File Name: notch signaling cell fate control and signal integration in development .zip
- An Overview of Notch Signaling in Adult Tissue Renewal and Maintenance
- Notch signaling pathway
- Glioma cell fate decisions mediated by Dll1-Jag1-Fringe in Notch1 signaling pathway
- Notch signaling: cell fate control and signal integration in development.
An Overview of Notch Signaling in Adult Tissue Renewal and Maintenance
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation.
We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. The Notch signaling pathway is present from nematodes to humans and carries out fundamental roles in a wide variety of developmental processes Lai, In Drosophila, the intercellular communication mediated by this pathway is performed by the Notch receptor N and its ligands Delta Dl and Serrate Ser ligands.
Binding of the ligands to Notch induces its proteolytic cleavage within the juxtamembrane domain, leading to the release of the intracellular domain N ICD.
N ICD translocates to the nucleus where it binds to the Suppressor of Hairless Su H transcription factor activating the expression of target genes Bray and Furriols, The best-characterized functions of this pathway in flies are related to neurogenesis lateral inhibition , dorso-ventral boundary formation during wing imaginal disc development, longitudinal vein specification and cell fate decisions after asymmetrical cell divisions Artavanis-Tsakonas, et al.
In all these processes Notch is subject to posttranslational modifications of the extracellular domain that regulate its function. Although 23 EGF Notch domains could be potentially glycosylated by these enzymes, only the EGF repeats are specific sites for ligand binding Rebay et al. Genetic evidence indicates that Fringe is not necessary for all aspects of Notch signaling, and so far dorso-ventral boundary formation in the wing and the eye primordium are the main events dependent on Fringe activity in Drosophila Irvine and Wieschaus, ; Panin et al.
In vivo and cell-based assays uncovered additional functions of OFUT1 related to Notch, mainly the regulation of its intracellular trafficking, endocytosis and degradation Sasamura et al. Hence, in the OFUT1 loss-of- condition, Notch is retained in the endoplasmic reticulum, and the resulting phenotypes are reminiscent of those caused by Notch null alleles Okajima and Irvine, ; Okajima et al.
In contrast, OFUT1 over-expression leads to Notch degradation, probably through the endocytic-lysosome pathway, which finally produces similar phenotypic outcomes Sasamura et al.
The effects observed in the OFUT1 gain and loss-of-function conditions are not dependent on altered O-fucosylation. Consequently, the expression of OFUT1 in a Gmd mutant background, therefore in cells depleted of GDP-fucose, is enough for Notch to reach the plasma membrane, and the only phenotypes observed are those related to Fringe function Okajima et al.
Together, these observations suggest that Notch does not need to be O-fucosylated when it is involved in Fringe-independent processes. Although Notch glycosylations have been extensively explored regarding their roles in regulating Notch-ligand interactions Shao et al. First, so far the focus of O-fucosylation has been centered on Notch EGF repeats, even though they are also present in other proteins i.
Second, an additional O-fucosyltransferase enzyme OFUT2 has been predicted in the Drosophila genome that could also function in these glycosylation processes CG; Roos et al. This behavior has been tentatively explained by the proposal that nucleotide-sugars i. GDP-fucose move from cell to cell through gap junctions. However, this has not been proven yet. Altogether these observations prompted us to reevaluate the role of fucose glycosylation using genetic approaches.
Our results indicate that correct GMD levels specifically modulate and are essential for the stability of wild type Notch, as well as for Abruptex mutant proteins. In both cases this function is absolutely dependent on OFUT1 activity. Drosophila melanogaster strains and phenotypic analysis. In a mutagenesis screening designed to identify genes affecting vein differentiation, we isolated one P-GS insertion Toba et al.
Medium and large Gmd clones or mosaic nota were generated in the following genetic backgrounds using the Minute technique: hsFLP1. Pictures were taken in an Axiophot microscope with a Spot digital camera and processed using Adobe Photoshop. Drosophila immunohistochemistry and in situ hybridization.
Third instar imaginal discs were dissected, fixed and stained as described in de Celis In situ hybridization of imaginal discs was carried out as described in de Celis To understand the role of fucose modifications in Drosophila development and in Notch signaling in particular, we analyzed the consequences of modifying the levels of GMD, the first enzyme in the de novo biosynthesis of GDP-fucose Roos et al.
