BIOLOGICAL OVERVIEW

The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors. Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions. At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate. This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship. This proposal has been tested by analyzing interactions between Delta- and Serrate-activated Notch signaling and Wingless signaling during wing development and patterning. Cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra. This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signaling but can also disrupt signaling through another pathway. One possibility is the Wingless signaling pathway, since the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signaling. Furthermore, the outcome of the interactions between Notch and Wingless signaling differs when Wingless signaling is activated by expressing either Wingless itself or an activated form of the Armadillo. For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone. This result suggests that signaling activated by Wingless leads to two effects: a reduction of Notch signaling and an activation of Armadillo (Brennan, 1999a).

Expression of a dominant negative Notch molecule (Extracellular Notch or ECN) throughout the developing wing mimics the effects of loss of Notch function. However, Nintra cannot rescue the cell death caused by overexpressing ECN. Since Nintra provides constitutive signaling for Delta and Serrate during wing development and the effects of ECN are mediated by the sequestration of extracellular molecules that can interact with Notch, this suggests that the ECN molecule is sequestering extracellular molecules other than Delta and Serrate and attenuating signaling through another pathway. One candidate pathway is the Wingless signaling pathway, since the cell death caused by expressing the ECN can be rescued by activating Wingless signaling. Therefore, it is possible that the ECN molecule is sequestering the Wingless protein. The possibility that Wingless can bind the extracellular domain of Notch is supported by the results that are presented here, in particular, by two observations: first, that some of the deleterious effects of ECN can be suppressed by Wingless, but not Wingless signaling in the form of a constitutively active Armadillo molecule; and second, that this interaction requires specific EGF-like repeats of Notch, namely repeats 17-19 and 24-26 but not 10-12. Evidence for a physical interaction between Notch and Wingless has also been provided recently by Wesley (1999) who finds that the Wingless protein is enriched in a biopanning assay designed to identify proteins that interact with the extracellular domain of the Notch protein and that Wingless can be immunoprecipitated with Notch from embryo extracts and cultured cells. These experiments also show that the association of Wingless with Notch requires the integrity of a region of Notch centered around EGF-like repeats 24-26 (Wesley, 1999) which these experiments indicate are essential for the interactions that are described between Wingless and ECN during wing development and patterning (Brennan, 1999a).

High levels of Wingless throughout the developing wing induce widespread development of sensory organs, an observation that correlates with the requirement for Wingless in this process during normal development. However, it is consistently observed that an activated form of Armadillo has a much weaker effect than Wingless on neural development. However, the difference is unlikely to be due to a weak UASarm* insert used in these experiments since in other instances where only a Wingless signal is required, such as the induction of the wing primordium during the early events of wing development, overexpressing Arm* or Wingless has very similar effects. A possible insight into the differences that the expression of Wingless and Arm* has on neurogenesis comes from the experiments where these two proteins are coexpressed with the ECN molecule. In these experiments the phenotypes generated by expressing UASECN with UASwg or UASarm* are very similar; namely, disrupting Notch signaling by expressing the ECN protein makes UASarm* and UASwg functionally equivalent. This suggests that the difference between the phenotypes generated by expressing Wingless and Arm* on their own might arise from the ability of Wingless to inhibit Notch signaling, which Arm* is unable to do; attenuating Notch signaling blocks lateral inhibition, which leads to increased numbers of sense organs. Since Wingless can activate Armadillo, overexpression of Wingless can achieve both effects simultaneously (Brennan, 1999a).

