Table of contents

Fish and Frog Notch homologs

An evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus has been conducted to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes results from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences has shown there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the C. elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal to the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function (Kortschak, 2001).

To facilitate analysis of vertebrate Notch gene function, cDNA fragments of three novel Notch genes were isolated from zebrafish (Danio rerio): Notch1b, Notch5 and Notch6. Notch1b is a second zebrafish Notch1 gene. From analysis of the Notch1b sequence, it is argued that the various vertebrate Notch gene subfamilies encode receptors with different signaling specificities. Notch5 and Notch6 represent novel vertebrate Notch gene subfamilies. Remarkably, Notch1b lacks expression in presomitic mesoderm; Notch5 is expressed in a metameric pattern within the presomitic mesoderm, while Notch6 expression is excluded from the nervous system. The expression patterns of these genes suggest important roles in gastrulation, somitogenesis, tail bud extension, myogenesis, heart development and neurogenesis (Westin, 1997).

her4 encodes a zebrafish bHLH protein of the Hairy-E(spl) family. The gene is transcribed in a complex pattern in the developing nervous system and in the hypoblast. During early neurogenesis, her4 expression domains include the regions of the neural plate from which primary neurons arise, suggesting that the gene is involved in directing their development. Indeed, misexpression of specific her4 variants leads to a reduction in the number of primary neurons formed. The amino-terminal region of her4, including the basic domain, and the region between the putative helix IV and the carboxy-terminal tetrapeptide WRPW are essential for this effect, since her4 variants lacking either of these regions are non-functional. However, the carboxy-terminal WRPW itself is dispensable. The interrelationships between deltaD, deltaA, notch1, her4 and neurogenin1 have been examined by means of RNA injections. her4 is involved in a regulatory feedback loop that modulates the activity of the proneural gene neurogenin, and as a consequence, the activity of deltaA and deltaD as well. Activation of notch1 leads to strong activation of her4, to suppression of neurogenin transcription and, ultimately, to a reduction in the number of primary neurons. These results suggest that her4 acts as a target of Notch-mediated signals that regulate primary neurogenesis (Takke, 1999a).

The main conclusion of this study is that her4 encodes a zebrafish homolog of the Drosophila E(Spl)-C proteins, which acts as a target of Notch to suppress primary neurogenesis. This conclusion is based on structural and functional considerations. HER4 shows considerable sequence identity in the bHLH domain, the region that binds DNA and is involved in target recognition. Furthermore it also exhibits the other characteristics of the Hairy-E(Spl) protein family, such as the carboxy-terminal tetrapeptide WRPW. her4 is expressed in the neural plate region in which the primary neurons are formed. In Drosophila, the E(Spl) proteins suppress the activity of the proneural genes. Misexpression of her4 suppresses ngn1 and the development of primary neurons. However, there are two major differences between HER4 and the E(Spl) proteins. In Drosophila the latter require Groucho for their function in neurogenesis whereas the data presented here do not provide evidence for interactions between HER4 and zebrafish Groucho2. Injections of groucho2 RNA do not have any apparent effect on the development of islet-1 cells, and co-injection of her4 and groucho2 RNA does not enhance the neural suppression mediated by her4 alone. In the association between Groucho and E(Spl)-C proteins in Drosophila, the WRPW domain plays an essential role in segmentation and neurogenesis; thus removal or mutation of this domain renders the protein non-functional. In contrast, the WRPW of HER4 is apparently not needed to suppress primary neurogenesis in zebrafish. It is important to note in this context that there is at least one precedent for the present result: it has been reported that the WRPW of Hairy is not required to suppress Scute in the sex determination pathway in Drosophila. The other important difference between the Drosophila E(Spl)-C proteins and HER4 concerns their amino-terminal regions, including the basic domain, which has been shown to bind a specific DNA sequence called the N-box. In misexpression experiments using the Gal4-UAS system in Drosophila, deletion of either the basic domain or both the basic and the HLH domains does not seriously affect the ability of E(Spl) proteins to suppress neural development. However, the amino-terminal domain of HER4 seems to be essential for activity, as its deletion resulted in non-functional proteins. Therefore, DNA binding of the HER4 protein might play a more important role during neurogenesis in the zebrafish than in the case of the E(SPL) proteins in Drosophila (Takke, 1999a and references).

