The Interactive Fly
Zygotically transcribed genes
Pattern formation takes place through a series of logical steps, reiterated many times over during the development of an organism. Viewed from a broader evolutionary perspective, across species, the same sorts of reiterative pattern formations are seen. The central dogma of pattern formation has been described by Lawrence and Struhl (1996) in an article entitled "Morphogens, Compartments and Pattern: Lessons from Drosophila." Three interlocking and overlapping steps are defined: first, positional information in the form of morphogen gradients allocate cells into nonoverlapping sets, each set founding a compartment. Second, each of these compartments acquires a genetic address, as a result of the function of active "selector" genes that specify cell fate within a compartment and also instruct cells and their descendents how to communicate with cells in neighboring compartments. The third step involves interactions between cells in adjacent compartments, initiating new morphogen gradients, which directly organize the pattern.
Taking these steps in greater detail, one finds the first step in patterning to be the definition of sets of cells in each primordium. Cells are allocated according to their positions with respect to both dorsoventral and anterior/posterior axes by morphogen gradients. Allocation of cells in the dorsoventral axis constitutes the germ layers, such as mesoderm or neurectoderm. This function is carried out by the gene dorsal and its targets. Subdivision of the anterior/posterior axis into segmental units known as parasegments is carried out by gap and pair rule genes.
In segmentation, the second step (the specification of cell fate in each compartment) is carried out by the gene engrailed and elements of the bithorax complex. engrailed defines anterior and posterior compartments both in segmentation and in limb specification.
The wing disc provides an excellent example of pattern formation in Drosophila. During the larval period, both anterior and posterior compartments are subdivided by the apterous selector gene, which is activated in dorsal and repressed in ventral cells. Selector genes do much more than specify the pattern and structures that the compartments will eventually make - they also specify, indirectly, a surface property termed cell affinity. Cells that share the same affinity can intermingle during growth, while cells in the neighboring compartment, with a different basis for affinity, also self-associate but minimize contact with cells in adjacent compartments; in this way, a well defined boundary forms between adjacent compartments. In wing compartment definition, alternative integrins function in different compartments determining mutual and exclusive affinity.
The third step in pattern formation, secretion of morphogens, functions to differentiate patterns within compartments (and thereby establish segment polarity). Initially all cells within a compartment are equipotent, but they become diversified to form pattern. Pattern formation depends on gradients of morphogens, gradients initiated along compartment boundaries. How are these gradients established? A short-range signal is induced in all the cells of the compartment in which a selector gene (engrailed) is active. For segment polarity this signal is Hedgehog. In the adjacent compartment the selector gene is inactive, ensuring that the cells are sensitive to the signal. The Hedgehog signal range is probably only a few rows of cells wide; responding cells become a linear source of a long-range morphogen that diffuses outward in all directions.
The long range signal in wing segment polarity is Decapentaplegic. Two targets of DPP are spalt and optomotor blind, both transcription factors activated by DPP. A graded distribution of DPP outside of cells organizes a graded distribution of the domains of spalt and omb, which in turn generate the patterning of elements such as bristles, arranged according to transcription factor concentration.
In the wing disc apterous functions as a selector gene that makes the dorsal surface distinct from the ventral surface. Apterous has at least two functions: first it is responsible for making the dorsal cell type distinct from the ventral, a property that may be due to its activation of gene Dorsal wing; second, it directs the expression of fringe and Serrate in the dorsal compartment and by its absence, Notch in the ventral compartment. It could be that wingless is the long-range morphogen induced by Serrate action on Notch. Fringe functions as a short range secreted signal. In embryonic segmentation, the long range signal is unknown, but may again be wingless.
Tissue organization requires the interplay between biochemical signaling and cellular force generation. The formation of straight boundaries separating cells with different fates into compartments is important for growth and patterning during tissue development. In the developing Drosophila wing disc, maintenance of the straight anteroposterior (AP) compartment boundary involves a local increase in mechanical tension at cell bonds along the boundary. The biochemical signals that regulate mechanical tension along the AP boundary, however, remain unknown. This study shows that a local difference in Hedgehog signal transduction activity between anterior and posterior cells is necessary and sufficient to increase mechanical tension along the AP boundary. This difference in Hedgehog signal transduction is also required to bias cell rearrangements during cell intercalations to keep the characteristic straight shape of the AP boundary. Moreover, severing cell bonds along the AP boundary does not reduce tension at neighboring bonds, implying that active mechanical tension is upregulated, cell bond by cell bond. Finally, differences in the expression of the homeodomain-containing protein Engrailed also contribute to the straight shape of the AP boundary, independently of Hedgehog signal transduction and without modulating cell bond tension. The data reveal a novel link between local differences in Hedgehog signal transduction and a local increase in active mechanical tension of cell bonds that biases junctional rearrangements. The large-scale shape of the AP boundary thus emerges from biochemical signals inducing patterns of active tension on cell bonds (Rudolf, 2015).
This study has analyzed the links between the determination of cell fate and the physical and mechanical mechanisms shaping the AP boundary of larval Drosophila wing discs. Previous work has shown a role for the transcription factors Engrailed and Invected and the Hedgehog signal transduction pathway in organizing the segregation of anterior and posterior cells of the wing disc. This study now shows that a difference in Hedgehog signal transduction between anterior and posterior cells significantly contributes to the straight shape of the AP boundary by autonomously and locally increasing mechanical cell bond tension that in turn biases the asymmetry of cell rearrangements during cell intercalations. Furthermore, Engrailed and Invected also contribute to maintaining the characteristic straight shape of the AP boundary by mechanisms that are independent of Hedgehog signal transduction and do not appear to modulate cell bond tension (Rudolf, 2015).
In the wild-type wing disc, anterior cells transducing the Hedgehog signal are juxtaposed to posterior cells that do not transduce the Hedgehog signal. Three cases were genereated to test whether this difference in Hedgehog signal transduction is important for the straight shape of the AP boundary, the morphological and molecular signature of cells along the AP boundary, and the local increase in cell bond tension. In case I, Hedgehog signal transduction was low (or absent) in both A and P cells. In case II, Hedgehog signal transduction was high in both A and P cells. And in case III, Hedgehog signal transduction was high in P cells, but low in A cells, reversing the normal situation. In cases I and II the AP boundary was no longer as straight as in the wild-type situation. Moreover, the increased apical cross-section area of cells along the AP boundary that is characteristic for the wild type was no longer seen. Finally, the levels of F-actin and cell bond tension were no longer increased along the AP boundary. In case III, it was found that the difference in Hedgehog signal transduction is sufficient to maintain the characteristic straight shape of the AP boundary, to induce the morphological signatures of cells along the AP boundary and to increase F-actin and mechanical tension. Taken together, these experiments establish that the difference in Hedgehog signal transduction between anterior and posterior cells plays a key role in increasing cell bond tension along the AP boundary, in maintaining the characteristic shape of the AP boundary, and in defining the molecular and morphological signatures of cells along the AP boundary. These findings account for the observation that while Hedgehog signal transduction is active within the strip of anterior cells, the increase in mechanical tension is confined to cell bonds along the AP boundary, where cells with highly different Hedgehog signal transduction activities are apposed. The small differences in Hedgehog signal transduction activity that might exist between neighboring rows of anterior cells in the vicinity of the AP boundary appear to be insufficient to increase cell bond tension. Importantly, Hedgehog signal transduction per se does not increase cell bond tension along the AP boundary. The role of Hedgehog signal transduction along the AP boundary thus differs from its roles during other morphogenetic processes in which all cells that transduce the Hedgehog signal, for example, respond by accumulation of F-actin and a change in shape. It will be interesting to elucidate the molecular mechanisms by which cells perceive a difference in Hedgehog signal transduction, and how such a difference in Hedgehog signal transduction results in increased cell bond tension (Rudolf, 2015).
F-actin and Myosin II are enriched along the AP boundary. Based on similar observations, the existence of actomyosin cables has been proposed for several compartment boundaries, including the AP boundary in the Drosophila embryonic epidermis, the DV boundary of Drosophila wing discs and the rhombomeric boundaries in zebrafish embryos. Actomyosin cables have been proposed to maintain the straight shape of compartment boundaries by acting as barriers of cell mixing between cells of the adjacent compartments. Actomyosin cables are also characteristic of additional processes, e.g. dorsal closure and germband extension in the Drosophila embryo, tracheal tube invagination and neural plate bending and elongation. During Drosophila germ band extension, it has been shown that mechanical tension is higher at cell bonds that are part of an actomyosin cable compared with isolated cell bonds, indicating that cell bond tension is influenced by higher-order cellular organization during this process. The results, based on laser ablation experiments, show that the increased cell bond tension along the AP boundary can be induced by single cells and does not depend on the integrity of the actomyosin cable. Thus, these data instead indicate that increased cell bond tension is autonomously generated cell bond by cell bond along the AP boundary. This suggests that differences in Hedgehog signal transduction activity regulate the structure and mechanical properties of cell junctions between adjacent cells and in particular upregulate an active mechanical tension, mediated by actomyosin contractility (Rudolf, 2015).
The cell cortex is a thin layer of active material that is under mechanical tension. In addition to viscous and elastic stresses, active stresses generated by actomyosin contractility are an important contribution. Adherens junctions are adhesive structures that include elements of the cell cortices of the adhering cells. Locally generated active tension, therefore, can largely determine the cell bond tension as long as cell bonds do not change length or rearrange. As a consequence, locally generated active tension also sets the cell bond tension at the actomyosin cable along the AP boundary. This view is consistent with experiments in which cell bond tension remains high even if the integrity of the actomyosin cable is lost. These mechanical properties of cell junctions along the AP boundary are thus different from those of a conventional string or cable in which elastic stresses are associated with stretching deformations. Such elastic stresses relax and largely disappear when the cable is severed. Thus, this work suggests that the mechanical properties of the actomyosin cable along the AP boundary are very different from those of a conventional cable, but fit well in the concepts of active tension studied in the cell cortex, e.g., in Caenorhabditis elegans. This active tension is a local property that can be set by local signals irrespective of the local force balances. Force balances rather determine movements and rearrangements, e.g. upon laser ablation (Rudolf, 2015).
How does a local increase in actively generated cell bond tension contribute to the straight shape of the AP boundary? Previous work showed that cell intercalations promote irregularities in the shape of compartment boundaries. The local increase in active cell bond tension enters the force balances during cell rearrangements. During cell intercalation, differences in active cell bond tension between junctions along the AP boundary and neighboring junctions are balanced by frictional forces associated with vertex movements. As a result, vertex movements are biased such that the AP boundary remains straight and cell mixing between neighboring compartments is suppressed. The observation that a local difference in Hedgehog signal transduction upregulates active cell bond tension leads to the prediction that cell rearrangements along the AP boundary should not be biased if there is no difference in Hedgehog signal transduction. This is indeed what was found in case II (Rudolf, 2015).
It has been previously suggested that the engrailed and invected selector genes play a role in maintaining the separation of anterior and posterior cells that is independent of Hedgehog signal transduction. Quantitative analysis of clone shapes in this study supports this notion. It is speculated that this Hedgehog-independent pathway contributes to the remarkably straight shape of the AP boundary in cases I and II, in which Hedgehog signal transduction activities between anterior and posterior cells have been nearly equalized. Two lines of evidence indicate that the Hedgehog-independent pathway shapes the AP boundary without modulating cell bond tension. First,several cases have been generated in which neighboring cell populations differed in the expression of Engrailed and Invected, but not in Hedgehog signal transduction activity. In none of these cases was an increase in cell bond tension detected along the interface of these two cell populations. Second, in cases in which a difference was created in Hedgehog signal transduction between two cell populations in the absence of differences in Engrailed and Invected expression, the same increase was detected in cell bond tension between these cell populations compared with the wild-type compartment boundary (Rudolf, 2015).
Previously studies have described several physical mechanisms that shape the DV boundary of wing discs. In addition to a local increase in mechanical tension along the DV boundary, evidence was provided that oriented cell division and cell elongation created by anisotropic stress contribute to the characteristic shape of the DV boundary. It is therefore conceivable that the Hedgehog-independent pathway influences the shape of the AP boundary by one or more of these mechanisms (Rudolf, 2015).
It is proposed that the AP boundary is shaped by mechano-biochemical processes that integrate signaling pathways with patterns of cell mechanical properties. In tjos model, Engrailed and Invected shape the AP boundary with the help of two different mechanisms. (1) Engrailed and Invected result in a difference in Hedgehog signal transduction between anterior and posterior cells. This difference leads to a cell-autonomous increase in F-actin and active cell bond tension along the AP boundary. The local increase in active cell bond tension then biases the asymmetry of cell rearrangements during cell intercalations and thereby contributes to maintaining the straight shape of the AP boundary. (2) Engrailed and Invected contribute independently of Hedgehog signal transduction to the straight shape of the AP boundary by an as yet unknown mechanism not involving the modulation of cell bond tension. The first mechanism uses biochemical signals to create mechanical patterns that subsequently guide junctional dynamics to organize a straight compartment boundary. It is speculated that the second mechanism also involves a mechano-chemical process, even though the nature of this process is currently unknown. The current work suggests that the large-scale shape of the AP boundary thus emerges from the collective behavior of many cells that locally exchange biochemical signals and regulate active mechanical tension (Rudolf, 2015).
Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. This study shows that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). This is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). This study further demonstrates that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking (Sanchez, 2016).