When Arm* is coexpressed with ECN, the dominant negative molecule reduces Notch signaling, providing the function of Wingless that is missing in Arm* and thus making this molecule functionally equivalent to Wingless. These results raise the question of how Wingless signaling inhibits Notch signaling and where in the Wingless signaling pathway the cross-talk between the two pathways occurs. The inability of Arm* to inhibit Notch signaling indicates that the cross-talk must occur upstream of Armadillo. One possibility is that the inhibition occurs through Wingless interacting with the extracellular portion of Notch, preventing the Notch protein from interacting with its ligands. However, it is more likely to occur through the interaction of Dishevelled with the intracellular domain of the Notch protein, which has been shown previously to inhibit Notch signaling (Axelrod, 1996). In keeping with this, it has been found that overexpressing the Dishevelled protein can induce sense organ development as effectively as overexpressing Wingless; this suggests that Dishevelled can also disrupt Notch signaling as effectively as Wingless. Finally, it is possible that the interaction of Notch with both Dishevelled and Wingless is required to inhibit Delta signaling through Notch, since it has been shown previously that the ability to overexpress Dishevelled, which induces supernumerary sense organs, requires Wingless function (Axelrod, 1996). The interference of Wingless signaling with Notch signaling can also provide an explanation for the effects of ectopic expression of Wingless on the patterning of the veins and its sensitivity to the concentration of Delta. Overexpression of Wingless would reduce the availability of Notch for lateral inhibition by causing Dishevelled to sequester Notch into complexes that are unable to transduce the Delta signal. This would reduce the effectiveness of lateral inhibition signaling, an effect which would be exaggerated in situations of limiting signaling, as is observe in Dl heterozygotes or when Wingless is coexpressed with ECN (Brennan, 1999a).

The interaction of Wingless and Notch signaling that has been observed might also be important during normal neural development. Wingless and Delta have opposite effects during neurogenesis; Wingless promotes while Delta suppresses the development of sense organs. Various experiments suggest that during the segregation of neural precursors a reduction of Notch signaling in the precursors themselves is as important as the Delta-mediated activation of Notch signaling in the surrounding cells. It is possible that, like the activation of Notch by Delta, the suppression of Notch signaling is an active process mediated by the interaction of Wingless and Dishevelled with Notch. If this were the case, since both Delta and Wingless have spatially and temporally regulated patterns of gene expression, their interactions with Notch could contribute to the well-documented bias in the appearance of precursors from clusters of cells with neural potential. This competitive interaction could also account for the observed increases in Wingless signaling associated with reductions in Notch signaling during lateral inhibition (Brennan, 1999a).

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, mutants were recovered that disrupt genes encoding serine palmitoyltransferase and Acetyl-CoA Carboxylase (ACC). Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EGFR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. This analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation (Sasamura, 2013).

The importance of lipid metabolism for the formation and maintenance of cell membranes is well established. Both serine palmitoyltransferase (SPT) and acetyl-CoA carboxylase (ACC) are critical enzymes that control different steps of lipid metabolism, and are highly conserved in diverse animal species. Genetic elimination of ACC1 or the SPT subunits Sptlc1 or Sptlc2 cause early embryonic lethality in mice, although the cellular basis for this lethality is unknown. In D. melanogaster, RNA-interfering disruption of ACC activity in the fat body results in reduced triglyceride storage and increased glycogen accumulation, and in oenocytes leads to loss of watertightness of the tracheal spiracles causing fluid entry into the respiratory system. This study demonstrates that D. melanogaster mutants lacking functional SPT or ACC exhibit endosomal trafficking defects, causing Notch, Wingless, EGFR, and Patched to accumulate abnormally in endosomes and lysosomes. These effects are accompanied by significant alterations in Notch and Wingless signaling, as revealed by changes in downstream target gene activation for both pathways. However, the mutants do not fully inactivate these developmental signaling pathways, and instead display phenotypes consistent with more complex, pleiotropic effects on Notch, Wingless, and potentially additional pathways in different tissues. These findings reinforce the importance of lipid metabolism for the maintenance of proper developmental signaling, a concept that has also emerged from studies demonstrating that: D. melanogaster mutants for phosphocholine cytidylyltransferase alter endosomal trafficking and signaling of Notch and EGFR; mutants for alpha-1,4-N-acetylgalactosaminyltransferase-1 affect endocytosis and activity of the Notch ligands Delta and Serrate; mutants for the ceramide synthase gene shlank disrupt Wingless endocytic trafficking and signaling, and mutants for the glycosphingolipid metabolism genes egghead and brainiac modify the extracellular gradient of the EGFR ligand Gurken (Sasamura, 2013).