In both Xenopus and zebrafish, differentiation of primary neurons has been shown to be perturbed following misexpression of Notch and Delta homologs. The results of these experiments, as well as those presented here, strongly support the idea that primary neurons are selected from equivalence groups within the neural plate, and that this process is mediated by lateral inhibition. her4 is one of the target genes of the Notch signaling cascade in the zebrafish. Transcription of her4 is activated by the constitutively active Notch1 variant encoded by nic, and misexpression of her4 leads to a reduction in the number of islet-1 positive cells. Similar observations have been made concerning ESR-1, a Xenopus homolog of the E(SPL) proteins. However, the effects of misexpression of her4 on the islet-1 cells are less severe than those caused by nic. It is suggested that, in analogy to Drosophila, this difference is due to the concomitant activation by Notch of additional bHLH genes involved in regulation of primary neurogenesis. The reduction in the number of islet-1 positive cells following misexpression of her4 might be due either to direct inhibition of proneural gene transcription, or to an effect on the target genes of the proneural proteins. Transcription of zebrafish ngn1 is modulated by misexpression of Delta variants that are assumed to activate or repress Notch. Inhibition of ngn1 transcription correlates with transcriptional activation of her4, thus suggesting a causal relationship between the two events (Takke, 1999a and references).

The existence of a feedback loop between Notch and Delta in zebrafish that is organized similar to one in Drosophila is strongly supported by the data. The activation of Notch has two detectable effects: (1) it leads to an increase in transcription of her4, resulting in repression of proneural activity and primary neural fate and (2) a feedback loop. Whether proneural activity is directly repressed by transcriptional suppression, and/or indirectly by posttranslational modifications, has to be analyzed in further detail. In any case, the second effect, a feedback loop, has been established; it leads to a reduction in the concentration of delta RNA in the cells in which Notch has been activated. This in turn reduces the intensity of Notch activation in the neighboring cells, and allows them to differentiate as primary neurons (Takke, 1999a).

Within the neural plate, the number of islet-1-positive cells is reduced following ngn1 RNA injection, while cells of this type develop ectopically outside the neural plate of the same animals. A similar behavior has been observed with XASH-3 in Xenopus. However, misexpression of Xenopus neurogenin leads in Xenopus to ectopic development of primary neurons in the neural plate. These apparently paradoxical effects of ngn1 can be understood when one considers that it activates transcription of deltaA, deltaD and her4 (i.e. the effectors responsible for lateral inhibition) only within the limits of the neural plate. This may explain the reduction in the number of islet-1 cells observed within the neural plate following injection of ngn1 RNA. ngn1 cannot activate deltaA, deltaD and her4; consequently, lateral inhibition outside the neural plate is not activated, thus permitting ectopic development of islet-1-positive cells in the non-neural ectoderm. The observation that the co-injection of her4 suppresses ngn1-mediated development of ectopic islet-1 cells supports this hypothesis. In a Drosophila embryo that overexpresses proneural genes the situation is remarkably similar to that of ngn1 misexpression in the zebrafish. Gal4-mediated overexpression of lethal of scute leads to ectopic development of neurons only within the amnioserosa; this effect is suppressed by the concomitant activation of Notch in the amnioserosa. However, selection of neural and epidermal progenitor cells takes place normally in the neuroectoderm -- in spite of the presence of large amounts of proneural gene products. The proneural gene products seem to activate lateral inhibition strongly, since a reduction in the complement of copies of the Notch+ gene from 2 to 1, leads to strong neurogenic phenotypes (Takke, 1999a and references).

Based on these findings, it is suggested that, like the E(Spl)-C genes in Drosophila, her4 constitutes the last link in the Notch signaling cascade in zebrafish neurogenesis. There are several other her genes in the zebrafish. Their complex patterns of expression during embryogenesis suggest their involvement in other processes besides differentiation of islet-1-positive cells. Since other genes encoding bHLH proteins of the same family are known to be expressed in other regions of the body, e.g. her1 and her5 in the presomitic mesoderm and in the midbrain anlage, respectively, they may represent different endpoints of this signaling pathway and regulate cell fate decisions other than those that result in the appearance of the primary neurons (Takke, 1999a and references).