This study demonstrates that the Hh pathway modulates the magnitude of the odorant response in adult Drosophila. The results show that the Hh pathway determines the level of the odorant response because it regulates the response in both the positive and negative directions. Loss of Ptc function increases the odorant response and the risk for long sustained responses, which shows that the Hh pathway limits the response potential of the OSNs and is crucial for maintaining the response at a physiological level. In addition, it was shown that the OSNs produce Hh protein, which regulates OR localization, which is interesting because autoregulation is one of the prerequisites for an adaptive mechanism. It was further shown that Hh signaling regulates the responses of OSNs that express different ORs, which demonstrates that the regulation is independent of OSN class and suggests that Hh signaling is a general regulator of the odorant response. It has been shown previously that Hh tunes nociceptive responses in both vertebrates and Drosophila (Babcock, 2011). It is not yet understood how Hh regulates the level of nociception. However, the regulation is upstream of the nociceptive receptors, which indicates that the Hh pathway is a general regulator of receptor transport and the level of sensory signaling (Sanchez, 2016).
The results show that OSN cilia have two separate OR transport systems, the Hh-regulated Cos2 and the intraflagellar transport complex B (IFT-B) together with the kinesin II system. The results show that Cos2 is required for OR transport to or within the distal cilium domain and suggest that the IFT system regulates the inflow to the cilium compartment. The two transport systems also are required for Smo cilium localization (Kuzhandaivel, 2014). This spatially divided transport of one cargo is similar to the manner in which Kif3a and Kif17 regulate distal and proximal transport in primary cilia in vertebrates. However, Cos2 is not required for the distal location of Orco or tubulin (Kuzhandaivel, 2014), indicating that, for some cargos, the IFT system functions in parallel to Cos2 (Sanchez, 2016).
Interestingly, the vertebrate Cos2 homolog Kif7 organizes the distal compartment of vertebrate primary cilia (He, 2014). Similar to the current results, Kif7 does so without affecting the IFT system, and its localization to the cilia is dependent on Hh signaling. However, the Kif7 kinesin motor function has been questioned (He, 2014). Therefore, it will be interesting to analyze whether Kif7-mediated transport of ORs and other transmembrane proteins occurs within the primary cilium compartment and whether the ciliary transport of ORs is also regulated by Hh and Smo signaling in vertebrates. To conclude, these results place the already well-studied Hh signaling pathway in the post-developmental adult nervous system and also provide an exciting putative role for Hh as a general regulator of receptor transport to and within cilia (Sanchez, 2016).
Primary cilia are non-motile sensory cell-organelle that are essential for organismal development, differentiation, and postnatal homeostasis. Their biogenesis and function are mediated by the intraflagellar transport (IFT) system. Pathogenic variants in IFT52, a central component of the IFT-B complex is associated with short-rib thoracic dysplasia with or without polydactyly 16 (SRTD16), with major skeletal manifestations, in addition to other features, this study sought to examine the role of IFT52 in osteoblast differentiation. Using lentiviral shRNA interference Ift52 was depleted in C3H10T1/2 mouse mesenchymal stem cells. This led to the disruption of the IFT-B anterograde trafficking machinery that impaired primary ciliogenesis and blocked osteogenic differentiation. In Ift52 silenced cells, Hedgehog (Hh) pathway upregulation during osteogenesis was attenuated and despite Smoothened Agonist (SAG) based Hh activation, osteogenic differentiation was incompletely restored. Further, IFT52 activity was investigated in Drosophila, wherein the only ciliated somatic cells are the bipolar sensory neurons of the peripheral nervous system. Knockdown of IFT52 in Drosophila neuronal tissues reduced lifespan with the loss of embryonic chordotonal cilia, and produced severe locomotion, auditory and proprioceptive defects in larva and adults. Together these findings improve knowledge of the role of IFT52 in various physiological contexts and its associated human disorder (Guleria, 2022).
The cell cycle is coordinated with differentiation during animal development. This study reports a cell-cycle-independent developmental role for a master cell-cycle regulator, the anaphase-promoting complex or cyclosome (APC/C), in the regulation of cell fate through modulation of Wingless (Wg) signaling. The APC/C controls both cell-cycle progression and postmitotic processes through ubiquitin-dependent proteolysis. Through an RNAi screen in the developing Drosophila eye, this study found that partial APC/C inactivation severely inhibits retinal differentiation independently of cell-cycle defects. The differentiation inhibition coincides with hyperactivation of Wg signaling caused by the accumulation of a Wg modulator, Drosophila Nek2 (dNek2). The APC/C degrades dNek2 upon synchronous G1 arrest prior to differentiation, which allows retinal differentiation through local suppression of Wg signaling. Evidence is provided that Decapentaplegic signaling may posttranslationally regulate this APC/C function. Thus, the APC/C coordinates cell-fate determination with the cell cycle through the modulation of developmental signaling pathways (Martins, 2017).
Cells heterozygously mutant for a ribosomal protein gene, called Minute/+ mutants, are eliminated from epithelium by cell competition when surrounded by wild-type cells. Whereas several factors that regulate Minute cell competition have been identified, the mechanisms how winner/loser status is determined and thereby triggers cell competition are still elusive. To address this, two assay systems were establised for Minute cell competition, namely (i) the CORE (competitive elimination of RpS3-RNAi-expressing cells) system in which RpS3-RNAi-expressing wing pouch cells are eliminated from wild-type wing disc and (ii) the SURE (supercompetition of RpS3-expressing clones in RpS3/+ tissue) system in which RpS3-over-expressing clones generated in RpS3/+ wing disc outcompete surrounding RpS3/+ cells. An ectopic over-expression screen using the CORE system identified Wg signaling as a critical regulator of Minute cell competition. Activation of Wg signaling in loser cells suppressed their elimination, whereas down-regulation of Wg signaling in loser cells enhanced their elimination. Furthermore, using the SURE system, it was found that down-regulation of Wg signaling in winner cells suppressed elimination of neighboring losers. These observations suggest that cellular Wg signaling activity is crucial for determining winner/loser status and thereby triggering Minute cell competition (Akai, 2018).
Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method,the regulation of canonical Wnt signaling was investigated during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2; see Drosophila Crptochrome) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila beta-catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of beta-catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. It is anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems (Kaur, 2017).
Adult stem cells provide a renewable source of differentiated cells for a wide variety of tissues and generally give rise to multiple cell types. Basic principles of stem cell organization and regulation underlying this behaviour are emerging. Local niche signals maintain stem cells, while different sets of signals act outside the niche to diversify initially equivalent stem cell progeny. This study shows that Drosophila ovarian follicle stem cells (FSCs) produced two distinct cell types directly. This cell fate choice was determined by the anterior-posterior position of an FSC and by the magnitude of spatially graded Wnt pathway activity. These findings reveal a paradigm of immediate diversification of stem cell derivatives according to stem cell position within a larger population, guided by a graded niche signal. It was also found that FSCs strongly resemble mammalian intestinal stem cells in many aspects of their organization, including population asymmetry and dynamic heterogeneity (Reilein, 2017).
The matricellular protein SMOC (Secreted Modular Calcium binding protein)
is conserved phylogenetically from vertebrates to arthropods. It has been
previously shown that SMOC inhibits bone morphogenetic protein (BMP)
signaling downstream of its receptor via activation of mitogen-activated
protein kinase (MAPK) signaling. In contrast, the most prominent effect of
the Drosophila orthologue, pentagone
(pent), is expanding the range of BMP signaling during wing patterning.
Using SMOC deletion constructs this study found that SMOC-∆EC, lacking the
extracellular calcium binding (EC) domain, inhibits BMP2 signaling,
whereas SMOC-EC (EC domain only) enhances BMP2 signaling. The SMOC-EC
domain binds HSPGs with a similar affinity to BMP2 and can expand the
range of BMP signaling in an in vitro assay by competition for
HSPG-binding. Together with data from studies in vivo the study proposes a
model to explain how these two activities contribute to the function of
Pent in Drosophila wing development and SMOC in mammalian joint
formation (Thomas, 2017). Fine-tuned Notch and Hedgehog signalling pathways via attenuators and dampers have long been recognized as important mechanisms to ensure the proper size and differentiation of many organs and tissues. This notion is further supported by identification of mutations in these pathways in human cancer cells. However, although it is common that the Notch and Hedgehog pathways influence growth and patterning within the same organ through the establishment of organizing regions, the cross-talk between these two pathways and how the distinct organizing activities are integrated during growth is poorly understood. An unbiased genetic screen in the Drosophila melanogaster eye has found that tumour-like growth was provoked by cooperation between the microRNA miR-7 and the Notch pathway. Surprisingly, the molecular basis of this cooperation between miR-7 and Notch converged on the silencing of Hedgehog signalling. In mechanistic terms, miR-7 silenced the interference hedgehog (ihog) Hedgehog receptor, while Notch repressed expression of the brother of ihog (boi) Hedgehog receptor. Tumourigenesis was induced co-operatively following Notch activation and reduced Hedgehog signalling, either via overexpression of the microRNA or through specific down-regulation of ihog, hedgehog, smoothened, or cubitus interruptus or via overexpression of the cubitus interruptus repressor form. Conversely, increasing Hedgehog signalling prevented eye overgrowth induced by the microRNA and Notch pathway. Further, it was shown that blocking Hh signal transduction in clones of cells mutant for smoothened also enhance the organizing activity and growth by Delta-Notch signalling in the wing primordium. Together, these findings uncover a hitherto unsuspected tumour suppressor role for the Hedgehog signalling and reveal an unanticipated cooperative antagonism between two pathways extensively used in growth control and cancer (Da Ros, 2013).
A challenge to understand oncogenesis produced by pleiotropic signalling pathways, such as Notch, Hh, and Wnts, is to unveil the complex cross-talk, cooperation, and antagonism of these signalling pathways in the appropriate contexts. Studies in flies, mice, and in human cell cultures have provided critical insights into the contribution of Notch to tumourigenesis. These studies highlighted that Notch when acting as an oncogene needs additional mutations or genes to initiate tumourigenesis and for tumour progression, identifying several determinants for such co-operation. The identification of these co-operative events has often been knowledge-driven, although unbiased genetic screens also identified known unanticipated tumour-suppressor functions. In this sense, this study describes a conserved microRNA that cooperates with Notch-induced overproliferation and tumour-like overgrowth in the D. melanogaster eye, miR-7. Alterations in microRNAs have been implicated in the initiation or progression of human cancers, although such roles of microRNAs have rarely been demonstrated in vivo. In addition, by identifying and validating functionally relevant targets of miR-7 in tumourigenesis, this study also exposed a hitherto unsuspected tumour suppressor role for the Hh signalling pathway in the context of the oncogenic Notch pathway. Given the conservation of the Notch and Hh pathways, and the recurrent alteration of microRNAs in human cancers, it is speculated that the genetic configuration of miR-7, Notch, and Hh is likely to participate in the development of certain human tumours (Da Ros, 2013).
In human cancer cells, miR-7 has been postulated to have an oncogene or a tumour suppressor functions that may reflect the participation of the microRNA in distinct pathways, due to the regulation of discrete target genes in different cell types, such as Fos, IRS-2, EGFR, Raf-1, CD98, IGFR1, bcl-2, PI3K/AKT, and YY1 in humans (Da Ros, 2013).
In Drosophila, multiple, cell-specific, targets for miR-7 have been previously validated via luciferase or in vivo eGFP-reporter sensors or less extensively via functional studiest. Although microRNAs are thought to regulate multiple target genes, when tested in vivo it is a subset or a given target that predominates in a given cellular context. Indeed, of the 39 predicted miR-7 target genes tested by direct RNAi, only downregulating ihog with several RNAi transgenes (UAS-ihog-IR) fully mimicked the effect of miR-7 overexpression in the transformation of Dl-induced mild overgrowth into severe overgrowth and even tumour-like growth. Moreover, it was confirmed that endogenous ihog is directly silenced by miR-7 and that this silencing involves direct binding of the microRNA to sequences in the 3'UTR of ihog both in vivo and in vitro (Da Ros, 2013).
Nevertheless, other miR-7 target genes may contribute to the cooperation with Dl-Notch pathway along with ihog, such as hairy and Tom. While miR-7 can directly silence hairy in the wing, this effect has been shown to be very modest, and thus, it is considered that while hairy may contribute to such effects, it is unlikely to be instrumental in this tumour model. Indeed, the loss of hairy is inconsequential in eye development, although retinal differentiation is accelerated by genetic mosaicism of loss of hairy and extramacrochaetae that negatively sets the pace of MF progression. It is unclear how Hairy might contribute to Dl-induced tumourigenesis (Da Ros, 2013).
The RNAi against Tom produced overgrowth with the gain of Dl albeit inconsistently and with weak penetrance, where one RNAi line did not modify the Dl-induced overgrowth and the other RNAi line caused tumours in less than 40% of the progeny. Tom is required to counteract the activity of the ubiquitin ligase Neuralized in regulating the Notch extracellular domain, and Dl in the signal emitting cells. These interactions are normally required to activate Notch signalling in the receiving cells through lateral inhibition and cell fate allocation. However, although it remains to be shown whether similar interactions are active during cell proliferation and growth, the moderate enhancement of Dl that is induced when Tom is downregulated by RNAi suggests that miR-7-mediated repression of Tom may contribute to the oncogenic effects of miR-7 in the context of Dl gain of function, along with other targets such as ihog (Da Ros, 2013).