Most strikingly, lace and ACC mutants also display prominent tissue overgrowth phenotypes. These tissue overgrowth effects are linked to changes in Notch and Wingless signaling outputs, and they involve gamma-secretase, Su(H), and Armadillo activities, suggesting that the overgrowth reflects an interplay of Wingless inactivation and Notch hyperactivation. Consistent with the findings, both Notch and Wingless regulate cell proliferation and imaginal disc size in D. melanogaster. Moreover, several observations indicate that Notch and Wingless are jointly regulated by endocytosis, with opposing effects on their respective downstream pathway activities, a dynamic process that might be especially sensitive to perturbations in membrane lipid constituents. Wingless itself exerts opposing effects on disc size that might depend on the particular developmental stage or disc territory. For example, hyperactivation of Wingless or inactivation of its negative regulators cause overproliferation, but Wingless activity can also constrain wing disc growth. Similar spatiotemporal effects might underlie the variability detected in studies with lace and ACC mutant clones, in which both tissue overgrowth and developmentally arrested discs were observed. Although no obvious changes were detected in downstream signaling for several other cell growth pathways that were examined, the trafficking abnormalities seen for other membrane proteins aside from Notch, Delta, and Wingless, as well as the incomplete suppression of the overgrowth phenotypes by blockage of Notch and Wingless signaling, suggest that other pathways might also be dysregulated in lace and ACC mutants, possibly contributing to the observed tissue overgrowth (Sasamura, 2013).

Wingless is modified by lipid addition, and lipoprotein vesicles have been suggested to control Wingless diffusion. In D. melanogaster embryos, endocytosis of Wingless limits its diffusion and ability to act as a long-range morphogen. Endocytosis can also affect Wingless signaling in receiving cells, where endocytosis both promotes signal downregulation and positively facilitates signaling. The apparently normal diffusion ranges for overaccumulated Wingless in lace and ACC mutant clones, yet reduced downstream target gene expression, is consistent with the idea that SPT and ACC act by promoting endocytic trafficking of Wingless in receiving cells rather than influencing the secretion and/or diffusion of Wingless from signal-sending cells (Sasamura, 2013).

The finding that lace and ACC mutant overgrowth phenotypes are also partially Notch-dependent is reminiscent of similar overproliferation phenotypes seen in certain D. melanogaster endocytic mutants, such as vps25, and tsg101. The overproliferation of disc tissue in these mutants is attributable to Notch hyperactivation, reflecting the fact that non-ligand-bound Notch receptors that are normally targeted for recycling or degradation are instead retained and signal from endosomes. Analogous effects are likely to contribute to the lace and ACC mutant overgrowth, where significant Notch overaccumulation was observed throughout the endosomal-lysosomal routing pathway. Some ectopic Notch signaling might emanate from the lysosomal compartment, which is enlarged and accumulates particularly high levels of Notch in lace and ACC mutant clones. Analysis of D. melanogaster HOPS and AP-3 mutants, which affect protein delivery to lysosomes, has identified a lysosomal pool of Notch that is able to signal in a ligand-independent, gamma-secretase-dependent manner (Sasamura, 2013).

How do SPT and ACC contribute to endosomal trafficking of Notch and other proteins? In the yeast SPT mutant lcb1, an early step of endocytosis is impaired due to defective actin attachment to endosomes, a phenotype that is suppressed by addition of sphingoid base. However, the trafficking abnormalities seen in lace and ACC mutants do not resemble those in the yeast lcb1 mutant, perhaps because endocytic vesicle fission is primarily dependent upon dynamin in D. melanogaster and mammals, instead of actin as in yeast. Nevertheless, the requirement for SPT and ACC in D. melanogaster endosomal compartments might reflect possible functions in endosome-cytoskeleton interactions. Another possibility is that the defective endosomal trafficking seen in lace and ACC mutants is caused by the inability to synthesize specific phospholipids needed for normal membrane homeostasis. Finally, lace and ACC might be important for the formation and/or function of lipid rafts, specialized membrane microdomains that have been implicated in both signaling and protein trafficking (Sasamura, 2013).