During vertebrate embryonic development, the paraxial mesoderm becomes subdivided into metameric units known as somites. In the zebrafish embryo, genes encoding homologs of the proteins of the Drosophila Notch signaling pathway are expressed in the presomitic mesoderm and expression is maintained in a segmental pattern during somitogenesis. This expression pattern suggests a role for these genes during somite development. Various zebrafish genes of this group were misexpressed by injecting mRNA into early embryos. RNA encoding a constitutively active form of NOTCH1a (notch1a-intra) and a truncated variant of deltaD [deltaD(Pst)], as well as transcripts of deltaC and deltaD, the hairy-E(spl) homologs her1 and her4, and groucho2 were tested for their effects on somite formation, myogenesis and on the pattern of transcription of putative downstream genes. In embryos injected with any of these RNAs, with the exception of groucho2 RNA, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment correctly. Activation of Notch results in ectopic activation of her1 and her4. This misregulation of the expression of her genes might be causally related to the observed mesodermal defects, since her1 and her4 mRNA injections led to effects similar to those seen with notch1a-intra. deltaC and deltaD seem to function after subdivision of the presomitic mesoderm, since the her gene transcription pattern in the presomitic mesoderm remains essentially normal after misexpression of delta genes. Whereas Notch signaling alone apparently does not affect myogenesis, zebrafish groucho2 is involved in differentiation of mesodermal derivatives (Takke, 1999b).

Results regarding the effect of misexpressing wild-type deltaD have suggested a function for this gene in somite development. Misexpression of a dominant negative variant of deltaD, wild-type deltaC, or an activated form of NOTCH1a, or misexpression of the hairy-E(spl) homolog her1 or her4, leads in all cases to considerable disruption of somitogenesis. However, whereas the mesodermal effects of perturbing deltaC and deltaD activity are similar, those observed following either Notch activation or coinjection of her1 and her4 appear to have a different basis. In the former case, patterning defects are evident, but the presomitic mesoderm seems to be subdivided into somitomeres, as incomplete somite borders are visible; in the latter case, somites apparently do not form, because no somite borders can be seen. Since both her1 and her4 are ectopically activated by notch1a-intra within the presomitic mesoderm and misexpression of both her genes causes defects similar to those seen with notch1a-intra mRNA, it is proposed that both her genes are targets of Notch during somitogenesis. It follows then that, during normal development, Notch-mediated activation of her genes may be causally related to the initial subdivision of the paraxial mesoderm into somitomeres. In contrast, in the case of deltaC or deltaD misexpression the defects seem to be independent of the activity of the two her genes because it fails to perturb the transcription pattern of her1 and her4. The same applies to misexpression of a truncated variant of DELTAD. This result is surprising and leads to two important corollaries: (1) it suggests that neither DELTAC nor DELTAD acts as a ligand to trigger NOTCH-dependent activation of her genes, and (2), it suggests that the DELTAC/D-dependent somitic defects do not depend directly on the activity of her genes. Accordingly, the delta function in somitogenesis that appears to operate downstream of the component of Notch function was assayed; namely, her gene activity. Double in situ hybridizations with her1 or her4 and MyoD probes following misexpression of delta variants suggest that the latter act within the somites once the presomitic mesoderm has been subdivided into somitomeres. By analogy to the situation in Drosophila, where the Notch regulatory network is required for maintenance of the epithelial state in several different instances, as well as for the formation of borders in the wing disc, it is proposed that DELTAC and DELTAD act during the definition and/or maintenance of somitic borders in zebrafish embryos (Takke, 1999b).

At least three important questions remain open in this scenario: (1) whereas the proposed function of Delta in controlling boundary development may rely on a mechanism similar to that operating in the wing margin of Drosophila, the mechanism by which Notch contributes to subdivide the presomitic mesoderm is unclear. (2) The ligand that activates the NOTCH1a receptor (and, consequently, the her genes) during the subdivision of the presomitic mesoderm is unknown. Although there are no less than four delta genes in the zebrafish, only deltaC and deltaD are expressed in the mesoderm and apparently neither one is capable of activating her genes under these experimental conditions. Therefore, there is no obvious candidate for this function. (3) The receptor required for the DELTAC/D-mediated function during later stages of somite development is also unknown (Takke, 1999b).

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. Loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, ectopic activation of Notch signaling leads to trepression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. These results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries (Lawson, 2001).

A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. It has been shown that Notch signaling inhibits the projection neuron fate. This study shows that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. Crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. These results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/ Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. These results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS (Quillien, 2011).

Bilateral symmetric tissues must interpret axial references to maintain their global architecture during growth or repair. The regeneration of hair cells in the zebrafish lateral line, for example, forms a vertical midline that bisects the neuromast epithelium into perfect mirror-symmetric plane-polarized halves. Each half contains hair cells of identical planar orientation but opposite to that of the confronting half. The establishment of bilateral symmetry in this organ is poorly understood. This study shows that hair-cell regeneration is strongly directional along an axis perpendicular to that of epithelial planar polarity. Compartmentalized Notch signaling was demonstrated in neuromasts, and directional regeneration was shown to depend on the development of hair-cell progenitors in polar compartments that have low Notch activity. High-resolution live cell tracking reveals a novel process of planar cell inversions whereby sibling hair cells invert positions immediately after progenitor cytokinesis, demonstrating that oriented progenitor divisions are dispensable for bilateral symmetry. Notwithstanding the invariably directional regeneration, the planar polarization of the epithelium eventually propagates symmetrically because mature hair cells move away from the midline towards the periphery of the neuromast. It is concluded that a strongly anisotropic regeneration process that relies on the dynamic stabilization of progenitor identity in permissive polar compartments sustains bilateral symmetry in the lateral line (Wibowo, 2011).