We reasoned that changing locally the levels of GMD should result in variations in the levels of GDP-fucose in mutant cells. Accordingly, this might uncover the requirements of GDP-fucose during development, as well as in Notch synthesis and signaling, irrespective of the O-fucosyltransferase enzymes involved in the transfer of this sugar OFUT1 or OFUT2 to their potential substrates.
Over-expression of GMD results in phenotypes similar to Notch loss-of-function. Gmd CG is located in the left arm of the second chromosome in the cytological position 25B5. In situ hybridization analysis revealed that Gmd is expressed ubiquitously in the wing disc throughout larval development Fig. We identified one of these insertions, the EP line Molnar et al.
Thus, over-expression of Gmd in this combination resulted in a reduction of the eye size and in ommatidial disorganization Fig. As expected, the co-expression of an interference RNA directed against Gmd Gmdi cancelled the phenotypes generated by Gmd over-expression in this territory Fig.
The expression of a dominant-negative form of Notch, consisting of its trans-membrane and extracellular domains, with these same Gal4 drivers produced very similar adult phenotypes Fig. These alterations were efficiently rescued by the co-expression of a weak activated form of Notch Fig. Although we have not monitored the levels of GDP-fucose in these combinations, these results suggest that correct Notch signaling requires appropriate amounts of GMD and, possibly, of GDP-fucose.
To further confirm that GMD over-expression interferes with Notch signaling, we monitored the expression of Enhancer of split-mb and wingless, two Notch target genes, in the wing disc of these experimental animals.
In addition, GMD over-expression induces ectopic wingless expression in cells dispersed throughout the wing blade Fig. It is likely that this ectopic Wg signal is the consequence of the cell death induced by the high level of GMD expression data not shown, Ryoo et al, ; Perez-Garijo et al, The elimination of Gmd function in homozygous null mutant larvae Gmd H78 or Gmd 1 inhibits the induction of wg expression along the dorso-ventral boundary suggesting a direct requirement of this gene for Notch activity in this region Fig.
We also confirmed that Gmd mutant cells display a considerable non-autonomous behavior, as the defects in wg expression are only detected when a large fraction of the wing disc is occupied by Gmd mutant cells arrowhead, Fig.
To further investigate the requirement of Gmd during development and in Notch signaling we generated transgenic flies that express RNA interference against Gmd Gmdi under the control of UAS sequences. These experiments allow us to study the consequences of changing the temporal and spatial accumulation of GMD in the developing wing disc.
Notch signaling is also involved in the formation of the sensory organs in the thorax, affecting both the number of sensory organ precursors SOP and its differentiation Schweisguth, This effect is also detected in the adult, where additional sensory organs appeared in several positions of the thorax arrows in Fig.
In these conditions the wing margin develops severe notching and the bristles show shaft duplications Fig. To confirm the impairment in Notch signaling detected upon Gmd knockdown we co-expressed Gmdi with an activated form of Notch N intra in the wing blade territory using the Gal4 driver. Interestingly, the reduction of GMD levels impaired both Fringe-dependent tarsal joint and wing margin formation and Fringe-independent processes sensory organ formation and vein thickness.
In summary, the reduction in GMD levels specifically modifies Notch signaling and it does so interfering with every aspect of Notch function in a similar way as reductions of the fucosyltransferase OFUT1 Okajima and Irvine ; Sasamura et al. The fact that the phenotypes caused by knocking down Gmd expression were suppressed by an activated version of Notch suggests that GDP-fucose is required either for receptor activation or for its synthesis.
Previously, it has been shown that decreasing the levels of OFUT1 influences Notch trafficking and subcellular localization, causing its retention and accumulation in the endoplasmic reticulum Okajima et al. Surprisingly, we found that reducing GMD expression strongly diminished the levels of Notch at the apical membrane in this territory Fig. This reduction was not the consequence of a failure of the antibody to detect unmodified Notch proteins, because it was also observed using antibodies that recognize both the extracellular Fig.
Furthermore, the reduction in Notch protein was independent of the transcription of the Notch gene, since the reduction of Gmd does not alter the expression of a Notch reporter consisting in a P-lacZ insertion in Notch that reveals Notch transcription Supplementary Fig. Finally, the effects produced by the reduction of GMD in Notch levels seem to be specific to this receptor, because other proteins with EGF motifs susceptible to be O-fucosylated, such as Delta or Crumbs, are detected at normal levels and with a normal distribution in discs with reduced Gmd levels Supplementary Fig.