A remarkable feature of the lace and ACC mutant phenotypes that suggests an underlying defect in lipid biogenesis is the non-autonomous effect in mutant tissue clones, wherein nearby wildtype cells generate a secreted activity that diffuses several cell diameters into the mutant tissue and rescues the trafficking and signaling defects. One possibility is that these secreted activities are diffusible lipid biosynthetic products of SPT and ACC, which enter the mutant cells and serve as precursors for further biosynthetic steps that do not require SPT or ACC. An intriguing alternative is that the SPT and ACC enzymes are themselves secreted and taken up by the mutant cells. A precedent for this mechanism has recently been demonstrated for D. melanogaster ceramidase, a sphingolipid metabolic enzyme that is secreted extracellularly, delivered to photoreceptors, and internalized by endocytosis to regulate photoreceptor cell membrane turnover (Sasamura, 2013).

Recent work has highlighted the importance of lipid metabolism for oncogenic transformation, and ACC has been advanced as a promising target for cancer drug development. ACC is upregulated in some cancers, possibly as a result of high demands for lipid biosynthesis during rapid cell divisions. Sphingolipids and their derivatives are also thought to influence the balance of apoptosis and cell proliferation during tissue growth, and thus have also garnered attention as potential cancer therapy targets. The current findings regarding the requirements of SPT and ACC for proper trafficking and signaling of key developmental cell-surface signaling molecules, including Notch and Wingless, provide insights into how lipid metabolic enzymes might influence cell proliferation and tissue patterning in multicellular animals. Complex lipid biosynthesis is essential for the creation of the elaborate, interconnected, and highly specialized membrane compartments in which developmental pathways operate, and perturbations in lipid biosynthesis that are tolerated by the cell might nevertheless exert significant pleiotropic effects on developmental patterning, cell proliferation, and other cellular processes. Exploration of lipid metabolic enzymes as pharmacological targets must therefore take into account potentially unfavorable effects on critical signaling pathways controlling development and organogenesis (Sasamura, 2013).

A re-examination of the selection of the sensory organ precursor of the bristle sensilla of Drosophila melanogaster

The bristle sensillum of the imago of Drosophila is made of four cells that arise from a sensory organ precursor cell (SOP). This SOP is selected within proneural clusters (PNC) through a mechanism that involves Notch signalling. PNCs are defined through the expression domains of the proneural genes, whose activities enables cells to become SOPs. They encode tissue specific bHLH proteins that form functional heterodimers with the bHLH protein Daughterless (Da). In the prevailing lateral inhibition model for SOP selection, a transcriptional feedback loop that involves the Notch pathway amplifies small differences of proneural activity between cells of the PNC. As a result only one or two cells accumulate sufficient proneural activity to adopt the SOP fate. Most of the experiments that sustained the prevailing lateral inhibition model were performed a decade ago. This study re-examined the selection process using recently available reagents. The data suggest a different picture of SOP selection. They indicate that a band-like region of proneural activity exists. In this proneural band the activity of the Notch pathway is required in combination with Emc to define the PNCs. A sub-group in the PNCs was found from which a pre-selected SOP arises. The data indicate that most imaginal disc cells are able to adopt a proneural state from which they can progress to become SOPs. They further show that bristle formation can occur in the absence of the proneural genes if the function of emc is abolished. These results suggest that the tissue specific proneural proteins of Drosophila have a similar function as in the vertebrates, which is to determine the time of emergence and position of the SOP and to stabilise the proneural state (Troost, 2015).

This study has re-examined the development of the SOP of the MC (macrochaetae) using recently available reagents. Evidence was found that strongly suggests that the range of the Notch signal is restricted to the next cell: The elevated expression of Notch activity reporter Gbe+Su(H) around the SOP is observed only in adjacent cells. In addition, cells of PNCs that are not able to receive the Notch signal, but can send a strong signal to adjacent wildtype cells, cannot prevent a wildtype cell from adopting the SOP fate at a distance of two cell diameters away. Likewise, cells that are not able to send a signal cannot be prevented by wildtype SOPs from adopting the SOP fate more than one cell diameter away. These results suggest that the discovered filopodia of the SOP, which contact more remotely located cells do not extend the range of the inhibitory signal to these cells (Troost, 2015).