X-MyT1 is a C2HC-type zinc finger protein that is involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, it is suggested that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation (Bellefroid, 1996).

The X-Notch-1 receptor, and its putative ligand, X-Delta-1, are thought to mediate an inhibitory cell-cell interaction, called lateral inhibition, that limits the number of primary neurons that form in Xenopus embryos. The expression of Xenopus ESR-1, a gene related to Drosophila Enhancer of split, appears to be induced by Notch signaling during this process. To determine how the activation of X-Notch-1 induces ESR-1 expression and regulates primary neurogenesis, the Xenopus homolog of Drosophila Suppressor of Hairless ) was isolated. X-Su(H) binds to the RAM21 region of X-Notch-1, just N-terminal of a ankyrin repeat domain and homologous to a similar site in Drosophila Notch. X-Su(H), when modified by the C-terminal covalent addition of ankyrin repeats, induces ESR-1 expression, perhaps directly. Using a DNA binding mutant of X-Su(H), it is shown that X-Su(H) activity is required for induction of ESR-1. Expression of the DNA binding mutant in embryos leads to a neurogenic phenotype (a higher density of primary neurons) as well as increased expression of both X-Delta-1 and XNGNR1, a proneural gene expressed during primary neurogenesis. These results suggest that activation of X-Su(H) is a key step in the Notch signaling pathway during primary neurogenesis in Xenopus embryos (Wettstein, 1997).

Xotch, the Xenopus homolog of Notch, is expressed in the eye in undifferentiated precursor cells of the ciliary marginal zone and late embryonic central retina. It is not expressed in stem cells or in differentiated neurons and glia. The final Xotch-positive precursor cells in the central retina mostly differentiate as Muller glia, suggesting that this is the last available fate of cells in the frog retina. Transfection of an activated form of Xotch into isolated retinal cells causes them to retain a neuroepithelial morphology, indicating that the continued activation of Xotch inhibits cell differentiation (Dorsky, 1995).

Expression of the Xenopus Neurogenin protein, Xenopus Neurogenin-related-1 (X-NGNR-1) and XNeuroD show a similar spatial overlap with temporal displacement. X-NGNR-1 induces ectopic neurogenesis and ectopic expression of X NeuroD mRNA, but not vice-versa. X-ngnr-1 expression precedes expression of X-Delta-1, and X-NGNR-1 can serve to activate expression of X-Delta-1. Expression of the intracellular domain of XNotch-1 inhibits both the expression and function of X-ngnr-1. Thus endogenous X-ngnr-1 expression becomes restricted to subsets of cells by lateral inhibition mediated by X-Delta-l and X-Notch. The properties of X-NGNR-1 are thus analogous to those of the Drosophila proneural genes, suggesting that it functions as a vertebrate neuronal determination factor (Ma, 1996).

The role of Notch signalling was analyzed during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway it was found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. It is proposed that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favors floor plate development at the expense of the notochord, preferentially before mid gastrula. Evidence is presented that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. Notch signalling is shown to require Presenilin to modulate this switch (López, 2003).

The Alagille Syndrome (AGS) is a heritable disorder affecting the liver and other organs. Causative dominant mutations in human Jagged 1 have been identified in most AGS patients. Related organ defects occur in mice that carry jagged 1 and notch 2 mutations. Multiple jagged and notch genes are expressed in the developing zebrafish liver. Compound jagged and notch gene knockdowns alter zebrafish biliary, kidney, pancreatic, cardiac and craniofacial development in a manner compatible with an AGS phenocopy. These data confirm an evolutionarily conserved role for Notch signaling in vertebrate liver development, and support the zebrafish as a model system for diseases of the human biliary system (Lorent, 2004).

The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. Monoclonal antibodies have been generated to identify the absorptive and secretory cells in the epithelium, and these antibodies were used to examine the role that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an after eight (aei) mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium (Crosnier, 2005).

