induce SMCs. Micromeres require nuclear beta-catenin to exhibit SMC induction activity. Transplantation studies show that much of the vegetal hemisphere is competent to receive the induction signal. The micromeres induce SMCs, most likely through direct contact with macromere progeny, or at most a cell diameter away. The induction is quantitative in that more SMCs are induced by four micromeres than by one. Temporal studies show that the induction signal is passed from the micromeres to macromere progeny between the eighth and tenth cleavage. If micromeres are removed from hosts at the fourth cleavage, SMC induction in hosts is rescued if they later receive transplanted micromeres between the eighth and tenth cleavage. After the tenth cleavage, addition of induction-competent micromeres to micromereless embryos fails to specify SMCs. For macromere progeny to be competent to receive the micromere induction signal, beta-catenin must enter macromere nuclei. The macromere progeny receive the micromere induction signal through the Notch receptor. Signaling-competent micromeres fail to induce SMCs if macromeres express dominant-negative Notch. Expression of an activated Notch construct in macromeres rescues SMC specification in the absence of induction-competent micromeres. These data are consistent with a model whereby beta-catenin enters the nuclei of micromeres and, as a consequence, the micromeres produce an inductive ligand. Between the eighth and tenth cleavage micromeres induce SMCs through Notch. In order to be receptive to the micromere inductive signal, the macromeres first must transport beta-catenin to their nuclei, and as one consequence the Notch pathway becomes competent to receive the micromere induction signal, and to transduce that signal. Since Notch is maternally expressed in macromeres, additional components must be downstream of nuclear beta-catenin in macromeres for these cells to receive and transduce the micromere induction signal (McClay, 2000).

Notch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more, with overlapping patterns of embryonic expression. The entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, has been cloned and the exon/intron organization has been obtained from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-12 repeats, a transmembrane region, and 6 ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic germ layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, in the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek’s kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several Wnt genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and Wnt pathways and brachyury interact. Although the Notch-signaling pathway has not been shown to be a direct target of Brachyury, in amphioxus, expression of Brachyury in the future blastoporal lip, before both Wnt1 and Notch are turned on in the same cells, suggests that Brachyury may act upstream of Notch either directly or via signaling through the wingless pathway (Holland, 2001).

In butterflies there is a class of 'intervein' wing patterns that have lines of symmetry halfway between wing veins. These patterns occur in a range of shapes, including eyespots, ellipses, and midlines, and were proposed to have evolved through developmental shifts along a midline-to-eyespot continuum. Notch (N) upregulation, followed by activation of the transcription factor Distal-less (Dll), is an early event in the development of eyespot and intervein midline patterns across multiple species of butterflies. A relationship between eyespot phenotype and N and Dll expression is demonstrated in a loss-of-eyespot mutant in which N and Dll expression is reduced at missing eyespot sites. A phylogenetic comparison of expression time series from eight moth and butterfly species suggests that intervein N and Dll patterns are a derived characteristic of the butterfly lineage. Furthermore, prior to eyespot determination in eyespot-bearing butterflies, N and Dll are transiently expressed in a pattern that resembles ancestral intervein midline patterns. In this study N upregulation is established as the earliest known event in eyespot determination, gene expression associated with intervein midline color patterns is demostrated, and molecular evidence is provided that wing patterns evolved through addition to and truncation of a conserved midline-to-eyespot pattern formation sequence (Reed, 2004).

Butterfly eyespots provide a prime example of how a novel character system may arise through the evolutionary recruitment of developmental genes and then diversify under the influence of natural selection. During development, eyespot pigment patterns are induced by a long-range signal that originates from a group of focal cells at the center of the eyespot. In late last-instar wing discs, several molecules normally associated with axis specification are expressed in focal cells, and it is proposed that these molecules are involved with activating the focal signal. Of these focal molecules, the transcription factor Dll is of particular interest because the geneencoding it is genetically linked to eyespot size. While gene expressi on studies have provided insight into eyespot development, little is known about how gene expression may be associated with the evolution and development of noneyespot patterns (Reed, 2004).