This study reveals the existence of a band of proneural activity. The PNCs are regions of elevated proneural activity in this band, rather than discrete clusters. In the band, the Notch pathway exerts an additional novel function, which defines the extent of the PNCs. In the absence of Notch function, most cells in the proneural band accumulate high levels of proneural activity that allows them to become SOPs. Thus, the pathway suppresses the proneural activity and the SOP fate in cells located between the PNCs in the proneural band. The short range of the Notch signal indicates that it is probably local mutual signalling among direct neighbours that generates the necessary Notch activity (mutual inhibition). The expression of Dl and Ser and the overall activity of Gbe+Su(H) (with exception of the halos) is unchanged in the absence of Ac and Sc. This suggests that the widespread activity of Notch in the notum that prevents most cells in the proneural band to become SOPs is not influenced by the proneural factors. It provides a baseline activity of Notch that suppresses the proneural activity in the band to prevent the formation of ectopic SOPs (Troost, 2015).

The presented results indicate that a subgroup within the PNCs exists, which is operationally defined via the requirement of the activity of Neur. The existence of a subgroup has previously been suggested on basis of experiments with a temperature sensitive allele of Notch. These data and the ones presented here, suggest that the cells of the subgroup require Notch activity that is stronger than the baseline activity to be inhibited from adopting the SOP fate. This increase in activity is generated by the nascent SOP through a Neur enhanced Dl signal: This study found that if only one cell in the subgroup is neur positive, it can prevent all other neur mutant members to adopt the SOP fate. Thus, initiating the expression of Neur first, is a critical step for a cell to adopt the SOP fate, since it allows a cell to strongly inhibit its neighbours. The inhibitory signal prevents the accumulation of sufficient proneural activity to also activate Neur in the neighbours. This inhibition is probably reflected in the observed halo of Gbe+Su(H) expression around SOPs. The findings are in good agreement with a previous study that showed that the level of Neur in a cell is a critical factor for the formation of the SOP of the microchaetae (mc) (Troost, 2015).

Loss of Notch activity results in expression of Neur and a dramatic increase in proneural activity in all cells of the PNC. Moreover, the nascent SOP, which contains the highest proneural activity, is the only cell that initiates Neur expression during normal development and expression of neur is abolished in ac sc mutant discs. These data indicate, that high proneural activity is required for the expression of Neur. Thus, the cell in the subgroup with the highest proneural activity is the cell that will express Neur first. The expression of Neur enables it to inhibit its neighbours from adopting the SOP fate by suppressing their proneural activity (Troost, 2015).

One generated through mutual signalling, which is not regulated by Ac and Sc and is sufficient to inhibit all cells in the proneural band outside the neur subgroup to become SOPs. This signalling requires the ubiquitously expressed Mib1 and antagonises the activity of Ac, Sc and Da. However, there is residual activity of Notch in mib1 mutants sufficient to prevent most cells from adopting the SOP fate. This residual activity is generated either independently of E3 ligases or by another unknown E3-ligase. In any case this component contributes to the baseline activity of the Notch pathway in addition to Mib1. The second activity on top of the baseline activity in the neur subgroup is generated by a Neur mediated strong signal from the nascent SOP. This signal suppresses the proneural activity of the other members of the neur subgroup. It is dependent on proneural activity, which initiates the expression of Neur. Thus, lateral inhibition is probably operating after the emerging SOP reaches a threshold of proneural activity. It serves to prevent the formation of supernumerary SOPs in the neur group and assures that other cells can generate the necessary SOP in case the selected one is lost (Troost, 2015).

How is the neur subgroup defined? It was found that the PNCs are small in their beginning, comprising the number of cells typical for the subgroup. These cells probably also constitute the small groups of SOPs observed in early third instar discs mutant for Psn. It is likely that E(spl)m8-SM expression defines this subgroup since this study shows that it is expressed in a small group of cells from which the SOP arises. This construct contains only one E box, the binding sites for Ac and Sc, and response to high proneural activity. It is therefore believed that the cells of the early PNC are the neur group and possess the highest proneural activity (Troost, 2015).