Conversely, while the target genes of the Notch pathway, E(spl)m3 and E(spl)m4 as well as E(spl)mγ, Bob, E(spl)m5, and E(spl)mδ, have been identified as direct targets of miR-7 in the normal wing disc via analysis of 3'UTR sensors, there was no evidence that HLHm3, HLHm4, HLHm5, Bob, and HLHmγ are biological relevant targets of miR-7 in the Dl overexpression context. HLHmδ RNAi produced inconsistent phenotypes in the two RNAi transgenic lines available, causing tumour-like growth at very low frequency in only one of the lines. No evidence was obtained that miR-7 provoked overgrowth by targeting the ETS transcription factor in the EGFR pathway AOP/Yan, a functionally validated target of the microRNA miR-7 during retinal differentiation. Neither was any evidence obtained that RNAi of atonal provoked eye tumours with Dl overexpression, although a strong inhibition via expression of a fusion protein Atonal::EN that converts Atonal into a transcriptional repressor has been shown to be sufficient to trigger tumorigenesis together with Dl. Thus, it was reasoned that given that microRNA influenced target genes only subtly (even when using ectopic expression), it is possible that downregulation of atonal contributes to the phenotype along with the other targets (Da Ros, 2013).
In conclusion, this study has identified cooperation between the microRNA miR-7 and Notch in the D. melanogaster eye and identified and validated ihog as a direct target of the miR-7 in this context and have identified boi as a target of Notch-mediated activity at the DV eye organizer, although it remains whether this regulation is direct or indirect. A hitherto unanticipated tumour suppressor activity was uncovered of the endogenous Hh signalling pathway in the context of gain of Dl-Notch signalling that is also apparent during wing development (Da Ros, 2013).
Hh tumour suppressor role is revealed when components of the Hh pathway were lost in conjunction with a gain of Dl expression in both the eye and wing discs. Hh and Notch establish signalling centres along the AP and DV axes, respectively, of the disc to organize global growth and patterning. Where the organizer domains meet, the Hh and Notch conjoined activities specify the position of the MF in the eye disc and the proximodistal patterning in the wing disc. This study also unvailed that in addition antagonistic interaction between the Hh and Notch signalling might help to ensure correct disc growth. Thus, it was shown that Hh signalling limits the organizing activity of Dl-Notch signalling. Although it is often confounded whether Dl-Notch signalling instructs overgrowth by autonomous or nonautonomous (i.e., DV organizers) mechanisms, these findings uncover that loss of Hh signalling enhances a non-cell autonomous oncogenic role of Dl-Notch pathway (Da Ros, 2013).
To date, Hh has not yet to be perceived as a tumour suppressor, although it is noteworthy that human homologs of ihog, CDO, and BOC were initially identified as tumour suppressors. Importantly, both CDO and BOC are downregulated by RAS oncogenes in transformed cells and their overexpression can inhibit tumour cell growth in vitro. Since human RAS regulates tumourigenesis in the lung by overexpressing miR-7 in an ERK-dependent manner, it is possible that RAS represses CDO and BOC via this microRNA. Indeed, the 3'UTR of both CDO and BOC like Drosophila ihog contains predicted binding sites for miR-7. There is additional clinical and experimental evidence connecting elements of the Hedgehog pathway with tumour-suppression. The function of Growth arrest specific gene 1 (GAS1), a Hh ligand-binding factor, overlaps that of CDO and BOC, while its overexpression inhibits tumour growth . More speculative is the association of some cancer cells with the absence of cilium, a structure absolutely required for Hh signal transduction in vertebrate cells (Da Ros, 2013).
Given the pleiotropic nature of Notch, Wnts, BMP/TGFβ, Ras, and Hh signalling pathways in normal development in vivo, it is speculated that competitive interplay as that described in this study between Notch and Hh may not be uncommon among core growth control and cancer pathways that act within the same cells at the same or different time to exert multiple outputs (such as growth and cell differentiation). Moreover, context-dependent tumour suppressor roles could explain the recurrent, unexplained, identification of somatic mutations in Hh pathway in human cancer samples. Indeed, the current findings stimulate a re-evaluation of the signalling pathways previously considered to be exclusively oncogenic, such as the Hh pathway (Da Ros, 2013).
Epithelial folding shapes embryos and tissues during development. This study investigated the coupling between epithelial folding and actomyosin-enriched compartmental boundaries. The mechanistic relationship between the two is unclear, because actomyosin-enriched boundaries are not necessarily associated with folds. Also, some cases of epithelial folding occur independently of actomyosin contractility. Shallow folds called parasegment grooves that form at boundaries between anterior and posterior compartments in the early Drosophila embryo were investigated. Formation of these folds requires the presence of an actomyosin enrichment along the boundary cell-cell contacts. These enrichments, which require Wingless signalling, increase interfacial tension not only at the level of the adherens junctions but also along the lateral surfaces. Epithelial folding is normally under inhibitory control because different genetic manipulations, including depletion of the Myosin II phosphatase Flapwing, increase the depth of folds at boundaries. Fold depth correlates with the levels of Bazooka (Baz), the Par-3 homologue, along the boundary cell-cell contacts. Moreover, Wingless and Hedgehog signalling have opposite effects on fold depth at the boundary that correlate with changes in Baz planar polarity (Urbano, 2018).
Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. MagT1 (CG7830) has been identified in the Drosophila genome and its protein product characterized by electrophysiological means. This study reports the generation of fly MagT1 mutants and shows that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, this study found that loss of MagT1 results enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, this study directly demonstrated that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila Kc167cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling (Xun, 2018).
Cell shape is known to influence the plane of cell division. In vitro, mechanical constraints can also orient mitoses; however, in vivo it is not clear whether tension can orient the mitotic spindle directly, because tissue-scale forces can change cell shape. During segmentation of the Drosophila embryo, actomyosin is enriched along compartment boundaries forming supracellular cables that keep cells segregated into distinct compartments. This study shows that these actomyosin cables orient the planar division of boundary cells perpendicular to the boundaries. This bias overrides the influence of cell shape, when cells are mildly elongated. By decreasing actomyosin cable tension with laser ablation or, conversely, ectopically increasing tension with laser wounding, this study demonstrates that local tension is necessary and sufficient to orient mitoses in vivo. This involves capture of the spindle pole by the actomyosin cortex. These findings highlight the importance of actomyosin-mediated tension in spindle orientation in vivo (Scarpa, 2018).
Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. This study shows that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/beta-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed definition of three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics (Suisse, 2019).
Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stromal interaction molecule (Stim), which encodes an endoplasmic reticulum-membrane protein that is an essential component of the store-operated calcium entry mechanism, which in neurons regulates flight. Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER. SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Suisse, 2019).
Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).
There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function. This study, shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).
Transmembrane proteins must pass through the secretory pathway to reach the cell surface, where they can interact with other cells and respond to signaling cues. Disrupting the environment in the first secretory compartment, the endoplasmic reticulum (ER), causes misfolding of transmembrane and secreted proteins and elicits a stress response that can either restore proteostasis or trigger apoptosis. The ER acts as a store of intracellular calcium (Ca2+) that can be rapidly released into the cytoplasm to trigger a variety of cellular responses. The sarcoplasmic-ER ATPase (SERCA) actively pumps Ca2+ into the ER, increasing its concentration to 1,000-fold higher than in the cytosol. Depletion of Ca2+ from the ER is sensed by Stim, which accumulates at ER-plasma membrane junctions and activates Orai, a Ca2+ channel in the plasma membrane that mediates store-operated calcium entry (SOCE). SERCA colocalizes with Stim-Orai complexes, allowing entering Ca2+ to be pumped directly into the ER (Alonso, 2012). SOCE maintains Ca2+ homeostasis in the ER so that Ca2+-binding proteins can fold correctly. In the absence of SERCA, the cell-surface receptor Notch, which has extracellular EGF and Lin-12/Notch repeats that interact with Ca2+, fails to mature (Suisse, 2019 and references therein).
Wnt signaling relies on the bifunctional β-catenin protein, which acts as an essential linker between E-cadherin (E-Cad) and α-catenin at adherens junctions (AJs), but also enters the nucleus and regulates target gene expression in cells that receive a Wnt signal. In the absence of Wnt, cytoplasmic β-catenin is phosphorylated within a destruction complex, leading to its ubiquitination and degradation. Junctional β-catenin is distinct from the pool available for Wnt signaling, and excess E-Cad can remove β-catenin from the signaling pool. The extracellular domain of E-Cad binds Ca2+ ions at the junctions between cadherin domains, giving it a rigid structure. The cadherin family also includes the large protocadherins Fat and Dachsous, which restrict growth by activating the Hippo signaling pathway and regulate planar cell polarity. The precise conformation of these molecules depends on Ca2+ binding by only a subset of their cadherin domain linkers (Suisse, 2019).
There has been significant interest in using SERCA inhibitors such as thapsigargin as cancer therapeutics due to their ability to induce ER stress and apoptosis. Their general toxicity means that they would need to be targeted to specific cancer cell types. However, activating mutations in Notch that are found in certain types of leukemia may make this receptor especially sensitive to reduced SERCA function (Roti, 2013). This study shows that a hypomorphic mutation in Drosophila SERCA preferentially affects signaling by the Wnt Wingless (Wg), because E-Cad is retained in the ER and sequesters bound Armadillo (Arm)/β-catenin. Complete loss of SERCA function leads to apoptosis, but an intermediate reduction in ER Ca2+ induced by mutating orai in the hypomorphic SERCA background disrupts Hippo signaling, leading to overgrowth and Notch signaling. These results imply that Wnt-driven cancers may be the most sensitive to SERCA inhibition but highlight the risk that inhibitors may activate cell proliferation through the Hippo pathway (Suisse, 2019).
Characterization of a hypomorphic SERCA mutant allele revealed that E-Cad trafficking is especially sensitive to reduced ER Ca2+ levels and that retention of E-Cad in the ER under these mild stress conditions sequesters Arm away from the pool available for Wg signaling. A similar ER retention of E-Cad and desmosomal cadherins, leading to the loss of cell adhesion, has been demonstrated in human keratinocytes in Darier disease, which results from a mutation in SERCA2. In addition, ER stress promotes the differentiation of mouse intestinal stem cells, suggesting that this may be a physiological mechanism to reduce the Wnt signaling that is required for stem cell maintenance. Ca2+ is essential for the homophilic binding of cadherin extracellular domains that mediates cell adhesion. Cadherin monomers contain multiple cadherin domains separated by hinge regions that can each bind three Ca2+ ions, stabilizing the molecule to form a rod-like structure that is resistant to protease cleavage. In larger cadherins, some of the linker regions are Ca2+ free and remain flexible. Cadherin folding into the correct conformation may thus be very sensitive to Ca2+ levels in the ER. In mammalian cells, Tg-induced ER stress leads to O-GlcNAc glycosylation of the E-Cad cytoplasmic domain, blocking its exit from the ER. However, this modification depends on caspase induction by ER stress-induced apoptosis, which does not occur in SERCAdsm mutant clones. It is also possible that E-Cad is not affected by ER Ca2+ levels directly, but is especially sensitive to the general reduction in secretion caused by the loss of SERCA (Suisse, 2019).
Arm that is bound to E-Cad at the ER membrane appears to be unavailable for Wg signaling. In mammalian cells, β-catenin forms a complex with E-Cad during co-translation in the ER and helps to transport E-Cad from the ER to the Golgi. Depleting ER Ca2+ levels may enhance the binding of Arm to E-Cad at the ER, as low extracellular Ca2+ induces rapid Arm recruitment to E-Cad at the plasma membrane. Because E-Cad competes with adenomatous polyposis coli and Axin to bind to the Arm domains, a stronger Arm-E-Cad interaction could both protect Arm from degradation and prevent it from translocating into the nuclei of Wg-receiving cells. The mechanism by which β-catenin enters the nucleus is poorly understood, and it is possible that mislocalization at the ER membrane would exclude it from docking with the partner proteins required for nuclear import (Suisse, 2019).
Using two SERCA alleles and a SERCA orai mutant combination, this study produced three distinct levels of ER Ca2+ that revealed the differential sensitivities of three oncogenic pathways. Wg signaling is the most sensitive, as it is disturbed by the weak allele SERCAdsm; while Notch trafficking is also abnormal in this mutant background, Notch target genes can still be activated. A further reduction in ER Ca2+ produced by disrupting SOCE prevents Notch and Hippo signaling, probably through effects on the trafficking of Notch and the large protocadherin Fat, but only complete loss of SERCA induces apoptosis. These findings have important implications for the use of SERCA inhibitors such as Tg as cancer therapeutics, even when targeted to specific cell types. Although it may be possible to selectively block Wnt-driven cancers with low doses of such inhibitors, the level of inhibition needed to prevent Notch signaling is likely to actually enhance tumor invasiveness by downregulating FAT family members and thus disrupting Hippo signaling (Suisse, 2019).
Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. Using a recently-developed glycoproteomic method, this study found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNAi knockdown and mutant clone analyses showed that loss of wdp leads to the upregulation of Hh signaling. Altogether, this study demonstrates a novel role of CSPGs in regulating Hh signaling (Takemura, 2020).
Spatial and temporal regulation of growth factor signaling pathways is essential to proper development and disease prevention. Cell surface signaling events, such as ligand-receptor interactions, are often modulated by proteoglycans. Proteoglycans are carbohydrate-modified proteins that are found on the cell surface and in the extracellular matrix. They are composed of a core protein and one or more glycosaminoglycans (GAGs) covalently attached to specific serine residues on the core protein. GAGs are long, unbranched, and highly sulfated polysaccharide chains consisting of a repeating disaccharide unit. Based on the composition of the disaccharide units, proteoglycans are classified into several types, including heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) (Takemura, 2020).
HSPGs function as co-receptors by interacting with a wide variety of ligands to modulate signaling activities. Drosophila offers a powerful model system to study the functions of HSPGs in vivo because of its sophisticated molecular genetic tools and minimal genetic redundancy in genes encoding core proteins and HS synthesizing/modifying enzymes. In vivo studies using the Drosophila model have shown that HSPGs orchestrate information from multiple ligands in a complex extracellular milieu and sculpt the signal response landscape in a tissue. However, the molecular mechanisms of co-receptor activities of HSPGs still remain a fundamental question. Previous studies predict that there are unidentified molecules involved in the molecular recognition events on the cell surface (Takemura, 2020).