Intervein pattern elements, those with centers of symmetry halfway between wing veins, occur in a range of shapes, including eyespots, tapered ellipses, and midlines, with gradients of intermediate shapes occurring both within and between species. Based on these adult phenotypes, it has been proposed that midline patterns are developmental precursors of circular eyespot patterns and that the observed gradient of intervein pattern morphologies can be explained by evolutionary changes in the timing of a common underlying developmental process. The observation that Dll expression passes through an intervein midline stage before forming an eyespot focus increases interest in this idea; however, there has been little comparative work done to test the molecular basis of this model across species. In this study the expression of Dll and the receptor molecule N in a variety of butterflies and moths were compared in order to explore the relationship between prepattern regulation and the evolution of wing patterns (Reed, 2004).

It was hypothesized that the N signaling pathway may be an upstream component of the focal determination process because ectopic expression of activated N in Drosophila melanogaster imaginal discs is sufficient to cause expression of Dll. N is a membrane bound receptor that plays several roles during Drosophila wing development. Its functions that are best understood in this context include defining the dorsoventral boundary and defining intervein tissue via a lateral inhibition interaction with its ligand Delta. In pupal butterfly wings, there is evidence that N-mediated lateral inhibition may be involved with organizing wing scales. During a lateral inhibition process, N expression tends to increase over time as a result of a local positive-feedback mechanism (Reed, 2004).

To test for an association between N and Dll expression and eyespots, the localization of N and Dll was examined in late last-instar wing imaginal discs of three species of eyespot-bearing nymphalid butterflies: Vanessa cardui, Junonia (Precis) coenia, and Bicyclus anynana. In all three of these species there is a perfect correlation between presence of forewing and hindwing eyespots and late last-instar N and Dll focal expression. To further characterize the relationship between N and Dll expression and eyespot phenotype, the effects of the B. anynana eyespot mutant missing were ascertained on levels of N and Dll. missing greatly reduces or eliminates two specific eyespots from the hindwing. In missing mutants, focal N and Dll expression is observed at lower levels than at corresponding positions in wild-type wings. N and Dll accumulation must therefore be regulated directly or indirectly by the missing locus, and localized inductions of N and Dll may serve as markers for the process of eyespot focus determination at a point downstream of missing activity (Reed, 2004).

To determine the spatiotemporal relationship between N and Dll in focal determination, a time series of N/Dll double stains was produced from V. cardui, J. coenia, and B. anynana. Focal N upregulation was found to precede Dll activation with a lag time of approximately 1.5 stages (equivalent to 12 to 24 hr, depending on species, temperature, and individual variation). In all three eyespot-bearing species, stages of gene expression were observed where N was upregulated in discrete focal patterns, while Dll was upregulated only in intervein midlines. The spatiotemporal relationship between N and Dll may be outlined in the four following primary phases. (1) Margin and intervein expression. N expression occurs at moderate levels across the wing disc, except for in the presumptive veins where N expression is relatively low. Early during this phase, Dll expression occurs only along the wing margin but progressively moves proximally after the upregulation of N along the intervein midline. (2) Midline prepatterning. Between the developing wing veins, N is upregulated in an intervein midline pattern along with an accompanying midline of Dll expression. In most species Dll expression in the midline tends to be more discretely focused than N. (3) Focal determination. N expression is increased in foci, which is subsequently mirrored by Dll. (4) Focal maturation. N and Dll express strongly in foci while fading sequentially from the intervein midline. During the focal maturation phase, expression of genes in the hedgehog pathway have been observed in foci of J. coenia and B. anynana (Reed, 2004).