During normal development, a cell with more proneural activity is already recognisable at the early phase of the PNCs. This suggests the existence of a pre-selection mechanism that assures that one cell in the neur-subgroup is advanced in its development. Evidence for such a mechanism has been also previously found during rescue experiments studying the function of the proneural genes Ac and Sc. This study has obtained additional experimental evidence for this pre-selecting mechanism: In neur clones one of the cells is advanced in its development towards the SOP fate. Moreover, clonal analysis of kuz and Psn mutants revealed that wildtype cells at positions in the PNC where the SOP arises cannot be prevented from adopting the SOP fate, even if a mutant SOP that cannot be inhibited (e.g., kuz mutant), is its neighbour. The mutant cells can generate a strong inhibitory Notch signal. This indicates that the pre-selecting mechanism renders the wildtype SOP immune to the signal. The nature of this mechanism is not clear, nor whether it is always the same cell in a cluster that is selected (Troost, 2015).

Recent work demonstrated that in the eye disc a regulatory loop between Da and Emc assures correct expression of both factors and results in their complementary expression. Consequently, loss of emc function results in an increase of expression of Da. The consequences of this up-regulation for the proneural state of the mutant cells have not been investigated in detail. A published study focused on the eye imaginal disc and revealed that a few of the mutant cells in clones could adopt the neural fate. The neural cells do not express Runt, a marker expressed in the normal neural cells. Thus, the loss of emc does not result in the complete determination of the neural fate. The state of the vast majority of the cells in clones remained unknown. This study observed up-regulation of proneural activity in emc clones already in early third instar wing imaginal discs, indicating that it is an immediate reaction to the loss of emc function. Some of these cells progress to become SOPs. The increase in proneural activity was also observed in emc clones of the leg disc. Thus, the cells of imaginal discs must be permanently inhibited from adopting a proneural state through the activity of Emc. It has to be pointed out that this situation is remarkably similar to that in the early vertebrate embryo, where all cells of the blastula adopt the proneural state unless they are inhibited through BMP signalling. The cells of the neural plate maintain the proneural state due to the presence of BMP antagonists (Troost, 2015).

In the eye disc and during oogenesis expression of Emc is regulated by the Notch pathway. This study failed to find evidence that supports a regulatory relationship between Emc and the pathway in the notum during SOP development, since the loss of Psn function did not affect the expression of EMC. However, it has been previously shown that the expression of Emc along the dorso-ventral boundary in the wing primordium depends on the activity of the Notch pathway. This correlates well with the finding that this domain is independent of the activity of Da. However, the genetic network of the wing is significantly different from that in the notum. For example Notch signalling induces the expression of Wg along the D/V boundary. However, its expression in the proximal wing and in the notum is independent of the activity of the Notch pathway. This appears to be true also for the different domains of expression of Emc (Troost, 2015).

This study found that the function of ac and sc is dispensable for bristle development in the absence of emc function. How is the SOP fate initiated in these emc ac sc triple mutant cells? It is believed that the activity of Da is sufficient for SOP development in this situation for the following reasons: 1) Da is expressed ubiquitously and is required for the formation of all external sense organs. 2) Strong over-expression of Da induces bristle formation in cells that lack the whole AS-C. In contrast, over-expression of Sc fails to induce SOP formation in the absence of Da. 3) Da can form homodimers that bind to the same DNA target sequences as Ac/Da and Sc/Da heterodimers in bend-shift assays. 4) Loss of emc activity increases the activity of Da. This study show that this increase is independent of the activity of Ac and Sc. 5) The results show that Da regulates the expression of sca independently of Ac and Sc. 6) It has been shown that the mammalian homologue of Da, E2A, acts without its class II partners during B-cell development. Thus, it is likely that in the absence of function of emc, ac and sc, Da forms active homo-dimers that initiate the required neural program (Troost, 2015).