In addition to HS, Drosophila produces CS, another type of GAG. CSPGs are well known as major structural components of the extracellular matrix. CSPGs have also been shown to modulate signaling pathways, including Hedgehog (Hh), Wnt, and fibroblast growth factor signaling. Given the structural similarities between CS and HS, CSPGs may have modulatory, supportive, and/or complementary functions to HSPGs. However, the mechanisms by which CSPGs function as a co-receptor are unknown. In contrast to a large number of studies on HSPGs, very few CSPGs have been identified and analyzed in Drosophila. Unlike HSPGs, CSPG core proteins are not well conserved between species. Therefore, the identification of CSPGs cannot rely on the sequence homology to mammalian counterparts (Takemura, 2020).
Recently, a glycoproteomic method was developed to identify novel proteoglycans. Briefly, this method includes trypsinization of protein samples, followed by enrichment of glycopeptides using strong anion exchange (SAX) chromatography. After enzymatic digestion of HS/CS chains, the glycopeptides bearing a linkage glycan structure common to HS and CS chains are identified using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This method has successfully identified novel CSPGs in humans (Takemura, 2020).
To study the function of CSPGs in signaling, the glycoproteomic method was used to identify previously unrecognized CSPGs in Drosophila. This study found that Windpipe (Wdp) is a novel CSPG and affects Hh signaling. Overexpression of wdp inhibits Hh signaling in the wing disc. This inhibitory effect of Wdp on Hh signaling is dependent on its CS chains and LRR motifs. Consistent with the overexpression analysis, loss of wdp increases Hh signaling: wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. This study highlights a novel function of CSPGs in cell signaling (Takemura, 2020).
Glycoproteomic analysis identified Wdp as a novel CSPG. Apart from Wdp, no additional novel core proteins were found in this study. However, some previously established core proteins were also identified, which were found with both CS- and / or HS- modifications. In a recent glycoproteomic study of C. elegans, 15 novel chondroitin core proteins were identfied, in addition to the 9 previously established. The reason for this discrepancy with the regard to the number of identified core proteins in the two model organisms is unclear, but it may suggest that optimization of sample preparation is necessary for identifying additional CSPGs in Drosophila (Takemura, 2020).
Although Wdp was found modified with CS in both wild-type and ttv backgrounds, general assessment of spectral intensities suggest that Wdp was present in higher abundance in the ttv samples. Earlier studies in Zebrafish, mammalian cells, and C. elegans indicated that reduced HS sulfation results in increased CS sulfation. Thus, it is not surprising to see a compensatory increase of CS synthesis in a strain lacking HS polymerase (ttv). It should be noted that Wdp modified with HS was not detected in wild-type flies, although this variant was explicitly looked for (Takemura, 2020).
Genetic analyses of Wdp showed that it acts as a negative modulator of Hh signaling in a CS- and LRR motif-dependent manner. It has also been reported that Wdp negatively regulates JAK-STAT signaling and controls adult midgut homeostasis and regeneration (Ren, 2015). The authors showed that Wdp interacts with the Dome receptor and promotes its endocytosis and lysosomal degradation. Although the mechanism by which Wdp regulates Hh signaling is not known, it is possible that Wdp modulates these two pathways via a similar mechanism: by controlling the stability of cell surface components of the pathways. Hh signaling is controlled by two key membrane proteins-Ptc and Smo. In the absence of Hh, Ptc inhibits the phosphorylation of Smo, which is internalized and degraded. In the presence of Hh, restriction of Ptc on Smo is relieved, allowing Smo to accumulate on the cell surface and activate Hh signaling. Preliminary observation showed that knockdown of wdp increases Smo protein levels. Thus, Wdp may downregulate Hh signaling by affecting Smo levels (e.g. disrupting Smo translocation to the cell membrane or the stability of Smo on the cell surface). However, this does not exclude other possibilities for Wdp action, such as sequestering the ligand, inhibiting Ptc in its Smo phosphorylation/activation, and competing with a HSPG co-receptor. In mice, sulfated CS is necessary for Indian hedgehog (Ihh) signaling in the developing growth plate. Although Ihh and Sonic hedgehog (Shh) have been shown to bind to CS, the molecular mechanisms of CSPG function in Hh signaling remain to be elucidated (Takemura, 2020).
It is worth noting that both JAK-STAT and Hh signaling, the two pathways negatively controlled by Wdp, are also regulated by HSPGs. Dally-like, a glypican family of HSPGs, positively regulates Hh signaling. In the developing ovary, Dally upregulates the JAK-STAT pathway. Given the importance of precise dosage control of oncogenic pathways, such as JAK-STAT and Hh signaling, this dual proteoglycan system could play an important role in fine-tuning of the signaling output in order to prevent cancer formation. In vertebrates, HSPGs and CSPGs show opposing effects in neural systems. For example, axon growth is typically promoted by HSPGs but inhibited by CSPGs. The current findings suggest that such competing effects of HSPGs and CSPGs may be a general mechanism for the precise control of signaling cascades and pattern formation (Takemura, 2020).
In addition to its functions in signaling, Wdp may play other roles. This study found that overexpression of wdp results in massive apoptosis in the wing disc, independent of Hh signaling inhibition. Since CSPGs are well known for structural functions, an excess amount of Wdp may affect the epithelial integrity of the wing disc, leading to subsequent apoptosis. The observation that Wdp is enriched on the basal side of the wing disc and adult midgut cells suggests that Wdp may interact with components of the basement membrane, which surrounds these organs (Takemura, 2020).
Previous studies also reported that wdp is associated with aggressive behaviors in Drosophila species. wdp is upregulated in the head of socially isolated male flies, which exhibit more aggressive behaviors than males raised in groups. Also, wdp expression is slightly higher in the brain of Drosophila prolongata, which is more aggressive compared to its closely-related species. Since CSPGs are important in neuronal patterning, it is interesting to define the molecular mechanisms by which Wdp affects Drosophila behavior.
In mammals, there is a class of CSPG molecules with LRR motifs (small leucine-rich proteoglycans, or SLRPs). A number of SLRP members are known as causative genes of human genetic disorders. Although Wdp does not have cysteine-rich regions that are commonly found in mammalian SLRPs, MARRVEL (ver 1.1) reports that wdp is a potential Drosophila ortholog of the human NYX gene (nyctalopin), a member of SLRPs (DIPOT score 1). Mutations in NYX cause X-linked congenital stationary night blindness. Further studies on Wdp will provide a novel insight into the function of these disease-related human counterparts (Takemura, 2020).
Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic Hh (Shh) responses in this way. To determine if this is a more general phenomenon, an RNAi screen targeting GPCRs was carried out in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. These results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals (Saad, 2021).
Growth and patterning of Drosophila wing depends upon the sequential organizing activities of Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways. The Hh signaling directly activates the expression of dpp through the transcription factor Cubitus interruptus (Ci). Dpp itself functions as a long-range morphogen to promote cell proliferation and differentiation through an essential transcription factor encoded by Mad. This study reports that the Mad1-2 allele exhibits phenotypes distinct from classical Dpp pathway mutants in the developing wing. The activity of Dpp signaling is attenuated in Mad1-2 mutant cells. However, activation of Dpp signaling is found in a subset of cells surrounding homozygous Mad1-2 clones when the clones are located at the anterior compartment of wing disc. Further analysis reveals that Mad1-2 mutant cells display high level of Hh signaling activity and accumulate significant amount of Ci. Unexpectedly, whole genome resequencing identifies multiple mutations in the 3'UTR region of Pka-C1 genomic loci in the Mad1-2 stock. Genetic and molecular evidence is provided that the Pka-C1 mutations carried by Mad1-2 likely underlies the observed Hh signaling defects. Therefore, the contribution of Pka-C1 mutation should be taken in consideration when analyzing Mad1-2 phenotypes. The isolation of independent Mad and Pka-C1 alleles from the Mad1-2 stock further supports these conclusions (Chen, 2021).
Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. This study shows that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, evidence is provided that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill (Roberto, 2022).
The Hedgehog (Hh) cascade is central to development, tissue homeostasis and cancer. A pivotal step in Hh signal transduction is the activation of glioma-associated (GLI) transcription factors by the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO). How SMO activates GLI remains unclear. This study showed that SMO uses a decoy substrate sequence to physically block the active site of the cAMP-dependent protein kinase (PKA) catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular and organismal models, it was demonstrated that interfering with SMO-PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism uncovered echoes one used by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, these findings define a mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases (Happ, 2022).
Graded Hedgehog (Hh) signaling is mediated by graded Cubitus interruptus (Ci)/Gli transcriptional activity, but how the Hh gradient is converted into the Ci/Gli activity gradient remains poorly understood. This study shows that graded Hh in Drosophila induces a progressive increase in Ci phosphorylation at multiple Fused (Fu)/CK1 sites including a cluster located in the C-terminal Sufu-binding domain. Fu directly phosphorylated Ci on S1382, priming CK1 phosphorylation on adjacent sites, and that Fu/CK1-mediated phosphorylation of the C-terminal sites interfered with Sufu binding and facilitated Ci activation. Phosphorylation at the N-terminal, middle, and C-terminal Fu/CK1 sites occurred independently of one another and each increased progressively in response to increasing levels of Hh or increasing amounts of Hh exposure time. Increasing the number of phospho-mimetic mutations of Fu/CK1 sites resulted in progressively increased Ci activation by alleviating Sufu-mediated inhibition. C-terminal Fu/CK1 phosphorylation cluster is conserved in Gli2 and contributes to its dose-dependent activation. This study suggests that the Hh signaling gradient is translated into a Ci/Gli phosphorylation gradient that activates Ci/Gli by gradually releasing Sufu-mediated inhibition (Zhou, 2022).
Hereditary spastic paraplegia (HSP) is a neurodegeneration disease, one of the reasons is caused by autosomal recessive missense mutation of the karyogene that encodes phenylalanyl-tRNA synthetase 2, mitochondrial (FARS2). However, the molecular mechanism underlying FARS2-mediated HSP progression is unknown. Mitochondrial phenylalanyl-tRNA synthetase gene (PheRS-m) is the Drosophila melanogaster homolog gene of human FARS2. This study constructed a Drosophila HSP missense mutation model and a PheRS-m knockout model. Some of the mutant fly phenotypes included developmental delay, shortened lifespan, wing-structure abnormalities and decreased mobility. RNA-sequencing results revealed a relationship between abnormal phenotypes and the hedgehog (Hh) pathway. A qRT-PCR assay was used to determine the key genes (ptc, hib, and slmb) of the Hh pathway that exhibited increased expression during different developmental stages. Hh signaling transduction was shown to be negatively regulated during the developmental stages of PheRS-m mutants but positively regulated during adulthood. By inducing the agonist and inhibitor of Hh pathway in PheRS-m larvae, the developmental delay in mutants can be partly salvaged or postponed. Collectively, these findings indicate that Hh signaling negatively regulates the development of PheRS-m mutants, subsequently leading to developmental delay (Mo, 2022).
Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. This study used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, this work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development (Nellas, 2022).
Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in Drosophila. Previous publications showed that the crystal structures of human and Drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, full-length SuFu's structure in solution was studied, both with circular dichroism and small angle X-ray scattering, comparing Drosophila, zebrafish and human species, to better understand this considerable difference. These studies show that, in spite of similar crystal structures restricted to ordered domains, Drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, this study showed that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal (Makamte, 2022).
The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3' untranslated region, this study analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, a genome-wide in vivo miRNA overexpression screen was performed in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, the miRNA sensor toolbox was used to show that Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. This study has used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, two different mutants were identified that mapped to the Hedgehog signalling pathway. Previously, a novel Ptc mutation was described, and this study adds to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma (Moore, 2022).
A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. This study found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and it, as well as other JNK pathway genes, were found to be essential for differentiation of FSC daughter cells. The data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, it was demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, these findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage (Tatapudy, 2021).
The segregation of FSC and daughter cell fates requires the orchestration of multiple developmental signaling pathways to ensure that cell fate specification and proliferation occurs at the correct time and place. This study has identified a new layer of regulation in which a single gene, ben, has multiple distinct roles in promoting differentiation and proliferation in the early FSC lineage (Tatapudy, 2021).
First, the findings demonstrate that ben functions along with the rest of the JNK signaling pathway to promote pFC (posterior follicle cell) differentiation. The phenotypes observed in JNK pathway mutants strongly suggest that the primary defect is a failure to differentiate into mature stalk cells. The range of tube-like and expanded stalk morphological phenotypes that were observed could both be explained by an inability of cells in the stalk regions between follicles to facilitate the pinching off of follicles from the germarium or to intercalate into a single row after the pinching off process has been initiated. The possibility cannot be ruled out that differentiation toward the polar cell fate or main body cell fate are also impaired upon loss of JNK signaling, but no evidence was seen for this with the markers used. Notably, although RNAi knockdown of ben or hep impaired the activity of a reporter for Notch signaling, NRE-GFP, in Region 2b pFCs and mature polar cells, this did not appear to interfere with the ability of mutant pFCs to differentiate into polar cells, so the significance of this effect on cell fate specification is unclear. Additional reporters of Notch signaling may help to determine the extent to which Notch signaling is disrupted in these mutant conditions. It was also found that pERK is retained in pFCs of ovarioles with impaired JNK signaling, and that constitutive activation of pERK phenocopies the differentiation defects observed in JNK pathway mutants. These observations suggest that JNK signaling promotes pFC differentiation into stalk cells by inhibiting the activation of ERK in pFCs (Tat in a JNK-independent manner to help shape the gradient of Hh signaling in FSCs and pFCs. Indeed, it was found that levels of Ptc-pelican-GFP, indicative of Hh signaling, are higher in ben mutant FSCs compared with wild type, and the Ptc-pelican-GFP reporter remains detectable throughout Regions 2b and 3 of tapudy, 2021).