Somitogenesis is the process by which the segmented precursors of the skeletal muscle and vertebral column are generated during vertebrate embryogenesis. While somitogenesis appears to be a serially homologous, reiterative process, there are differences between the genetic control of early/anterior and late/posterior somitogenesis. Point mutations can cause segmentation defects in either the anterior, middle, or posterior somites in the zebrafish. Mutations in zebrafish integrinα5 disrupt anterior somite formation, giving a phenotype complementary to the posterior defects seen in the notch pathway mutants after eight/deltaD and deadly seven/notch1a. Double mutants between the notch pathway and integrinα5 display somite defects along the entire body axis, with a complete loss of the mesenchymal-to-epithelial transition and Fibronectin matrix assembly in the posterior. The data suggest that notch- and integrinα5-dependent cell polarization and Fibronectin matrix assembly occur concomitantly and interdependently during border morphogenesis (Julich, 2005).

The early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Evidence has been found that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone (Lopez, 2005 ).

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps, although the mechanism by which this modulation occurs remains unknown. Notch signaling also delays the onset of LMC in whole embryos, as it does in animal caps. To better understand this effect and the mechanism of LMC itself, an investiation was carried out to discover at which step of activin signal transduction pathway the Notch signaling acts to affect the timing of the LMC. In this system, ALK4 (activin type I receptor) maintains the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 can bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. It is concluded that LMC is induced because C-terminal-phosphorylated smad2 loses its ability to bind to smad4, and consequently can not accumulate in the nucleus. Notch signal activation restores the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC (Abe, 2005).

Subdividing the embryo: a role for Notch signaling during germ layer patterning in Xenopus laevis

The development of all vertebrate embryos requires the establishment of a three-dimensional coordinate system in order to pattern embryonic structures and create the complex shape of the adult organism. During the process of gastrulation, the three primary germ layers are created under the guidance of numerous signaling pathways, allowing cells to communicate during development. Cell-cell communication, mediated by receptors of the Notch family, has been shown to be involved in mediating diverse cellular behaviors during development and has been implicated in the regulation of cell fate decisions in both vertebrate and invertebrate organisms. In order to investigate a role for Notch signaling during boundary formation between the mesoderm and endoderm during gastrulation, Notch signaling was manipulated in gastrula stage Xenopus embryos and gene expression was examined in resultant tissues and organs. The findings demonstrate a much broader role for Notch signaling during germ layer determination than previously reported in a vertebrate organism. Activation of the Notch pathway, specifically in gastrula stage embryos, results in a dramatic decrease in the expression of genes necessary to create many different types of mesodermal tissues while causing a dramatic expansion of endodermal tissue markers. Conversely, temporally controlled suppression of this pathway results in a loss of endodermal cell types and an expansion of molecular markers of mesoderm. Thus, these data are consistent with and significantly extend the implications of prior observations suggesting roles for Notch signaling during germ layer formation and establish an evolutionarily conserved role for Notch signaling in mediating mesoderm-endoderm boundaries during early vertebrate development (Contakos, 2005).

The development of the vertebrate face relies on the regionalization of neural crest-derived skeletal precursors along the dorsoventral (DV) axis. This study shows that Jagged-Notch signaling ensures dorsal identity within the hyoid and mandibular components of the facial skeleton by repressing ventral fates. In a genetic screen in zebrafish, a loss-of-function mutation in jagged 1b (jag1b) was identified that results in dorsal expansion of ventral gene expression and partial transformation of the dorsal hyoid skeleton to a ventral morphology. Conversely, misexpression of human jagged 1 (JAG1) represses ventral gene expression and dorsalizes the ventral hyoid and mandibular skeletons. It was further shown that jag1b is expressed specifically in dorsal skeletal precursors, where it acts through the Notch2 receptor to activate hey1 expression. Whereas Jagged-Notch positive feedback propagates jag1b expression throughout the dorsal domain, Endothelin 1 (Edn1) inhibits jag1b and hey1 expression in the ventral domain. Strikingly, reduction of Jag1b or Notch2 function partially rescues the ventral defects of edn1 mutants, indicating that Edn1 promotes facial skeleton development in part by inhibiting Jagged-Notch signaling in ventral skeletal precursors. Together, these results indicate a novel function of Jagged-Notch signaling in ensuring dorsal identity within broad fields of facial skeletal precursors (Zuniga, 2010).

Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue

Mind bomb1 (Mib1)-mediated endocytosis of the Notch ligand DeltaD is essential for activation of Notch in a neighboring cell. Although most DeltaD is localized in cytoplasmic puncta in zebrafish neural tissue, it is on the plasma membrane in mib1 mutants because Mib1-mediated endocytosis determines the normal subcellular localization of DeltaD. Knockdown of Notch increases cell surface DeltaA and DeltaD, but not DeltaC, suggesting that, like Mib1, Notch regulates endocytosis of specific ligands. Transplant experiments show that the interaction with Notch, both in the same cell (in cis) and in neighboring cells (in trans), regulates DeltaD endocytosis. Whereas DeltaD endocytosis following interaction in trans activates Notch in a neighboring cell, endocytosis of DeltaD and Notch following an interaction in cis is likely to inhibit Notch signaling by making both unavailable at the cell surface. The transplantation experiments reveal a heterogeneous population of progenitors: in some, cis interactions are more important; in others, trans interactions are more important; and in others, both cis and trans interactions are likely to contribute to DeltaD endocytosis. It is suggested that this heterogeneity represents the process by which effective lateral inhibition leads to diversification of progenitors into cells that become specialized to deliver or receive Delta signals, where trans and cis interactions with Notch play differential roles in DeltaD endocytosis (Matsuda, 2009).

Notch destabilises maternal β-catenin and restricts dorsal-anterior development in Xenopus

The blastula chordin- and noggin-expressing centre (BCNE) is the predecessor of the Spemann-Mangold's organiser and also contains the precursors of the brain. This signalling centre comprises animal-dorsal and marginal-dorsal cells and appears as a consequence of the nuclear accumulation of β-catenin on the dorsal side. This study proposes a role for Notch that was not previously explored during early development in vertebrates. Notch initially destabilises β-catenin in a process that does not depend on its phosphorylation by GSK3. This is important to restrict the BCNE to its normal extent and to control the size of the brain (Acosta, 2011).

Studies in the epithelium of the Drosophila wing disc have identified another endocytic pathway for Notch which requires neither the classical ligands nor cleavage by γ-secretase. Although it depends on structural motifs present in the intracellular domain of Notch, this pathway does not involve CSL-mediated transcription. Immunoprecipitation assays demonstrated that Notch and Arm/β-catenin are associated in the same protein complex. Notch associates near the adherens junctions with hypophosphorylated Arm/β-catenin, which has escaped tagging by GSK3 for ubiquitin-proteasome degradation. Because this complex enters endosomal trafficking and becomes degraded, this non-canonical, non-transcriptional function of Notch would be important to buffer activated Arm/β-catenin in order to keep low levels of spontaneous Wnt activity in the system. This antagonistic interaction of Notch and Wnt was proposed as an example of how biological systems decrease their own noise. This is in agreement with the results shown in this study during early Xenopus development (Acosta, 2011).

Chicken Notch homologs

The importance of lateral inhibition mediated by Notch signaling is well demonstrated to control neurogenesis both in invertebrates and vertebrates. The chicken homolog of Drosophila numb, which suppresses Notch signaling, has been identified. NUMB (c-NUMB) protein is localized to the basal cortex of mitotic neuroepithelial cells, suggesting that c-NUMB regulates neurogenesis by the modification of Notch signaling through asymmetrical cell division. Consistent with this suggestion, it has been shown (1) that c-NUMB interferes with the nuclear translocation of activated c-NOTCH-1 through direct binding to the PEST sequence in the cytoplasmic domain of c-NOTCH-1 and (2) that c-NUMB interferes with c-NOTCH-1-mediated inhibition of neuronal differentiation (Wakamatsu, 1999).

Published studies on the distribution of Numb and Notch in asymmetrically dividing neuroepithelial cells in vertebrates have challenged the understanding of Notch function in neurogenesis. Thus, it has been reported that NUMB-IR is localized on the apical side of mitotic neuroepithelial cells in mice. Further, NOTCH-IR has been reported to be localized basally in mitotic neuroepithelial cells in the developing ferret neocortex. Since the basal daughter cells of asymmetrically dividing neuroepithelial cells appear to undergo neuronal differentiation in these vertebrate systems, it has been proposed that apical daughter cells that receive Numb remain undifferentiated. Activation of Notch signaling in the basal daughter cells has also been proposed to be responsible for causing the postmitotic, but nondifferentiated, state of migratory daughter cells. This unprecedented role for Notch in promoting a nondividing, but nondifferentiated, intermediate neuronal phenotype and the implied role of Numb in preventing neuronal differentiation by repressing Notch function in apical cells seem paradoxical and difficult to reconcile with the Drosophila literature. In contrast, the basal localization of Numb that has been observed in the current study suggests a more parsimonious model in which Numb inhibits Notch signaling and thereby permits neuronal differentiation in the basal daughter cells. This model is consistent both with the perceived function of Notch to inhibit neuronal differentiation in vertebrate neurogenesis and with the role of Numb to suppress that inhibition in the development of the Drosophila nervous system. At present, both of these models remain to be tested further. Perhaps, future experiments using m-NUMB targeted loss-of-function mutations will be useful to elucidate the issue. At present the apparent discrepancy between the data demonstrating basal c-NUMB localization and the data reporting apical localization of m-NUMB cannot be explained (Wakamatsu, 1999).

