The Interactive Fly

Zygotically transcribed genes

Segment Polarity Genes

  • Segment polarity in Drosophila: Cell-cell signaling and the origins of patterning
  • Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo

    Hedgehog signaling
  • A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary
  • Hedgehog signaling regulates the ciliary transport of odorant receptors in Drosophila
  • Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis
  • Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila
  • Drosophila melanogaster Hedgehog cooperates with Frazzled to guide axons through a non-canonical signalling pathway
  • Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway

    Wingless signaling
  • The APC/C coordinates retinal differentiation with G1 arrest through the Nek2-dependent modulation of Wingless signaling
  • Wingless signaling regulates winner/loser status in Minute cell competition
  • Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling
  • Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis
  • Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila
  • MagT1 is essential for Drosophila development through the shaping of Wnt and Dpp signaling pathways
  • Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum
  • Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila
  • Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts
  • Wnt regulation: is exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex
  • Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome
    Transcription factors of the posterior compartment
    Genes regulated by engrailed and invected
    Secreted proteins acting in segment polarity
    Genes downstream of hedgehog
    Genes functioning in wingless pathway

    Other

    Segmentation in Drosophila: Cell-Cell Signaling and the Origin of Patterning

    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.

    A local difference in Hedgehog signal transduction increases mechanical cell bond tension and biases cell intercalations along the Drosophila anteroposterior compartment boundary

    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 signaling regulates the ciliary transport of odorant receptors in Drosophila

    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).

    The APC/C coordinates retinal differentiation with G1 arrest through the Nek2-dependent modulation of Wingless signaling

    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).

    Wingless signaling regulates winner/loser status in Minute cell competition

    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).

    Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis

    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).

    Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling

    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).

    SMOC can act as both an antagonist and an expander of BMP signaling

    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).

    Dampening the signals transduced through Hedgehog via microRNA miR-7 facilitates Notch-induced tumourigenesis

    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).

    Suppression of epithelial folding at actomyosin-enriched compartment boundaries downstream of Wingless signalling in Drosophila

    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).

    MagT1 is essential for Drosophila development through the shaping of Wnt and Dpp signaling pathways

    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).

    Actomyosin-driven tension at compartmental boundaries orients cell division independently of cell geometry in vivo

    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).

    Reduced SERCA function preferentially affects Wnt signaling by retaining E-Cadherin in the endoplasmic reticulum

    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).

    Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila

    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).

    Drosophila melanogaster Hedgehog cooperates with Frazzled to guide axons through a non-canonical signalling pathway
    This study reports that the morphogen Hedgehog (Hh) is an axonal chemoattractant in the midline of D. melanogaster embryos. Hh is present in the ventral nerve cord during axonal guidance and overexpression of hh in the midline causes ectopic midline crossing of FasII-positive axonal tracts. In addition, Hh influenced axonal guidance via a non-canonical signalling pathway dependent on Ptc. These results reveal that the Hh pathway cooperates with the Netrin/Frazzled pathway to guide axons through the midline in invertebrates (Ricolo, 2015).

    Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway

    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).

    Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila

    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).

    Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

    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 regulation: is exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

    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).

    Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome

    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).

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    Zygotically transcribed genes

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