Eyespot patterns have been gained and/or lost multiple times throughout butterfly evolution, and eyespots are even seen in some moths. While a rigorous phylogenetic treatment is required to infer the specific pattern of eyespot evolution throughout the Lepidoptera, some questions many nevertheless be answered about eyespot evolution by using selected exemplar taxa. Specifically, it was of interest to determining if a secondary loss of eyespots in a lineage is associated with a change in the N/Dll prepatterning process. To address this two species were examined from the nymphalid subfamily Heliconiinae: Agraulis vanillae and Heliconius melpomene. These species belong to a monophyletic subtribe called the Heliconiiti, in which there are no obvious eyespot-bearing species, although intervein midline patterns are common throughout the group. Eyespots are found in non-Heliconiiti heliconiines, as well as throughout the rest of the Nymphalidae, suggesting that the Heliconiiti represent a secondary loss of eyespots (Reed, 2004).

In A. vanillae the margin and intervein expression and midline prepattern phases appear similar to those in other butterflies. In this species, however, midline definition occurs relatively slowly, and development only reaches the midline prepattern phase by pupation. Furthermore, intervein midline gene expression was not observed to fade as it does during focal maturation in eyespot-bearing species. The midline expression patterns correspond with orange midline pigment patterns on both the hindwing and forewing (Reed, 2004).

In last-instar H. melpomene, N and Dll are expressed in an intervein midline pattern similar to A. vanillae, although they are extended proximally. There is an association between gene expression and pigment pattern in H. melpomene, where a recessive gene from the Ecuadorian race plesseni reveals a melanic midline pattern in the forewing that matches N and Dll expression. It is notable that even though expressivity of the intervein midline pigment pattern varies throughout the genus Heliconius, the N/Dll expression pattern appears to be similar between species both bearing and lacking these patterns (including H. cydno, H. erato, and H. hecale). These observations suggest that N and Dll expression is, in itself, not sufficient for midline pigment patterns in Heliconius (Reed, 2004).

In order to gain insight into the origin of the N/Dll prepatterning process, the expression patterns of these proteins were determined in the outgroup pierid butterfly Pieris rapae and two 'higher' (ditrysian) moths: the sphingid Manduca sexta, and the gelichiid Pectinophora gossypiella (Reed, 2004).

A time series of N/Dll stains in P. rapae resembles the time series from A. vanillae in that N and Dll form persisting intervein midline patterns. Interestingly, however, P. rapae does not display a midline pigment pattern in the adult. Midline pigment patterns are found in many pierid species, suggesting that as with Heliconius, N/Dll midline expression may be associated with, but is not sufficient for, pigment midlines in adults. No gene expression was observed associated with the black chevrons on the P. rapae forewing (Reed, 2004).

In the moths M. sexta and P. gossypiella, early N and Dll expression resembles initial margin and intervein expression in butterflies. In late stage M. sexta wing discs, Dll forms vaguely defined proximal extensions along the wing veins themselves. In P. gossypiella no expression of Dll was observed in intervein tissue. Given the species sampling in this study, it is most parsimonius to infer that the N/Dll intervein midline originated sometime after the divergence of the sphingid lineage and before the divergence of the pierid lineage. Further sampling of gene expression patterns from basal butterfly families and moth outgroups would help clarify the point of origin of the midline prepattern. It should be noted that published expression patterns for the monarch Danaus plexippus and the B. anynana Cyclops mutant do not show Dll midline expression; however, gene expression time series have not been described from either of these species, so it remains unknown if midlines may be expressed earlier or later than the sampled time points (Reed, 2004).