S1 and data not shown. In these sections, we failed to detect Notch accumulation in any subcellular compartment, and only a very weak signal was detected at the apical cell membranes Fig. The failure to detect Notch was not the result of a general disturbance in the apico-basal polarity of the cell, as other proteins, like Scribble, Delta, Discs large and Crumbs were normally localized at apical regions of Gmdi -expressing cells Fig.
Therefore, we suggest that the normal processing of Notch during its transport to the apical cell membrane fails when the level of GMD is lowered, or alternatively, that Notch endocytosis and its subsequent degradation are augmented under these conditions.
However, in these discs apico-basal polarity was severely disturbed and the epithelial integrity is lost as evidenced by the extrusion of dying cells. These observations suggest a more general effect of GMD excess in protein trafficking that could be caused by lowering the amount of GDP-L-mannose, the substrate of GMD, which plays key roles in the secretory pathway. Thus, OFUT1 reduction leads to Notch accumulation at the ER, blocking its processing and apical localization and, in consequence, preventing its potential activation Okajima et al.
Likewise, loss of GMD function also reduces Notch signaling and causes a vein thickening phenotype, although in this case the defect is caused by a decrease in Notch protein levels arrows Fig. Co-expression of the two RNAi constructs, iOfut1 and Gmdi, also resulted in adult wing phenotypes compatible with reductions of Notch signaling Fig.
These observations indicate that an unbalance of GDP-fucose in the cell facilitates Notch degradation, likely through an endocytic-lysosome pathway assisted by OFUT1, further supporting the notion that OFUT1, acting from the extracellular milieu, directs the endocytosis and degradation of Notch Sasamura et al. Abruptex alleles are caused by point mutations within EGF repeats 24 to 29 of the Notch extracellular domain and modify Notch activation in a ligand-dependent manner Kelley et al.
In addition, in vitro binding assays showed that the Abruptex region specifically interacts with the ligand binding region of Notch, and this has been proposed to serve as a barrier for the ligand-induced activation of Notch Pei and Baker, Ax viable hemizygotes show higher levels of Notch signaling, specifically affecting wing growth and vein development Fig.
Moreover, the veins appeared thickened, suggesting that reductions in GMD function produce a Notch deficiency even in the presence of hyper-activable Notch proteins Fig. Notch is a glycosylated transmembrane receptor with central roles during development Artavanis-Tsakonas et al. The Notch protein suffers several glycosylation steps that affect Notch-ligand interactions Xu et al.
Classical sugar modifications occur in the ER and Golgi apparatus by a battery of glycosyltransferases Helenius and Aebi, So far, two of these enzymes have been involved in Notch synthesis. A recent report has uncovered a quality control mechanism that affects Notch synthesis, which unexpectedly, relies on a non-enzymatic activity of OFUT1 Okajima et al.
Besides this function, Sasamura et al. In addition, our results show that it is the balance of GDP-fucose and OFUT1 what modulates Notch signaling by regulating the amount of functional Notch protein at the cell surface. The phenotypes caused by changes in GMD activity are related to loss of Notch signaling.
Gmd null alleles and OFUT1 mutant forms have been used to assess the specific dependency of Notch signaling on O-fucosylation Okajima et al.
This has lead to the conclusion that O-fucosylation is exclusively required for Fringe-dependent processes Okajima et al. We found that over-expression of Gmd leads to a reduction of Notch activity. This effect is reverted by a weak constitutively active form of Notch, indicating that GMD over-expression interferes upstream of Notch activation. Clonal analysis has shown a non cell-autonomous requirement of Gmd, preventing the analysis of GMD function in mosaics.
In this background there is a strong reduction of Notch protein levels that was not detected in mosaic Gmd tissues data not shown. Our GMD knockdown conditions reduce Notch protein levels without noticeable changes in Notch gene expression, Delta levels or apico-basal polarity. Thus, appropriate levels of GDP-fucose are specifically essential for Notch stability, but not for the accumulation or function of other proteins that contain EGF domains.
But how is Notch destabilized under our experimental conditions? Sasamura et al. They showed that OFUT1 acting from the extracellular space enhances Notch endocytosis promoting its degradation Sasamura et al. Okajima et al.