Second, ben functions in the germarium. Likewise, it was found that RNAi knockdown of ben caused the main body follicle cells to retain zfh1 expression, a downstream target of Hh signaling, throughout the germarium and even into Stage 2 in some cases, where it is completely absent in wild-type tissue. However, it was also found that ben mutant main body follicle cells were able to mature into a Cas+, Eya- state, indicating proper differentiation with respect to these two markers. Thus, although loss of ben does not fully impair main body follicle cell differentiation, it may influence the process through its role as an upstream regulator of zfh1 expression. Further study will be required to understand the functional significance of decreased cas and zfh1 expression during main body follicle cell differentiation (Tatapudy, 2021).
JNK signaling has been found to be involved in a wide variety of biological processes in a cell type- and context-dependent manner. Some of the most well-known functions of JNK signaling involve responses to non-homeostatic conditions, such as stress, cellular damage, infection and tumor growth. However, JNK signaling has also been found to be important for processes that occur during normal development or adult homeostasis. Indeed, hep mutants were originally described for their role in dorsal closure of the epidermis in the embryo, and subsequent studies have clearly established the importance of the JNK signaling pathway in this process. Likewise, JNK signaling is also important during adult homeostasis in mammalian stem cell lineages, for example to promote self-renewal of human hematopoietic stem cells and differentiation in the mouse intestinal stem cell and human neural stem cell lineages. In line with this type of role for JNK signaling, the current findings reveal new pathway interactions for JNK signaling in the regulation of cell fate decisions in an epithelial stem cell lineage (Tatapudy, 2021).
In addition to regulating cell fate decisions, a JNK-independent role was identified for Ben in the regulation of proliferation rate and FSC niche competition. Previous studies have established that increased proliferation causes hypercompetition, and that the proliferative response to Hh signaling is the key mediator of the FSC niche competition phenotypes in Hh pathway mutants. Therefore, the hypercompetition phenotype of ben mutant clones is likely caused, in large part, by the increased Hh signaling levels and proliferation rates in these mutants. The findings that overexpression of dap or RNAi knockdown of smo was sufficient to suppress the hypercompetition phenotype of benA mutant clones are consistent with this. It is possible that delayed differentiation in newly-produced ben mutant pFCs also contributes to the hypercompetition phenotype. However, the observation that hepG0107 mutant clones are not hypercompetitive indicates that the hypercompetition phenotype of ben mutant clones is not caused by a block in the JNK-dependent functions of Ben in pFC differentiation (Tatapudy, 2021).
Taken together, these findings indicate that Ben has JNK-dependent and -independent roles in regulating pFC cell fate decisions as they differentiate into polar, stalk and main body follicle cells. Specifically, the findings support a model in which JNK signaling promotes the differentiation of pFCs during normal homeostasis by helping to downregulate EGFR signaling in early pFCs. Previous studies found that pERK inhibits Groucho and that Groucho is required for the upregulation of Notch signaling in pFCs. Thus, the retention of pERK in pFCs may explain why Notch signaling is not upregulated in JNK pathway mutants. Separately, Ben inhibits Hh signaling in a JNK-independent manner, thereby controlling pFC proliferation, regulating FSC niche competition, and possibly contributing to main body follicle cell differentiation. Thus, this study has revealed a new layer of regulation within the signaling network that controls cell behavior in the early FSC lineage. The recently published single-cell atlases of the adult Drosophila ovary revealed a continuum of changes in gene expression that occur during cellular differentiation in the early FSC lineage, and provide the tools for characterizing this signaling network in a cell-type specific manner. In future studies, it will be interesting to apply these new single-cell approaches to mutants such as those described in this study to further elucidate the molecular basis of cell fate decisions in the FSC lineage (Tatapudy, 2021).
Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, a genome-wide in vivo RNAi screen was performed for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. These analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. 29 dual regulators were identified that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, this study established that Cdc37 and Gilgamesh/CK1gamma inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease (Swarup, 2015).
Divergent disease states have been attributed to be a cause or consequence of aberrant protein phosphorylation. Wnt signaling is phosphor-regulated both in its silent and active states, but thus far understanding of kinases, phosphatases and associated factors of the pathway has been limited. In this study, the first genome-wide in vivo screen was performed under physiological conditions in the Drosophila wing disc for phospho-regulators of the Wnt pathway. 54 high-confidence regulators were identified, 22 of which are novel. The results of these analyses do not indicate whether a high-confidence regulator has a direct or indirect effect on signaling. However, as ~60% of the high-confidence regulators identified have been previously validated to have a direct effect on Wnt signaling, it is predictd that at least some of the novel high-confidence regulators identified would also have a direct effect on the pathway. Indeed, subsequent analyses of Myopic revealed a novel role in regulating Wg secretion. Although the mechanism and components of the Wnt pathway are for the most part conserved between Drosophila and humans, there are possibly vertebrate-specific phospho-regulators of signaling that would not have been identified in these analyses. The dataset represents the largest list of putative phospho-regulators of the Wnt pathway identified to date, almost all of which have identified human orthologs and are therefore likely to be functionally conserved (Swarup, 2015).
As part of this study, previously unknown relationships were established between the Wnt and Hh pathways in vivo by identifying 12 novel dual regulators that are proposed to function at analogous levels of signaling. As proof of concept, the roles of Cdc37 and Gish/CK1γ were biochemically characterized to demonstrate that their functions are conserved from Drosophila to mammalian cells. An initial analysis is reported of candidate regulators of Notch signaling during wing disc development. Although these findings are preliminary, they highlight an emerging theme of phospho-regulation of Notch that likely hold parallels in vertebrate biology. The comparison of signaling pathways in vivo and the identification of specific versus shared phospho-regulators facilitate understanding of human development and disease states (Swarup, 2015).
Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein 'destruction complex', assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation (Yang, 2016).
The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas. Two multimeric protein complexes with opposing functions -- the cytoplasmic destruction complex and the plasma membrane-associated signalosome -- control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli (APC), and two kinases: casein kinase α (CK1α) and glycogen synthase kinase 3 (GSK3). Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated components, GSK3 and Dishevelled (Dvl). Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the transcriptional regulation of Wnt pathway target genes (Yang, 2016).
As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex. Following Wnt exposure, the rapid association of phospho-Axin with phospho-LRP6 triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin's interaction with both the destruction and signalosome complexes. Axin is subsequently degraded; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin's essential role during the initial activation of the Wnt pathway (Yang, 2016).
The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models, Wnt stimulation induces Axin's dissociation from the destruction complex, thereby promoting its interaction with the signalosome. As Wnt stimulation induces Axin dephosphorylation, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex; however, recent work revealed that the interaction of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction (Kim, 2013) Furthermore, some findings have challenged prevailing models, providing evidence that Axin's interaction with the destruction complex is not diminished upon Wnt stimulation. Thus, whereas the rapid switch in Axin function following Wnt stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain (Yang, 2016).
During investigation of this critical process, an unanticipated role was discovered for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis, small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers. This study identified a novel mechanism through which Tnks regulates Axin: by promoting Axin's central role in rapid Wnt pathway activation. Wnt stimulation was found to modulate Axin levels biphasically in both Drosophila and human cells. Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin's association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin function following Wnt stimulation. These results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6, suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin's central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors (Yang, 2016).
Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. These findings lead to three major revisions of the current model for the role of Tnks in the activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components. Second, the results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process. These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation, through mechanisms yet to be elucidated. Finally, pharmacologic inactivation of Tnks was shown to diminish the interaction of Axin with LRP6, revealing a previously unknown mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex inhibitors (Yang, 2016).
In the absence of Wnt stimulation, the concentration-limiting levels of Axin regulate its scaffold function in the destruction complex. As components of the destruction complex participate in other signalling pathways, the low levels of Axin were proposed to maintain modularity of the Wnt pathway. The new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis, the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. The findings reveal that Wnt exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in Tnks null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, it is proposed that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid response to Wg stimulation (Yang, 2016).
It is postulated that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction, whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were responsive to the relative change in β-catenin levels, rather than their absolute value. This dependence was proposed to impart robustness and resistance to noise and cellular variation. The current data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in β-catenin levels (Yang, 2016).
The current findings have relevance for the context-specific in vivo roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a number of cultured cell lines, but in vivo studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or developmental stages. In mice, functional redundancy exists between the two Tnks homologues, such that Tnks single mutants are viable and fertile, whereas double mutants display embryonic lethality without overt Wnt-related phenotypes. However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex roles in mouse embryonic development. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development; however, the regeneration of injured fins in adults, a process that requires Wnt signalling, was disrupted (Yang, 2016).
Similarly, the finding that Drosophila Tnks null mutants are viable (Wang, 2016a; Wang, 2016b; Feng, 2014) was unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, the current studies reveal that a less than twofold increase in Axin levels uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, it is postulated that Tnks loss can be compensated during development unless Axin levels are increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels. Furthermore, Drosophila Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration. Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described in this study, which reveal that the Wnt-induced association of Axin with LRP6 occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. It is postulated that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves to amplify the initial response to Wnt stimulation, and thus is essential in a subset of in vivo contexts (Yang, 2016).
The recent discovery that Tnks enhances signalling in Wnt-driven cancers has raised the possibility that Tnks inhibitors will offer a promising new therapeutic option. Indeed, preclinical studies have supported this possibility. Tnks inhibitors were thought previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, the current findings indicate that the degree to which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some in vivo contexts. Instead, the results reveal that Tnks inhibition simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, it is proposed that the efficacy of Tnks inhibitors results from their combined action at both of these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, these results suggest that in contrast with the current focus on tumours in which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in RNF43, a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (Yang, 2016).
Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. This study investigated developmental regulation of Chk1 activation. This study reports Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. Canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, this study shows that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation (Kizhedathu, 2020).
Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector beta-catenin. The multiprotein destruction complex binds and phosphorylates beta-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing beta-catenin. Recent work clarified underlying mechanisms, but important questions remain. This study explored beta-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. This study found that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. Elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When Dishevelled levels were elevated, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high (Schaefer, 2020).
During embryonic development, cells must choose fate based on their position within the unfolding body plan. One key is cell-cell signaling, by which cells communicate positional information to neighbors and ultimately direct downstream transcriptional programs. A small number of conserved signaling pathways play an inordinately important role in these events in all animals. These include the Hedgehog, Notch, Receptor Tyrosine kinase, BMP/TGFβ, and Wnt pathways, which influence development of most tissues and organs. These same signaling pathways regulate tissue stem cells during tissue homeostasis and play critical roles in most solid tumors. Due to their powerful effects on cell fate and behavior, evolution has shaped dedicated machinery that keeps each signaling pathway definitively off in the absence of ligand (Schaefer, 2020).
In the Wnt pathway, signaling is turned on and off by regulating stability of the key effector β-catenin (βcat). In the absence of Wnt ligands, newly synthesized βcat is rapidly captured by the multiprotein destruction complex . Within this complex, the protein Axin acts as a scaffold, recruiting multiple partners. Axin and adenomatous polyposis coli (APC) bind βcat and present it to the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3) for sequential phosphorylation of a series of N-terminal serine and threonine residues on βcat (Schaefer, 2020).
It has become increasingly clear that the destruction complex is not a simple four-protein entity. Instead, Axin directs assembly of destruction complex proteins into what the field originally described as 'puncta.' These are now recognized as examples of supermolecular, nonmembrane bound cellular compartments, referred to as biomolecular condensates. Condensate formation is driven by Axin polymerization via its DIX domain, by APC function, and by other multivalent interactions (Schaefer, 2020).
Ubiquitination by E3 ubiquitin ligases is a key mechanism for regulating protein stability. Once the destruction complex templates βcat phosphorylation, the most N-terminal phosphorylated serine forms part of the core of a recognition motif for a Skp-Cullin-F-box (SCF)-class E3 ubiquitin ligase. This E3 ligase ubiquitinates βcat for proteasomal destruction. SCF-class E3 ligases include Cullin1 (Cul1), Skp1, F-box proteins, and Ring box (RBX) subunits, which work together to bind substrates and attach multiple ubiquitin moieties. Cul1 is the scaffold of the complex, at one end binding Rbx1 and its associated E2-Ubiquitin proteins and at the other end binding Skp1. Skp1(SkpA in Drosophila) links Cul1 and the F-box protein-in this case, βTrCP. βTrCP (Slimb in Drosophila) contains the substrate recognition domain of the E3 ligase. The βcat recognition site spans the WD40 repeats on the C-terminal end of βTrCP. This domain forms a propeller structure with a pocket that binds only to phosphorylated proteins. βTrCP can bind multiple phospho-proteins and thus regulate diverse cell signaling pathways (e.g., NFκB and Hedgehog signaling). After βTrCP-βcat binding, βcat is poly-ubiquitinated and can now be recognized by the proteasome. While down-regulation of βcat levels via protein degradation is a key function of the destruction complex, understanding of how βcat is transferred from the complex to the SCF E3 ligase is a key unanswered question (Schaefer, 2020).