The roles of Notch, Delta, and Serrate were studied in vertebrate epithelial appendage morphogenesis using the feather as a model. The following observations were made:

  1. C-Notch-1, C-Delta-1, and C-Serrate-1 are not expressed at the early placode stage and are therefore not involved in the determination of bud versus interbud compartments.
  2. From symmetric short buds to asymmetric long buds, C-Delta-1 and C-Serrate-1 are expressed in the posterior bud mesenchyme in a nested fashion, while C-Notch-1 is expressed as a stripe perpendicular to the anterior-posterior (A-P) axis and positioned posterior to the midpoint.
  3. Epithelial-mesenchymal recombination with rotation leads to the disappearance of protein products of these genes followed by their reappearance with new positions appearing to predict their new morphological orientation.
  4. Conditions leading to branched buds (e.g., recombination of later buds) show polarized staining patterns before branching occurs.
  5. The condition leading to symmetrical round buds (e.g., treatment with the protein kinase A agonist forskolin) suppresses expression of all three genes.
These results have led to the hypothesis that Notch, Delta, and Serrate are involved in establishing the A-P asymmetry of feather buds (C. Chen, 1997).

Chick embryonic feather buds arise in a distinct spatial and temporal pattern. Although many genes are implicated in the growth and differentiation of the feather buds, little is known about how the discrete pattern of the feather array is formed and which gene products may be involved. Possible candidates include Notch and its ligands, Delta and Serrate, as they play a role in numerous cell fate decisions in many organisms. Notch-1 and Notch-2 mRNAs are expressed in the skin in a localized pattern prior to feather bud initiation. In the early stages of feather bud development, Delta-1 and Notch-1 are localized to the forming buds while Notch-2 expression is excluded from the bud. Thus, Notch and Delta-1 are expressed at the correct time and place to be players in the formation of the feather pattern. Once the initial buds form, expression of Notch and its ligands is observed within each bud. Notch-1 and -2 and Serrate-1 and -2 are expressed throughout the growth and differentiation of the feathers, whereas Delta-1 transcripts are downregulated. When chick Delta-1 is misexpressed in either large or small patches using a replication competent retrovirus, Notch-1 and-2 are induced, accompanied by a loss of feather buds from the embryo. In large regions of Delta-1 misexpression, feathers are lost throughout the infected area. In contrast, in small regions of misexpression, Delta-1 expressing cells differentiate into feather buds more quickly than normal and inhibit their neighbors from accepting a feather fate. A dual role is proposed for Delta-1 in promoting feather bud development and in lateral inhibition. These results implicate the Notch/Delta receptor ligand pair in the formation of the feather array (Crowe, 1998).

Neurons of the vertebrate central nervous system (CNS) are generated sequentially over a prolonged period from dividing neuroepithelial progenitor cells. Some cells in the progenitor cell population continue to proliferate while others stop dividing and differentiate as neurons. The mechanism that maintains the balance between these two behaviors is not known, although previous work has implicated Delta-Notch signaling in the process. In normal development, the proliferative layer of the neuroepithelium includes both nascent neurons that transiently express Delta-1 (Dl1), and progenitor cells that do not. Using retrovirus-mediated gene misexpression in the embryonic chick retina, it has been shown that where progenitor cells are exposed to Dl1 signaling, they are prevented from embarking on neuronal differentiation. A converse effect is seen in cells expressing a dominant-negative form of Dl1, Dl1(dn), which renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1(dn), essentially all progenitors stop dividing and differentiate prematurely as neurons, which can be of diverse types. Thus, Delta-Notch signaling controls a cell's choice between remaining as a progenitor and differentiating as a neuron. It is concluded that nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. It is proposed that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues (Henrique, 1997).

Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos

Using avian embryos, mechanisms were studied underlying the three-dimensional assembly of the dorsal aorta, the first-forming embryonic vessel in amniotes. This vessel originates from two distinct cell populations, the splanchnic and somitic mesoderms. A role was unveiled for Notch signaling in the somitic contribution. Upon activation of Notch signaling, a subpopulation of cells in the posterior half of individual somites migrates ventrally toward the primary dorsal aorta of splanchnic origin. After reaching the primary aorta, these somitic cells differentiate into the definitive aortic endothelial cells. This Notch-induced ventral migration is mediated by EphrinB2 and by an attractant action of the primary aorta. Furthermore, long-term chasing of cells by transposon-mediated gene transfer reveals that the segmentally provided endothelial cells of somitic origin in the dorsal aorta ultimately populate the entire region of the vessel. This study demonstrates the molecular and cellular mechanisms underlying the formation of embryonic blood vessels from mesenchymal cells (Sato, 2008).

A novel function of DELTA-NOTCH signalling mediates the transition from proliferation to neurogenesis in neural progenitor cells

A complete account of the whole developmental process of neurogenesis involves understanding a number of complex underlying molecular processes. Among them, those that govern the crucial transition from proliferative (self-replicating) to neurogenic neural progenitor (NP) cells remain largely unknown. Due to its sequential rostro-caudal gradients of proliferation and neurogenesis, the prospective spinal cord of the chick embryo is a good experimental system to study this issue. This study reports that the NOTCH ligand DELTA-1 is expressed in scattered cycling NP cells in the prospective chick spinal cord preceding the onset of neurogenesis. These Delta-1-expressing progenitors are placed in between the proliferating caudal neural plate (stem zone) and the rostral neurogenic zone (NZ) where neurons are born. Thus, these Delta-1-expressing progenitors define a proliferation to neurogenesis transition zone (PNTZ). Gain and loss of function experiments carried by electroporation demonstrate that the expression of Delta-1 in individual progenitors of the PNTZ is necessary and sufficient to induce neuronal generation. The activation of NOTCH signalling by DELTA-1 in the adjacent progenitors inhibits neurogenesis and is required to maintain proliferation. However, rather than inducing cell cycle exit and neuronal differentiation by a typical lateral inhibition mechanism as in the NZ, DELTA-1/NOTCH signalling functions in a distinct manner in the PNTZ. Thus, the inhibition of NOTCH signalling arrests proliferation but it is not sufficient to elicit neuronal differentiation. Moreover, after the expression of Delta-1 PNTZ NP continue cycling and induce the expression of Tis21, a gene that is upregulated in neurogenic progenitors, before generating neurons. Together, these experiments unravel a novel function of DELTA-NOTCH signalling that regulates the transition from proliferation to neurogenesis in NP cells. It is hypothesized that this novel function is evolutionary conserved (Hammerle, 2007).

Notch mediates Wnt and BMP signals in the early separation of smooth muscle progenitors and blood/endothelial common progenitors

During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. This study shows that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. These findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs (Shin, 2009).

The precise function of the Notch pathway in the process of muscle and BC/EC lineage separation remains to be elucidated. The data suggest that, during chick ventral mesoderm differentiation, the Notch pathway acts together with the BMP and Wnt pathways, and that it plays a 'permissive', rather than an 'instructive', role in mediating the separation of SMCs and BC/ECs. The Notch pathway does not control the induction of but rather the balance between these two populations. Evidence is provided that the induction of these lineages is controlled by the activities of both the BMP pathway, as a general ventral mesoderm inducer, and the canonical Wnt pathway, as a strong SMC lineage inducer. Ectopic activation of the BMP pathway can induce both SMC and BC/EC lineages, with the balance of SMCs and BC/ECs being regulated by Notch activity. It is not clear whether the induction of SMCs by the BMP pathway is a direct or indirect process, or whether it requires an active Wnt pathway. In this analysis, a stronger and wider ectopic dHand induction was observed by CA-β-Catenin than by CA-ALK6 around the anterior primitive streak where BMP antagonists are highly expressed, suggesting that the induction of SMCs by the Wnt pathway does not require active BMP signaling. A recent in vitro study suggested that Notch activity promotes the degradation of Scl by facilitating its ubiquitination, and that this process requires the transcriptional regulation of Notch pathway activity through Suppressor of Hairless. Although there is no direct evidence in support of a similar phenomenon in the current system, it could in principle act as a possible mechanism for the Notch activity-mediated segregation of SMCs and BC/ECs. Furthermore, Nrarp (an ankyrin-repeat protein that is transcriptionally regulated by the Notch signaling pathway), in addition to serving as a Notch-activity readout and a feedback regulator of the Notch pathway, has also been shown to positively regulate the canonical Wnt pathway by blocking the ubiquitination and increasing the stability of Lef1 in zebrafish. This might also serve as a possible mechanism for the Notch and Wnt pathway-mediated SMC specification observed in this system (Shin, 2009).

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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