Notch signaling pathway
Cell-cell interactions mediated by the Notch signaling pathway occur throughout C. These interactions have major roles in specifying cell fates and in tissue morphogenesis. The network of Notch interactions is linked in part through the Notch-regulated expression of components of the pathway, allowing one interaction to pattern subsequent ones. The Notch signal transduction pathway is highly conserved in animal embryogenesis. The Notch signaling pathway plays a central role in patterning metazoan development, and is used in remarkably diverse cell fate decisions for general review, see Artavanis-Tsakonas et al. Cell interactions mediated by these receptors have been documented throughout embryonic and postembryonic development of C. For example, Notch signaling controls mesoderm induction during embryonic development, and controls germ cell mitosis during postembryonic development see below and Austin and Kimble,
The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism that is required for embryonic development, cell fate specification, and stem cell maintenance. Discovered and studied initially in Drosophila melanogaster , the Notch pathway is conserved and functionally active throughout the animal kingdom. In this paper, we summarize the biochemical mechanisms of Notch signaling and describe its role in regulating one particular developmental pathway, oogenesis in Drosophila. Utilized by the simplest metazoans through mammals, Notch signaling is an evolutionarily conserved signaling pathway that is required for embryonic development, cell fate specification, and stem cell maintenance [ 1 — 5 ]. Notch signaling selects among preexisting cellular potentials to specify different cell fates and activate different programs through either promoting or suppressing differentiation, proliferation, survival, and apoptosis [ 6 , 7 ]. In humans, mutations in this pathway cause inherited genetic diseases such as Alagille syndrome, spondylocostal dysostosis, Hadju-Cheney syndrome, Tetralogy of Fallot, familial aortic valve disease, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.
Glioma cell fate decisions mediated by Dll1-Jag1-Fringe in Notch1 signaling pathway
King , The unicellular ancestry of animal development , Dev Cell , vol. Thakur, H. Ushijima, A.
Notch Signaling in Embryology and Cancer pp Cite as. In humans and other species, Notch-signaling is of critical importance for carcinogenesis in several organs, including the skin. Interestingly, Notch-signaling appears to exert opposite roles in skin carcinogenesis as compared to carcinogenesis in other tissues. While the Notch1 receptor Notch1 acts as a proto-oncogene in most tissues, it has been shown that Notch1 deletion in epidermal keratinocytes causes skin carcinogenesis.
Metrics details. The Notch family of proteins plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. It has been shown that Notch1 and its ligands, Dll1 and Jag1, are overexpressed in many glioma cell lines and primary human gliomas.
Notch signaling: cell fate control and signal integration in development.
Wing margin formation in Drosophila requires the Notch receptor and, in the dorsal compartment, one of its ligands, Serrate. We provide evidence that Delta, the other known ligand for Notch, is also essential for this process. Moreover, ectopic Delta expression induces wingless, vestigial, and cut and causes adult wing tissue outgrowth in the dorsal compartment. The effect is mediated by Notch, because loss of Notch activity suppresses Delta-induced ectopic wing outgrowth. Whereas ectopic expression of Notch or the truncated activated Notch induces cut in both dorsal and ventral compartments, ectopic Delta expression induces cut only in the dorsal compartment and ectopic Serrate induces cut only in the ventral compartment.
Louis, Missouri, USA The Notch pathway is a critical mediator of short-range cell-cell communication that is reiteratively used to regulate a diverse array of cellular processes during embryonic development and the renewal and maintenance of adult tissues. This releases the Notch intracellular domain, which translocates to the nucleus to activate transcription. The Notch pathway is one of several conserved signaling pathways that regulate cell proliferation, cell fate decisions and induction of differentiation during embryonic and postnatal development [ 1 - 4 ].
The Notch signaling pathway is a highly conserved cell signaling system present in most animals. It is a hetero-oligomer composed of a large extracellular portion, which associates in a calcium -dependent, non-covalent interaction with a smaller piece of the notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region. Notch signaling promotes proliferative signaling during neurogenesis , and its activity is inhibited by Numb to promote neural differentiation. It plays a major role in the regulation of embryonic development. In , John S. Dexter noticed the appearance of a notch in the wings of the fruit fly Drosophila melanogaster. The alleles of the gene were identified in by American evolutionary biologist Thomas Hunt Morgan.
Notch signaling defines an evolutionarily ancient cell interaction mechanism, which plays a fundamental role in metazoan development. Signals exchanged.
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. The Notch signaling pathway is present from nematodes to humans and carries out fundamental roles in a wide variety of developmental processes Lai, In Drosophila, the intercellular communication mediated by this pathway is performed by the Notch receptor N and its ligands Delta Dl and Serrate Ser ligands.
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