Two classes of models seem plausible. In the first class of models, the E3 ligase is a physical entity separate from the destruction complex-this would fit with the many roles for the SCFSlimb E3 ligase, which binds and ubiquitinates diverse phospho-proteins, ranging from the Hedgehog effector Ci/Gli to the centrosome assembly regulator PLK4 . However, given the abundance of cellular phosphatases, this model has a potential major problem. Phosphorylated βcat released free from the destruction complex into the cytoplasm would likely be rapidly dephosphorylated, preventing its recognition by the E3 ligase. Consistent with this, earlier work revealed that APC helps prevent βcat dephosphorylation within the destruction complex. In a second class of models, the SCFSlimb E3 ligase might directly dock on or even become part of the destruction complex, either by direct interaction with destruction complex proteins or by using phosphorylated βcat as a bridge. In this model, once βcat is phosphorylated it could be directly transferred to the E3 ligase, thus preventing dephosphorylation of βcat by cellular phosphatases during transit. Immunoprecipitation (IP) experiments in animals and cell culture revealed that βTrCP can co-IP with Axin, APC, βcat, and GSK3, and that Wnt signals reduce Axin:βTrCP co-IP. However, these studies did not examine whether βTrCP or other components of the E3 are recruited to the destruction complex, leaving both models an option, especially if βTrCP acts as a shuttling protein between complexes (Schaefer, 2020).
A second set of outstanding questions concerns the mechanisms by which Wnt signaling down-regulates βcat destruction. Wnt signaling is initiated when Wnt ligands interact with complex multiprotein receptors, comprised of Frizzled family members plus LRP5/6. This receptor complex recruits the destruction complex to the plasma membrane via interaction of Axin with the phosphorylated LRP5/6 tail and with the Wnt effector Dishevelled (Dvl in mammals/Dsh in Drosophila). This leads to down-regulation of the destruction complex, reducing the rate of βcat destruction. Current data suggest destruction complex down-regulation occurs via multiple mechanisms, some rapid and others initiated more slowly. These include direct inhibition of GSK3 by the phosphorylated LRP5/6 tail, inhibition of Axin homo-polymerization by competition with hetero-polymerization with Dsh, competition between Dsh and APC2 for access to Axin, targeting Axin for proteolytic destruction, and blockade of βcat transfer to the E3 ligase. Recent work has explored the role of Dsh. Overall protein levels of Axin, APC2 and Dsh in Drosophila embryos experiencing active Wnt signaling are within a fewfold of one another, suggesting that competition is a plausible mechanism for destruction complex down-regulation (Schaefer, 2018). The competition model is also consistent with the effects of elevating Axin levels, which makes the destruction complex more resistant to turn-down. However, somewhat surprisingly, elevating Dsh levels had only modest consequences on cell fate choices, and Dsh only assembled into Axin puncta in cells receiving Wingless signals, suggesting that Dsh may need to be 'activated' by Wnt signals in order to effectively compete with APC for Axin and thus mediate destruction complex down-regulation. Candidate phosphorylation sites and kinases potentially involved in this activation have been identified. Intriguingly, when Axin, APC, and Dvl were expressed in mammalian cells, potential competition between APC and Dvl for interaction with Axin was revealed. This study examined in vivo the effects of simultaneously altering levels of Dsh and Axin, testing aspects of the competition model, and combined this with analysis of how Dsh and Axin affect one another's assembly into puncta in a simple cell culture model, using structured illumination superresolution microscopy (Schaefer, 2020).
Wnt signaling plays key roles in development and disease by regulating the stability of its effector βcat. In the absence of Wnt signals, βcat is phosphorylated by the Wnt-regulatory destruction complex, ubiquitinated by an SCF-class E3 ubiquitin ligase, and destroyed by the proteasome. Binding of Wnt ligands to their Frizzled/LRP receptors stabilizes βcat via the cytoplasmic effector Dsh. This study explored two important questions in the field: Is there a direct transfer of βcat from the destruction complex to the E3 ligase, and how does Dsh interaction with the destruction complex protein Axin regulate destruction complex function (Schaefer, 2020)?
Regulating the stability of βcat is the key step in Wnt signaling. The SCFSlimb E3 ligase was first identified as the relevant E3 regulating βcat levels in 1998. It specifically recognizes βcat after its sequential phosphorylation by CK1 and GSK3, and the most N-terminal phosphoserine is a key part of the binding site for the F-box protein Slimb/βTrCP. Phosphatase activity in the cytoplasm can rapidly dephosphorylate this residue, raising the question of how βcat is transferred to the E3 ligase without being dephosphorylated. Earlier work offered two clues. First, βTrCP can co-IP with Axin and APC, suggesting it may associate, at least transiently, with the destruction complex, providing a potential transfer mechanism. Consistent with this, stabilizing Axin using Tankyrase inhibitors led to colocalization of βTrCP and Tankyrase with the destruction complexes that assemble in response. However, it was not clear if this occurred by a direct interaction of βTrCP with destruction complex components via bridging by phosphorylated βcat or occurred because other components of the SCFSlimb E3 ligase were recruited more directly, with βTrCP recruited as a secondary consequence. A second clue emerged from analyses revealing that one role for APC is to prevent dephosphorylation of βcat while it is in the destruction complex, protecting the βTrCP binding site (Schaefer, 2020).
Two plausible models were suggested by these data. In the first, the entire SCFSlimb E3 ligase might be recruited to the destruction complex, allowing direct transfer of phosphorylated βcat between the two complexes. In a second model, βTrCP could serve as a shuttle, binding to phosphorylated βcat at the destruction complex and shuttling it to a place where the E3 assembled and ubiquitinated βcat (Schaefer, 2020).
This study explored interactions of the E3 ligase with the destruction complex using cell biological assays in SW480 cells. Ready recruitment was observed of the βTrCP homologue Slimb to destruction complex puncta by Axin, butrecruitment by APC2 was not observed, consistent with earlier assays by co-IP. Slimb recruitment did not require the βcat-binding site of Axin, making it less likely that recruitment occurs solely via bridging by βcat. However, it was enhanced by the RGS domain of Axin-future work to assess whether this involves a direct interaction or whether an indirect one is warranted. There are conserved residues in the RGS domain that are not necessary for the APC-Axin interaction, some of which form a pi helix, and it will be interesting to further explore the function of these residues. Both the region containing the N-terminus plus the F-box of Slimb and that including its WD40 repeats could be separately recruited into Axin puncta, suggesting it may be recruited by multiple interactions-in the case of the WD40 repeats, this could include bridging by phosphorylated βcat. Once again, direct binding assays in vitro would provide further insights, building on earlier assays suggesting a multipartite binding interaction. Superresolution imaging suggests the interaction between Slimb and Axin is intimate, consistent with direct binding. FRAP data, on the other hand, reveal that Slimb can come in and out of the complex, similar to the behavior of Axin and APC (Schaefer, 2020).
In contrast to the strong recruitment of Slimb to destruction complex puncta, two other core components of the SCFSlimb E3 ligase, Skp1 and Cul1, were not avidly recruited. The occasional recruitment seen could reflect interactions with endogenous βTrCP in the puncta. Coexpression of SkpA or Cul1 with Slimb slightly enhanced recruitment, but this was still not as robust as the recruitment of Slimb itself. IP/mass spectroscopy data and earlier work are consistent with the presence of all three core SCFSlimb E3 ligase proteins in the destruction complex, but suggest they may be present at lower levels than core destruction complex proteins. One possibility is that Slimb/βTrCP usually acts as a shuttle, but its presence occasionally recruits the other E3 proteins. Another possibility is that the entire SCFSlimb E3 ligase docks on the destruction complex transiently to accept phosphorylated βcat, ubiquitinate it, and then transfer it to the proteasome. Consistent with this possibility, inhibiting Tankyrase not only stimulates association of βTrCP with Axin but also leads to recruitment of the proteasome itself to the destruction complex-intriguingly, proteasome inhibition reduces destruction complex assembly, though this effect appears to be indirect due to effects on Axin2 levels. Further analyses will be needed to discriminate between these possibilities (Schaefer, 2020).
Additional work is also needed to explore how βcat transfer to the E3 ligase is regulated. Direct targeting of βcat to the E3, by fusing the F-box of Slimb with the βcat-binding sites of Tcf4 and E-cadherin, is sufficient to stimulate βcat destruction, independent of the destruction complex, but in vivo the destruction complex plays a critical role. Several pieces of data are consistent with the idea that transfer of βcat to the E3 ligase is the step regulated by Wnt signaling, rather than phosphorylation of βcat, with APC having an important role. Further exploration of this process will be welcome (Schaefer, 2020).
It has been clear for more than two decades that Dsh is a key effector of Wnt signaling. However, its precise mechanisms of action are complex and not fully understood. Current data suggest that Dsh is recruited to activated Frizzled receptors via its DEP domain. Dsh then helps ensure the Wnt-dependent phosphorylation of LRP5/6, leading to receptor clustering, facilitating Axin recruitment, and thus inhibiting GSK3. Dsh homo-polymerization, via its DIX domain, and hetero-polymerization with Axin, along with DEP-domain dependent Dsh cross-linking, are then thought to lead to down-regulation of the destruction complex and thus stabilization of βcat (Schaefer, 2020).
Intriguingly, in Drosophila embryos Dsh, Axin, and APC are present at levels within a few-fold of one another. Many current models suggest that relative ratios of these three proteins are critical to the signaling outcome, with APC and Dsh competing to activate or inhibit Axin, respectively. Consistent with this, substantially elevating Axin levels in vivo, using Drosophila embryos as a model, renders the destruction complex immune to down-regulation by Wnt signaling. Subsequent work revealed that the precise levels of Axin are critical-elevating Axin levels by two- to fourfold has little effect, while elevation by ninefold is sufficient to constitutively inactivate Wnt signaling. One might then predict that elevating Dsh levels would have the opposite effect, sequestering Axin and thus stabilizing βcat and activating Wnt signaling. While very high levels of Dsh overexpression can have this effect, it was previously surprising to learn that sevenfold elevation of Dsh levels only had a subtle effect on Wnt signaling and thus had little effect on embryonic viability (Schaefer, 2018). The data further suggested that Dsh is only recruited into Axin puncta in cells that received Wg signal, in which puncta are recruited to the plasma membrane, even though seemingly similar levels of Dsh were present in Wnt-OFF cells (Schaefer, 2018). This opened the possibility that a Wnt-stimulated activation event, such as Dsh phosphorylation, might be required to facilitate Dsh interaction with Axin and thus Axin inactivation. In this scenario, elevating Dsh levels in cells without this activation event, for example, in Wnt-OFF cells, would not alter signaling output (Schaefer, 2020).
The simplest versions of the antagonism model, involving competition between formation of Axin/APC versus Axin/Dsh complexes, would also suggest that elevating Dsh levels should alleviate effects of elevating Axin. This was tested directly, expressing high levels of Dsh maternally and lower levels of Axin zygotically. It was anticipated that elevating Dsh levels would blunt the effects of elevating levels of Axin. Instead, a substantial surprise was in store: elevating levels of Dsh enhanced the ability of Axin to resist turndown by Wnt signaling, thus leading to global activation of the destruction complex and inactivation of Wnt signaling. This was true whether effects were assessed on cell fate choice, Arm levels, or expression of a Wnt-target gene. Intriguingly, the data were also consistent with the possibility that elevating Dsh levels may alter Axin:APC interactions in Wnt-ON cells-this might provide a clue to an underlying mechanism (Schaefer, 2020).
What could explain this paradoxical result? The current data do not provide a definitive answer but do open some intriguing possibilities and new questions. In the view of the authors, part of the explanation will be that Wg-dependent 'activation' of Dsh is required for it to interact with and thus down-regulate Axin. Consistent with this, Dsh phosphorylation can regulate its ability to homopolymerize. By elevating Dsh levels, the capacity of this activation system might have been exceeded. High levels of 'nonactivated' Dsh, while unable to interact with Axin, might still interact with other key proteins involved in destruction complex down-regulation, sequestering them in nonproductive complexes. For example, Dsh can bind CK1, which has complex roles in Wnt regulation With key proteins sequestered, the system might become less able to inactivate the slightly elevated levels of Axin present, thus leading to constitutive activity of the destruction complex. In this speculative scenario, it is not the relative levels of Axin and Dsh that are key but the relative levels of Axin and 'active Dsh' (Schaefer, 2020).
The results of our SIM experiments may also provide insights. The ability of Axin and Dsh to both homo- and hetero-polymerize means free monomers must make a choice. It is likely this is a regulated choice, though the mechanism of regulation remains unclear. The experiments with SW480 cells, while overly simple, may provide an illustration of how the homo-/hetero-polymerization balance can shift. In cells in which both Axin and Dsh were expressed at relatively low levels, puncta contained both proteins, and internal structure was consistent with some level of hetero-polymerization. In contrast, when levels of Dsh were significantly higher, Axin and Dsh tended to segregate into separate, adjoining puncta, suggesting the balance was shifted to homo-polymerization, though the polymers retained the ability to dock on one another. If similar events occur on elevating Dsh expression in Drosophila embryos, segregation could allow Axin to remain in functional destruction complexes, even in Wnt-ON cells, while Dsh localized to separate puncta sequestered other Wnt-regulating proteins, potentially explaining how elevating Dsh expression could paradoxically down-regulate Wnt signaling. Elevating Dsh levels may also lead it to preferentially associate with itself, as was suggested by the SIM data in SW480 cells-this could recruit endogenous Dsh away from its normal localization with the destruction complex, thus preventing it from participating in inactivating Axin. Defining the mechanisms that determine the relevant affinities of each protein for itself versus for its partner will be informative. Intriguingly, a similar docking was observed rather than coassembly behavior when the puncta formed by Axin and those formed by the Arm repeat domain of APC2 were imaged-this may be another example where relative affinities of proteins for themselves versus their binding partners differ (Schaefer, 2020).
