Compensatory flux changes within an endocytic trafficking network maintain thermal robustness of Notch signaling.

Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals.

Reversible regulation of stem cell niche size associated with dietary control of Notch signalling.

BACKGROUND: Stem cells can respond to environmental and physiological inputs to adaptively remodel tissues. Little is known about whether stem cell niches are similarly responsive. The Drosophila ovary germline stem cell (GSC) niche is a well-studied model, which is comprised of cap cells that provide anchorage and maintenance signals for GSCs to maintain oogenesis. Previous studies have shown a strong link between diet and the regulation of oogenesis, making this a useful model system in which to investigate dietary regulation of the niche and its associated stem cells. RESULTS: We show that the Drosophila ovary GSC cap cell niche is a dynamic structure, which can contract and expand in fluctuating dietary conditions. Cap cells are lost when adult flies are shifted to nutrient poor diet and are restored after returning flies to nutrient-rich medium. Notch signalling in cap and escort cells is similarly reduced and restored by dietary shifts to nutrient poor and rich media. In old flies decreased Notch signalling is associated with decreased robustness of the niche to dietary changes. We demonstrated using a Notch temperature sensitive allele that removal and restoration of Notch signalling also leads to a reduction and re-expansion of the niche. Changes in niche size were not associated with apoptosis or cell division. We identified two distinct roles for Notch in the adult germarium. Notch can act in cap cells to prevent their loss while activation of Notch in the flanking escort cells results in expansion of the niche. CONCLUSIONS: We provide evidence that dietary changes alone are sufficient to alter Notch signalling and reversibly change niche size in the adult in wild type flies. We show Notch acts in different cells to maintain and re-expand the niche and propose a model in which cell fate transitions between cap cells and flanking somatic cells accounts for niche dynamics. These findings reveal an unexpected reversible plasticity of the GSC niche whose responses provide an integrated read out of the physiological status of the fly that is modulated by diet and age.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

Roles of Drosophila deltex in Notch receptor endocytic trafficking and activation.

Cell signaling mediated by the Notch receptor (N) regulates many cell-fate decisions and is partly controlled by the endocytic trafficking of N. Drosophila deltex (dx) encodes an evolutionarily conserved regulator of N signaling, an E3-ubiquitin ligase, which ubiquitinates N's intracellular domain. Although Dx was shown to function in N endocytosis in studies of dx over-expression, the roles of endogenous Dx have remained hidden. Here, we investigated N endocytosis in a dx-null Drosophila mutant and found that endogenous Dx is required for at least two steps of N trafficking: the incorporation of N into endocytic vesicles from the plasma membrane and the transport of N from early endosomes to lysosomes. In the absence of Dx functions, N was stabilized in unknown endocytic compartments, where it was probably insulated from transport to lysosomes. We also found that canonical N signaling and Dx-mediated N signaling are activated in two different endocytic compartments, before N is incorporated into multivesicular body (MVB) interluminal vesicles and after N is transported from MVBs, respectively. The endocytic compartment in which Dx-mediated N signaling is activated appears to coincide with the activity of endogenous Dx in N trafficking. These findings extend our understanding of how N's trafficking and activation are correlated.

Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins.

The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation.

The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. We found that the Drosophila ZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation both in vivo and in cell culture assays. In vivo we confirmed the physiological relevance and direction of the Pyd/Dx interaction by showing that the expanded ovary stem cell niche phenotypes of pyd mutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells we found that Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, our results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.

A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition.

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Endocytic regulation of Notch activation and down-regulation (review).

Notch receptor signalling plays a central role in development and its misfunction has been linked to a number of diseases. In the cannonical Notch signalling pathway, ligand binding to Notch activates a series of proteolytic cleavages that release the Notch intracellular domain for trafficking to the nucleus, where it activates the transcription factor, Suppressor of Hairless (Su(H)). A number of recent papers have demonstrated the importance of endocytic trafficking of Notch and its ligands for both the activation and the down-regulation of the Notch receptor. These reports highlight uncertainty regarding the whereabouts in the cell where Notch activation occurs, and the form of the ligand that can induce signalling. In this review we speculate that, decision points between alternative trafficking pathways represent important regulatory nodes that may allow Notch signalling levels to be modulated by other developmental signals, providing context-dependency to Notch activation. We also review data that suggest that key proteolytic events, associated with Notch activation, may occur within the endocytic pathway or require prior endocytosis and recycling of Notch and its ligands to the cell surface. Sorting within the endocytic pathway, regulated by several different ubiquitin ligase proteins, may be involved in ensuring whether ligand and receptor are competent to signal. Furthermore, the utilisation of an alternative mechanism of Notch signalling, independent of Su(H), may depend on driving endocytic Notch into a specific compartment, in response to the activity of the ring finger domain protein, Deltex.

Down-regulation of Notch target gene expression by Suppressor of deltex.

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.