Gene expression data was mapped onto a phylogeny of the Lepidoptera used in this study and several conclusions were drawn regarding the evolution of intervein and focal prepatterning in butterfly wings. (1) The temporal order of gene expression states is conserved in all the taxa examined. (2) As outlined above, the formation of a discrete intervein midline appears to be a synapomorphy of the butterfly lineage. What, then, can be concluded about the origin of focal gene expression? Given the species sampling, there are two equally parsimonious hypotheses for the evolution of focal expression patterns: (1) there were two independent origins of foci in the lineages leading to the Satyrinae and Nymphalinae, respectively, or (2) there was a gain of foci in the lineage leading to the Nymphalidae and a loss of foci in the lineage leading to the Heliconiiti. Although a greater species sampling is required to rigorously distinguish between these possibilities, at this point the latter model is favored because of (1) the rarity or absence of eyespots (i.e., concentric circular or oval pigment patterns consistent with a focal induction model) among the basal butterfly families Pieridae, Papilionidae, and Hesperiidae, and (2) the occurrence of putative inductive eyespots in basal heliconiine genera such as Vindula and possibly Cethosia. It would be of great interest to determine the expression patterns of N and Dll in eyespot-bearing lepidopteran lineages not closely related to the Nymphalidae, such as the papilionid genus Parnassius or the eyespot-bearing saturniid and sphingid moths. These lineages potentially represent origins of inductive eyespot patterns evolutionarily independent from the nymphalid clade, and studying them could provide insight into the developmental basis of parallel pattern evolution (Reed, 2004).

The gene expression patterns reported provide useful markers for the wing pattern-formation process; however, it remains unknown what the developmental significance of the observed prepatterning sequence is. It is striking that in the eyespot-bearing butterflies examined, midline gene expression occurs prior to focal determination and that midlines always terminate proximally at the eyespot foci. These observations suggest a noncoincidental relationship between formation of midlines and foci; however, with the current data it cannot be determined if the midline/focus relationship is causal or if these prepatterns are both downstream of an as-of-yet unknown coordinate system (Reed, 2004).

The data presented in this study establish N upregulation as the earliest known event in the development of butterfly eyespots. Furthermore, finding that eyespots and midlines share a similar prepatterning process supports earlier modelsthat these intervein pattern elements are developmentally related. The observation in eyespot-bearing species that N and Dll pass through a transient, and apparently ancestral, phase of midline expression prior to focal determination raises the possibility that this developmental sequence represents a kind of evolutionary heterochrony at the level of molecular pattern formation. In sum, the data illustrate how a discrete morphological character may evolve through temporal changes in a conserved molecular pattern-formation process (Reed, 2004).

In the development of most arthropods, the caudal region of the elongating germ band (the growth zone) sequentially produces new segments. Previous work with the spider Cupiennius salei suggested involvement of Delta-Notch signaling in segmentation. This study reports that, in the spider Achaearanea tepidariorum, the same signaling pathway exerts a different function in the presumptive caudal region before initiation of segmentation. In the developing spider embryo, the growth zone becomes morphologically apparent as a caudal lobe around the closed blastopore. Preceding caudal lobe formation, transcripts of a Delta homolog, At-Delta, are expressed in evenly spaced cells in a small area covering the closing blastopore and then in a progressively wider area of the germ disc epithelium. Cells with high At-Delta expression are likely to be prospective mesoderm cells, which later express a twist homolog, At-twist, and individually internalize. Cells remaining at the surface begin to express a caudal homolog, At-caudal, to differentiate as caudal ectoderm. Knockdown of At-Delta by parental RNA interference results in overproduction of At-twist-expressing mesoderm cells at the expense of At-caudal-expressing ectoderm cells. This condition gives rise to a disorganized caudal region that fails to pattern the opisthosoma. In addition, knockdown of Notch and Suppressor of Hairless homologs produces similar phenotypes. It is suggested that, in the spider, progressive activation of Delta-Notch signaling from around the blastopore leads to stochastic cell fate decisions between mesoderm and caudal ectoderm through a process of lateral inhibition to set up a functional caudal lobe (Oda, 2007).

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, the functions of the Delta (Gb'Delta) and Notch (Gb'Notch) genes were analyzed in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. Gb'Delta and Gb'Notch were expressed in developing legs, and RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. The results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, Gb'Delta was found to be expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries (Mito, 2011).

Table of contents

Notch continued: Biological Overview | Regulation | Protein Interactions | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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