Very recent work provides important new insights in this regard. Yamanishi (2019) determined the structure of the heterodimer of the DIX domains of Dsh and Axin and also measured their relative affinities for one another. Another study used cryo-electron microscopy to solve the structure of Dsh filaments and also measured affinities of DIX domains of Dsh and Axin. The results of the second group contrast, with the first group suggesting Dsh homodimerization is an order of magnitude more favorable than Axin homodimerization, while heterodimerization is intermediate in affinity, and the other suggesting Axin homodimerization is most favorable. Resolution of this will be important, as how Dsh acts to turn down destruction complex activity by heterodimerization is assessed. It also is interesting given in vivo observations that APC may help stabilize Axin homo-polymerization. These data also may help explain the results in SIM, where segregation of Dsh and Axin is favored in some circumstances. Defining the in vivo regulatory mechanisms that modulate homo- and heteropolymerization will be an important goal. Together the results leave more questions than answers but suggest that there are important features of Wnt signaling in vivo yet to be uncovered. Further cell biological and biochemical experiments in vivo, combined with new mathematical models of the suspected competition, will be extremely useful (Schaefer, 2020).
Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS malformations derive from the deregulation of developmental pathways, inclusive of the canonical WNT pathway. This study has evaluated MRI anomalies and behavioral and neurological clinical manifestations in CdLS patients. Importantly, a significant association was observed between behavioral disturbance and structural abnormalities in brain structures of hindbrain embryonic origin. Considering the cumulative evidence on the cohesin-WNT-hindbrain shaping cascade, possible ameliorative effects of chemical activation of the canonical WNT pathway with lithium chloride was explored in different models: (I) Drosophila melanogaster CdLS model showing a significant rescue of mushroom bodies morphology in the adult flies; (II) mouse neural stem cells restoring physiological levels in proliferation rate and differentiation capabilities toward the neuronal lineage; (III) lymphoblastoid cell lines from CdLS patients and healthy donors restoring cellular proliferation rate and inducing the expression of CyclinD1. This work supports a role for WNT-pathway regulation of CdLS brain and behavioral abnormalities and a consistent phenotype rescue by lithium in experimental models (Grazioli, 2021).
Wnt signaling is one of the major signaling pathways that regulate cell differentiation, tissue patterning and stem cell homeostasis and its dysfunction causes many human diseases, such as cancer. It is of tremendous interests to understand how Wnt signaling is regulated in a precise manner both temporally and spatially. Naked cuticle (Nkd) acts as a negative-feedback inhibitor for Wingless (Wg, a fly Wnt) signaling in Drosophila embryonic development. However, the role of Nkd remains controversial in later fly development, particularly on the canonical Wg pathway. This study shows that nkd is essential for wing pattern formation, such that both gain and loss of nkd result in the disruption of Wg target expression in larvae stage and abnormal adult wing morphologies. Furthermore, it was demonstrated that a thirty amino acid fragment in Nkd, identified previously in Wharton lab, is critical for the canonical Wg signaling, but is dispensable for Wg/planar cell polarity pathway. Putting aside the pleiotropic nature of nkd function, i.e. its role in the Decapentaplegic signaling, it is concluded that Nkd universally inhibits the canonical Wg pathway across a life span of Drosophila development (Wang, 2021).
Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, this study used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L (2) mutant and GMR-hid, GMR-GAL4 eye. L (2) exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L (2) and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. These data highlight that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability (Mehta, 2021).
Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. This study identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. It is proposed that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development (Kim, 2021).
Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, this study analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. A dominant loss of wing margin bristles and Senseless expression was observed in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, this study showa that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. These data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. These findings represent a novel regulatory role for Arf6 in Wingless signalling (Marcetteau, 2021).
Patients with Alzheimer's disease suffer from a decrease in brain mass and a prevalence of amyloid-β plaques. These plaques are thought to play a role in disease progression, but their exact role is not entirely established. This study developed an optogenetic model to induce amyloid-β intracellular oligomerization to model distinct disease etiologies. This study examined the effect of Wnt signaling on amyloid in an optogenetic, Drosophila gut stem cell model. It was observed that Wnt activation rescues the detrimental effects of amyloid expression and oligomerization. The gene expression changes downstream of Wnt that contribute to this rescue was analyzed, and changes were found in aging related genes, protein misfolding, metabolism, and inflammation. It is proposed that Wnt expression reduces inflammation through repression of Toll activating factors. It was confirmed that chronic Toll activation reduces lifespan, but a decrease in the upstream activator Persephone extends it. It is proposed that the protective effect observed for lithium treatment functions, at least in part, through Wnt activation and the inhibition of inflammation (Kaur, 2022).
Across the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod. This study identified the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. This study shows radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exdv, Meis/hthv, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. This study assessed the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and found the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss. This regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, it is suggested that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures (Neal, 2022).
Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. This study shows that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. These results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, it was demonstrated the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species (Chen, 2022).
The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/β-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, this study examined the cellular determinants of ISC proliferation, harnessing Kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation and kramer regulates Wnt signaling in this context by antagonizing Kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes Kramer as a physiological regulator of Wnt/β-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling (Sun, 2023).
The adult Drosophila melanogaster intestine is a powerful model to study stem cell proliferation.1-5 The fly gut has many important physiological functions, most notably nutrient absorption, acting as a physical barrier and providing immunity to enteric pathogens and chemical insults.1,2 A conserved hallmark of the gut is the dynamic nature of its architecture.3-5 The Drosophila midgut is the largest and central portion of the intestines, and it is analogous to the mammalian small intestine in function and, to some extent, cellular architecture and composition (Sun, 2023).
The signature feature of the midgut is its epithelium, a single cell layer acting as a barrier to separate the lumen from internal tissues. In Drosophila, the midgut epithelium comprises four principal cell types. Intestinal stem cells (ISCs) can self-renew and also give rise to progenitor cells termed enteroblasts (EBs), which in turn can fully differentiate into enterocytes (ECs) that comprise the bulk of the epithelium. The specification of the fourth cell type, enteroendocrine cells (EEs), has not been fully elucidated, despite the importance of these cells in mediating important paracrine signaling events. EBs have been proposed to differentiate into EEs; however, recent studies demonstrated that ISCs, when Prospero-positive, divide into a distinct progenitor type termed pre-EEs, which subsequently differentiate into EEs. Disruption of ISC function can lead to either excessive proliferation or precocious differentiation, often resulting in disease. Therefore, a detailed understanding of the pathways and mechanisms regulating ISC proliferation is an important long-term goal with therapeutic implications (Sun, 2023 and references therein).
Numerous studies have shown that Wnt/β-catenin signaling, a morphogen signaling pathway that is highly conserved in animals, promotes ISC proliferation and differentiation under both physiological conditions and upon challenges such as enteric infection or chemical insults, both of which can damage the gut epithelium. More broadly, Wnt/β-catenin signaling, also known as canonical Wnt signaling, controls diverse cellular processes during animal development and homeostasis, including stem cell maintenance, cell fate specification, neural patterning, spindle orientation, cell migration, cell polarity, and gap junction communication. Dysregulation of canonical Wnt signaling caused by mutations of core components of this pathway is frequently linked to birth defects and many types of cancer. During tissue development and homeostasis, canonical Wnt signaling is thought to be the main pathway for regulating ISC proliferation and self-renewal, which drives massive renewal processes of intestinal epithelial cells (Sun, 2023).
Despite the fundamental importance of Wnt signaling in regulating ISC proliferation and subsequent tissue renewal in the gut epithelium, understanding of how Wnt signaling in different cell types within the gut contributes to these effects on ISCs is still rudimentary. Furthermore, recent studies have elucidated roles for β-catenin-independent, or non-canonical Wnt signaling pathways, which share some upstream components in the Wnt-receiving cell but do not activate β-catenin-dependent gene expression, in regulating ISC proliferation in the Drosophila midgut. Thus, knowledge of which cell types exhibit canonical and non-canonical Wnt signaling that collectively contribute to ISC proliferation and thus tissue maintenance in the midgut are major fundamental and unanswered questions (Sun, 2023).
A key shared player in all Wnt signaling pathways is Dishevelled (Dsh/DVL), which is recruited to the plasma membrane upon activation of Wnt receptors and co-receptors from the Frizzled and LRP families. Such recruitment catalyzes the disassembly of a multiprotein complex that facilitates proteasomal degradation of β-catenin, enabling its accumulation and subsequent translocation to the nucleus to activate TCF/LEF-dependent gene expression in the canonical pathway. Dsh recruitment to the plasma membrane also activates planar cell polarity and other non-canonical Wnt pathways, including the Wnt/Ca2+ pathway, by activation of Frizzled receptors. Thus, regulation of Dsh levels, which occurs via the ubiquitin-proteasome system and involves the action of several distinct E3 ubiquitin ligases, is a key control point in all Wnt signaling pathways (Sun, 2023).
Notably, a mammalian multi-subunit phosphoinositide-binding protein, pleckstrin homology domain-containing family A number 4 (PLEKHA4), promotes both Wnt/β-catenin and non-canonical Wnt signaling in human cell lines by antagonizing DVL polyubiquitination by the Cullin-3 (CUL3)-Kelch-like protein 12 (KLHL12) E3 ubiquitin ligase. This study found as well that Wnt/β-catenin signaling and subsequent cell proliferation in mouse models of melanoma was dependent upon PLEKHA4 expression (Shah, 2021). In Drosophila, knockout of the closest fly ortholog of PLEKHA4, kramer (kmr), impairs planar cell polarity in the adult wing, larval wing imaginal disc, and pupal wing disc epithelium. The absence of any discernable defects in canonical Wnt signaling in kmr knockout flies led the authors to question whether kmr indeed controlled Wnt/β-catenin signaling in this organism. It is proposed that the extent to which PLEKHA4/kmr loss affected canonical or non-canonical Wnt pathways might depend on cellular and tissue contexts, where expression of other factors downstream of DVL/Dsh might govern how tuning of DVL/Dsh levels would differentially affect outcomes from these pathways (Sun, 2023).
To test this prediction, this study investigated the role of kmr in controlling ISC proliferation in the Drosophila midgut, a physiological process dependent upon canonical Wnt signaling and recently linked to non-canonical Wnt signaling pathways as well. The experimental model used in this study involved challenge of adult flies with E. carotovora carotovora 15 (Ecc15), a gram-negative bacterium that produces non-lethal infection, to damage the midgut epithelium and induce repair pathways dependent upon ISC proliferation. Global knockout and cell type-specific knockdown of kmr was performed and effects on tissue pathophysiology were compared to those induced by knockdown of other established components of Wnt signaling pathways. As such, this study accomplished several goals. First, roles for kmr were established in controlling canonical Wnt signaling in Drosophila. Second, kmr was used as a tool to elucidate roles for canonical and non-canonical Wnt signaling within different cell types in the Drosophila midgut. These studies reveal not only that kmr-dependent canonical Wnt signaling controls ISC proliferation in the midgut but that such signaling occurs in several cell types, including Prospero-positive cells, suggesting that EEs, which these studies support derive from pre-EE progenitors, may play an unexpectedly important role in these processes (Sun, 2023).
Proliferation and differentiation of intestinal stem cells in the adult Drosophila midgut is essential to maintain the balance of tissue homeostasis and prevent excessive proliferation in this tissue. Wnt/β-catenin signaling plays central roles in tissue maintenance during development, including in the midgut. However, how canonical Wnt signaling is activated within intestinal stem cell progenitors and by fully differentiated progeny cells is still not well understood. This study discovered a new player, kramer (kmr), that regulates canonical Wnt signaling in the Drosophila midgut using a challenge with the non-lethal pathogen Erwinia carotovora carotovora (Ecc15) to induce massive ISC proliferation required to rebuild the damaged gut epithelium (Sun, 2023).
Using this system, kmr was established as a positive regulator of ISC proliferation and Wnt/β-catenin signaling. Inducible, cell type-specific kmr knockdown was used as a tool to interrogate the requirements for Wnt signaling within each cell type in the gut for controlling ISC proliferation. In addition to known effects of Wnt signaling within intestinal stem cells (ISCs), their immediate downstream progenitors termed enteroblasts (EBs), and fully differentiated enterocytes (ECs) in governing this process, this study unexpectedly points to roles for Wnt signaling within enteroendocrine cells (EEs) as a mechanism controlling stem cell proliferation in the Drosophila midgut (Sun, 2023).
It was first shown that interruption of kmr function decreases the expression of a canonical Wnt signaling target gene, fz3, in the posterior midgut, consistent with studies demonstrating fz3RFP expression in both ISCs and ECs showing that knockout of Wnt signaling components led to loss of fz3 expression in midgut region R5. Though ECs are the primary cell type in which Wnt signaling is activated, studies using kmr knockdown in multiple cell types suggest that Wnt signaling is also active in ISCs/EBs and EEs in posterior end of the midgut. These studies also revealed that loss of kmr in multiple cell types results in fewer EE cells and decreased ISC proliferation in the posterior midgut, indicating a role for kmr in EE differentiation (Sun, 2023).
Further, kmr knockdown in all cell types caused a decrease in proliferating, phospho-H3 positive (pH3+) cells, though staining with Armadillo/Prospero antibodies, which enables identification of ISCs/EBs and EEs, suggests that kmr knockdown in EE cells specifically downregulates stem cell proliferation. This distinction is important because pH3+ cells include all dividing progenitor cells, including ISCs/EBs and pre-EEs, and the data showed that kmr knockdown in all cell types significantly decreased dividing stem cells. Notably, kmr knockdown in ISCs/EBs and in ECs did not result in a defect in number of Arm+/Pro- cells (i.e., ISCs/EBs); However, kmr knockdown in Pro+ cells led to a reduction in number of ISCs/EBs, suggesting that kmr expression in EE cells regulates the proliferation of neighboring ISCs in a non-autonomous manner. These data also provide support to a model wherein EEs derive not from EBs but instead from a distinct set of progenitors termed pre-EEs (Sun, 2023).