While it is clear that the activity of Ac and Sc is required during normal development, the formation of normal bristles in their absence after concomitant loss of emc function raises the question about their function. The data suggest that an important function is the neutralisation of Emc through formation of heterodimers with it or with Da. This releases Da from inactive heterodimers with Emc. The neutralisation of Emc by Ac and Sc, which are expressed in precise spatial and temporal regulated patterns, allows the differentiation of neural precursors at the correct position and time. The recent finding that a Sc variant without its transactivation domain is fully active fits well to this view of the function of Ac and Sc. Thus, through their intricate and dynamic expression, Ac and Sc and other tissue specific proneural factors determine when and where a neural precursor cell develops. In this view the function of the tissue-specific proneural genes of Drosophila, is similar to that in mammals where their orthologs also promote differentiation of neural precursors in a proneural field, the neural plate, at correct positions and time (Troost, 2015).

Based on the current results, a working model is suggested for the selection of the SOP of the MC: The differential expression of Emc defines a proneural band in the notum with changing proneural activity. The PNCs in this band are determined and positioned through the cluster-like expression of Ac and Sc, which increases the proneural activity at these positions. A baseline of activity of the Notch pathway generated by mutual inhibition prevents cells between the PNCs to accumulate high levels of proneural activity. In addition, it prevents cells located in the PNC, but outside the neur group, to accumulate high proneural activity required for adopting the SOP fate (Troost, 2015).

In the PNCs, expression of Ac and Sc neutralise Emc. Consequently, the proneural activity increases dramatically, since the released Da can form homodimers and/or heterodimers with Ac or Sc. The cells of the initial small PNCs later constitute the neur subgroup. The cells of this subgroup have the highest level of proneural activity and experience this activity also for the longest time. Within this subgroup a cell is pre-selected to become the SOP by a so far unidentified mechanism. Hence, it is the first to reach the threshold level of proneural activity required to initiate the expression of Neur. The expression of Neur enables it to efficiently inhibit the other cells of the subgroup through lateral inhibition. As a consequence these cells never accumulate sufficient proneural activity to activate Neur expression and to become a SOP. The strong signal also further activates the expression of Brd proteins that inhibit the activation of Neur, which might be accidentally activated weakly in one of the neighbours. This activation contributes to the precision of determination process. Thus, a combination of mutual and lateral inhibition mediated by the Notch pathway operates in the PNC during the determination of the SOP. Only the lateral inhibition component depends on proneural activity through transcriptional activation of expression of Neur (Troost, 2015).

The model differs from the lateral inhibition model in the following points: No feedback loop between expression of Dl and proneural activity and, hence, no differential Dl expression is required. Instead the future SOP is pre-selected and advanced in its development. Subgroups within a proneural band defined through its requirement of Neur exist. In this subgroup the activation of the expression of Neur is critical for SOP development since it enables a cell to potently inhibit its neighbours. The pre-selection mechanism favours a cell at the right position to initiate the expression of Neur before the others of the Neur group and therefore secures its development as SOP. Moreover, the existence of mutual signalling explains the inhibition of cells in the proneural band outside the subgroup without the necessity of signalling of Dl over longer distances (Troost, 2015).

Intra-lineage fate decisions involve activation of Notch receptors basal to the midbody in Drosophila sensory organ precursor cells

Notch receptors regulate cell fate decisions during embryogenesis and throughout adult life. In many cell lineages, binary fate decisions are mediated by directional Notch signaling between the two sister cells produced by cell division. How Notch signaling is restricted to sister cells after division to regulate intra-lineage decision is poorly understood. More generally, where ligand-dependent activation of Notch occurs at the cell surface is not known, as methods to detect receptor activation in vivo are lacking. In Drosophila pupae, Notch signals during cytokinesis to regulate the intra-lineage pIIa/pIIb decision in the sensory organ lineage. This study identified two pools of Notch along the pIIa-pIIb interface, apical and basal to the midbody. Analysis of the dynamics of Notch, Delta, and Neuralized distribution in living pupae suggests that ligand endocytosis and receptor activation occur basal to the midbody. Using selective photo-bleaching of GFP-tagged Notch and photo-tracking of photo-convertible Notch, this study showed that nuclear Notch is indeed produced by receptors located basal to the midbody. Thus, only a specific subset of receptors, located basal to the midbody, contributes to signaling in pIIa. This is the first in vivo characterization of the pool of Notch contributing to signaling. A simple mechanism of cell fate decision based on intra-lineage signaling is proposed: ligands and receptors localize during cytokinesis to the new cell-cell interface, thereby ensuring signaling between sister cells, hence intra-lineage fate decision (Trylinski, 2017).