This study also sheds light on the mechanisms by which kmr expression in distinct midgut cell types might regulate the physiological response to Ecc15 infection. Prior work on the mammalian ortholog of kmr, PLEKHA4, revealed that it promotes Wnt signaling pathways via physical interaction with KLHL12, a CUL3 E3 ubiquitin ligase substrate-specific adaptor and negative regulator of DVL. By binding to KLHL12 and sequestering it in plasma membrane-associated clusters, PLEKHA4 prevents DVL polyubiquitination by CUL3-KLHL12, ultimately causing elevated DVL levels and enhanced Wnt signaling in Wnt-receiving cells (Sun, 2023).
Because of the pleiotropic roles for DVL in both canonical Wnt/β-catenin and non-canonical β-catenin-independent pathways, this study found that PLEKHA4 knockdown affected both of these pathways. Previous studies on kmr in Drosophila, focusing on hair patterning, identified defects in planar cell polarity (PCP), a Frizzled- and Dsh-dependent pathway in flies. However, the present study identifies for the first time a role for kmr in promoting canonical Wnt/β-catenin signaling in vivo. By analyzing knockdown of the two fly KLHL12 orthologs, kelch and diablo, this study found that kmr acts in opposition to kelch, much as PLEKHA4 does with KLHL12 in mammalian cells, supporting the established mechanism of action. Finally, Wnt/β-catenin and PCP signaling were independently blocked and ISC proliferation following Ecc15 challenge was assessed; only blockade of Wnt/β-catenin signaling was found to exactly phenocopy loss of kmr (Sun, 2023).
In conclusion, this study reveals that the Dsh regulator kmr is a new, important regulator of Wnt/β-catenin signaling and ISC proliferation in vivo. As well, kmr was harnessed as a tool to systematically investigate the role of Wnt signaling in each of the major cell types in the Drosophila midgut in controlling ISC proliferation and differentiation during epithelial repair following challenge with an enteric pathogen. These studies suggest that Wnt signaling within enteroendocrine cells can control this process, adding a new layer of regulation to this important physiological process. Future studies will be necessary to identify downstream mechanisms by which EEs may non-autonomously regulate ISC proliferation in the midgut, as well as the existence of similar pathways in mammalian systems (Sun, 2023).
Akai, N., Igaki, T. and Ohsawa, S. (2018). Wingless signaling regulates winner/loser status in Minute cell competition. Genes Cells 23(3): 234-240. PubMed ID: 29431244
Babcock, D. T., Shi, S., Jo, J., Shaw, M., Gutstein, H. B. and Galko, M. J. (2011). Hedgehog signaling regulates nociceptive sensitization. Curr Biol 21: 1525-1533. PubMed ID: 21906949
Binh, T. D., Nguyen, Y. D. H., Pham, T. L. A., Komori, K., Nguyen, T. Q. C., Taninaka, M. and Kamei, K. (2022). Dysfunction of lipid storage droplet-2 suppresses endoreplication and induces JNK pathway-mediated apoptotic cell death in Drosophila salivary glands. Sci Rep 12(1): 4302. PubMed ID: 35277579
Chen, T. A., Lin, K. Y., Yang, S. M., Tseng, C. Y., Wang, Y. T., Lin, C. H., Luo, L., Cai, Y. and Hsu, H. J. (2022). Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation. Front Cell Dev Biol 10: 877047. PubMed ID: 35517512
Chen, Y., Liu, T., Shen, J. and Zhang, J. (2021). Phenotypical and genetical characterization of the Mad(1-2) allele during Drosophila wing development. Cells Dev 169: 203761. PubMed ID: 34875394
Da Ros, V. G., Gutierrez-Perez, I., Ferres-Marco, D. and Dominguez, M. (2013). Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis. PLoS Biol 11(5): e1001554. PubMed ID: 23667323
Grazioli, P., Parodi, C., Mariani, M., Bottai, D., Di Fede, E., Zulueta, A., Avagliano, L., Cereda, A., Tenconi, R., Wierzba, J., Adami, R., Iascone, M., Ajmone, P. F., Vaccari, T., Gervasini, C., Selicorni, A. and Massa, V. (2021). Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome. Cell Death Discov 7(1): 34. PubMed ID: 33597506
Guleria, V. S., Parit, R., Quadri, N., Das, R. and Upadhyai, P. (2022). The intraflagellar transport protein IFT52 associated with short-rib thoracic dysplasia is essential for ciliary function in osteogenic differentiation in vitro and for sensory perception in Drosophila. Exp Cell Res 418(2): 113273. PubMed ID: 35839863
Happ, J. T., Arveseth, C. D., Bruystens, J., Bertinetti, D., Nelson, I. B., Olivieri, C., Zhang, J., Hedeen, D. S., Zhu, J. F., Capener, J. L., Brockel, J. W., Vu, L., King, C. C., Ruiz-Perez, V. L., Ge, X., Veglia, G., Herberg, F. W., Taylor, S. S. and Myers, B. R. (2022). A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. Nat Struct Mol Biol 29(10): 990-999. PubMed ID: 36202993
He, M., Subramanian, R., Bangs, F., Omelchenko, T., Liem, K. F., Jr., Kapoor, T. M. and Anderson, K. V. (2014). The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment. Nat Cell Biol 16: 663-672. PubMed ID: 24952464
He, T., Fan, Y., Wang, Y., Liu, M. and Zhu, A. J. (2022). Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila. Front Cell Dev Biol 10: 866491. PubMed ID: 35573695
Hu, D. J., Yun, J., Elstrott, J. and Jasper, H. (2021). Non-canonical Wnt signaling promotes directed migration of intestinal stem cells to sites of injury. Nat Commun 12(1): 7150. PubMed ID: 34887411
Kaur, P., Saunders, T. E. and Tolwinski, N. S. (2017). Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis. Sci Rep 7(1): 16636. PubMed ID: 29192250
Kaur, P., Chua, E. H. Z., Lim, W. K., Liu, J., Harmston, N. and Tolwinski, N. S. (2022). Wnt Signaling Rescues Amyloid Beta-Induced Gut Stem Cell Loss. Cells 11(2). PubMed ID: 35053396
Kim, J. Y., Tsogtbaatar, O. and Cho, K. O. (2021). Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster. J Dev Biol 9(4). PubMed ID: 34698231
Kizhedathu, A., Kunnappallil, R. S., Bagul, A. V., Verma, P. and Guha, A. (2020). Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts. Elife 9. PubMed ID: 32876044
Kuzhandaivel, A., Schultz, S. W., Alkhori, L. and Alenius, M. (2014). Cilia-mediated hedgehog signaling in Drosophila. Cell Rep 7: 672-680. PubMed ID: 24768000
Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments, and pattern: lessons from drosophila? Cell 85: 951-961. PubMed ID: 8674123
Makamte, S., Thureau, A., Jabrani, A., Paquelin, A., Plessis, A., Sanial, M., Rudenko, O., Oteri, F., Baaden, M. and Biou, V. (2022). A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study. J Struct Biol 214(2): 107853. PubMed ID: 35364288
Marcetteau, J., Matusek, T., Luton, F. and Therond, P. P. (2021). Arf6 is necessary for senseless expression in response to Wingless signalling during Drosophila wing development. Biol Open. PubMed ID: 34779478
Martins, T., Meghini, F., Florio, F. and Kimata, Y. (2017). The APC/C coordinates retinal differentiation with G1 arrest through the Nek2-dependent modulation of Wingless signaling. Dev Cell 40(1): 67-80. PubMed ID: 28041905
Mehta, A. S., Deshpande, P., Chimata, A. V., Tsonis, P. A. and Singh, A. (2021). Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye. iScience 24(10): 103166. PubMed ID: 34746690
Mo, L., Li, R., He, C., Chen, Q., Xu, C., Shen, L., Chen, K. and Wu, Y. (2022). Hedgehog pathway is negatively regulated during the development of Drosophila melanogaster PheRS-m (Drosophila homologs gene of human FARS2) mutants. Hum Cell. PubMed ID: 36205831
Moore, S. L., Adamini, F. C., Coopes, E. S., Godoy, D., Northington, S. J., Stewart, J. M., Tillett, R. L., Bieser, K. L. and Kagey, J. D. (2022). Patched and Costal-2 mutations lead to differences in tissue overgrowth autonomy. Fly (Austin) 16(1): 176-189. PubMed ID: 35468034
Neal, S., McCulloch, K. J., Napoli, F. R., Daly, C. M., Coleman, J. H. and Koenig, K. M. (2022). Co-option of the limb patterning program in cephalopod eye development. BMC Biol 20(1): 1. PubMed ID: 34983491
Nellas, I., Iyer, K. V., Iglesias-Artola, J. M., Pippel, M., Nadler, A., Eaton, S. and Dye, N. A. (2022). Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc. EMBO Rep: e202154025. PubMed ID: 36134875
Reilein, A., Melamed, D., Park, K. S., Berg, A., Cimetta, E., Tandon, N., Vunjak-Novakovic, G., Finkelstein, S. and Kalderon, D. (2017). Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling. Nat Cell Biol 19(5):433-444. PubMed ID: 28414313
Ricolo, D., Butí, E. and Araújo, S.J. (2015). Drosophila melanogaster Hedgehog cooperates with Frazzled to guide axons through a non-canonical signalling pathway. Mech Dev [Epub ahead of print]. PubMed ID: 25936631
Roberto, N., Becam, I., Plessis, A. and Holmgren, R. A. (2022). Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels. Development 149(6). PubMed ID: 35290435
Rudolf, K., Umetsu, D., Aliee, M., Sui, L., Julicher, F. and Dahmann, C. (2015). A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary. Development 142: 3845-3858. PubMed ID: 26577205
Saad, F. and Hipfner, D. R. (2021). Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway. Development. PubMed ID: 33653875
Sanchez, G. M., Alkhori, L., Hatano, E., Schultz, S. W., Kuzhandaivel, A., Jafari, S., Granseth, B. and Alenius, M. (2016). Hedgehog signaling regulates the ciliary transport of odorant receptors in Drosophila. Cell Rep 14: 464-470. PubMed ID: 26774485
Scarpa, E., Finet, C., Blanchard, G. B. and Sanson, B. (2018). Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo. Dev Cell. PubMed ID: 30503752
Schaefer, K. N., Bonello, T. T., Zhang, S., Williams, C. E., Roberts, D. M., McKay, D. J. and Peifer, M. (2018). Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo. PLoS Genet 14(4): e1007339. PubMed ID: 29641560
Schaefer, K. N., Pronobis, M., Williams, C. E., Zhang, S., Bauer, L., Goldfarb, D., Yan, F., Major, M. B. and Peifer, M. (2020). Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex. Mol Biol Cell: mbcE19110647. PubMed ID: 32129710
Suisse, A. and Treisman, J. E. (2019). Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum. Cell Rep 26(2): 322-329. PubMed ID: 30625314
Sun, H., Shah, A. S., Bonfini, A., Buchon, N. S. and Baskin, J. M. (2023). Wnt/β-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation. bioRxiv. PubMed ID: 36865263
Swarup, S., Pradhan-Sundd, T. and Verheyen, E. M. (2015). Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila. Development 142(8): 1502-1515. PubMed ID: 25852200
Tatapudy, S., Peralta, J. and Nystul, T. (2021). Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation. Development 148(22). PubMed ID: 35020878
Takemura, M., Noborn, F., Nilsson, J., Bowden, N., Nakato, E., Baker, S., Su, T. Y., Larson, G. and Nakato, H. (2020). Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila. Mol Biol Cell: mbcE19060327. PubMed ID: 32049582
Thomas, J.T., Eric Dollins, D., Andrykovich, K.R., Chu, T., Stultz, B.G., Hursh, D.A. and Moos, M. (2017). SMOC can act as both an antagonist and an expander of BMP signaling. Elife 6. PubMed ID: 28323621
Urbano, J. M., Naylor, H. W., Scarpa, E., Muresan, L. and Sanson, B. (2018). Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila. Development 145(8). PubMed ID: 29691225
Wang, R., Xie, H., Yang, L., Wang, P., Chen, M. M., Wu, H. Y., Liao, Y. L., Wang, M. Y., Wang, Q., Gong, X. X., Cheng, Q., Cheng, L., Xie, F. Y., Bi, C. L. and Fang, M. (2021). Naked cuticle inhibits wingless signaling in Drosophila wing development. Biochem Biophys Res Commun 576: 1-6. PubMed ID: 34474244
Xun, Q., Bi, C., Cui, X., Wu, H., Wang, M., Liao, Y., Wang, R., Xie, H., Shen, Z. and Fang, M. (2018). MagT1 is essential for Drosophila development through the shaping of Wnt and Dpp signaling pathways. Biochem Biophys Res Commun. PubMed ID: 29959918
Yamanishi, K., Fiedler, M., Terawaki, S. I., Higuchi, Y., Bienz, M. and Shibata, N. (2019). A direct heterotypic interaction between the DIX domains of Dishevelled and Axin mediates signaling to beta-catenin. Sci Signal 12(611). PubMed ID: 31822591
Yang, E., Tacchelly-Benites, O., Wang, Z., Randall, M. P., Tian, A., Benchabane, H., Freemantle, S., Pikielny, C., Tolwinski, N. S., Lee, E. and Ahmed, Y. (2016). Wnt pathway activation by ADP-ribosylation. Nat Commun 7: 11430. PubMed ID: 27138857
Zhou, M., Han, Y., Wang, B., Cho, Y. S. and Jiang, J. (2022). Dose-dependent phosphorylation and activation of Hh pathway transcription factors. Life Sci Alliance 5(11). PubMed ID: 36271509
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