Several methods are currently available to monitor in vivo the signaling activity of Notch by measuring the level and/or activity of NICD. By contrast, in vivo reporters for ligand-receptor interaction, conformational change of Notch in response to mechanical force, and S2 cleavage of Notch are lacking. Consequently, the subcellular location of Notch receptor activation in vivo and the relative contribution of the different pools of Notch to signaling remain unknown. Two complementary fluorescent-based approaches have been developed in this study to track where NICD comes from. Notch receptors present basal to the midbody along the pIIa-pIIb interface were shown to contribute to the accumulation of NICD, whereas receptors located apical to the midbody did not significantly contribute to NICD production. This study provides the first in vivo analysis of ligand-dependent Notch receptor activation at the cell surface. Moreover, the photo-bleaching and photo-conversion approaches used in this study should be broadly applicable in model organisms that can be genetically engineered and easily imaged (Trylinski, 2017).

Other sites of Notch activation had previously been proposed in pIIa. In one model, based on the specific requirements for Arp2/3 and WASp activities for both Notch signaling and actin organization, Dl at apical microvilli in pIIb would activate Notch located apically in pIIa. However, loss of Arp2/3 activity also disrupted cortical actin along the basal pIIa-pIIb interface, suggesting that regulation of the actin cytoskeleton at this location, rather than at microvilli, may be key for receptor activation. In a second model, Dl-Notch signaling was proposed to occur at the new apical pIIa-pIIb junction. This model was largely based on the detection of Notch at this location. The current study, however, indicated that this pool of Notch did not significantly contribute to the production of NICD in pIIa. In a third model, Notch activation was proposed to occur in specific Sara-positive endosomes in pIIa. Whereas the possible contribution of these endosomes to NICD production could not be directly addressed by photo-tracking, two lines of evidence suggest that their contribution can only be minor. First, live imaging of Notch failed to detect this pool indicating that this pool represents a minor fraction of Notch in pIIa. Second, symmetric partitioning of Sara endosomes did not affect the pIIa-pIIb decision, indicating that this proposed pool is not essential for fate asymmetry. Finally, the nature of the mechanical force acting on Notch at the limiting membrane of the Sara-positive endosomes remains to be addressed. In summary, all available data are fully consistent with the conclusion that receptor activation occurs mostly basal to the midbody (Trylinski, 2017).

Whereas these experiments identified the signaling pool of Notch along the pIIa-pIIb, they did not, however, address whether S3 cleavage takes place at the cell surface or intracellularly following endocytosis. Indeed, the photo-tracking approach used in this study did not inform whether the activation of Notch by Delta, i.e., s2 cleavage, is followed by S3 cleavage at the same location or whether S2-processed Notch is internalized to be further processed in signaling endosomes. It is noted, however, that the accumulation of lateral Notch observed in Psn mutant cells is consistent with S3 cleavage taking place, at least in part, at the cell surface (Trylinski, 2017).

This work also sheds new light on the general mechanism whereby Notch signaling is specifically restricted to sister cells within a lineage. In several tissues, including the gut, lung, and CNS, Notch regulates intra-lineage decisions between sister cells soon after mitosis. In this study it is proposed that Notch-mediated intra-lineage decisions are directly linked to division. Indeed, it is suggested that ligands and receptors localize to the lateral membranes that separate the two sister cells at cytokinesis so that Dl-Notch signaling is primarily restricted to sister cells. Thus, neighboring cells - belonging to other cell lineages - would not interfere with intra-lineage fate decisions. The current data indicating that Neur-dependent activation of Notch by Dl predominantly occurs along the pIIa-pIIb lateral interface, basal to the midbody during cytokinesis, fully support this model. Also, the observation that core components of the secretory machinery, e.g., Sec15, are specifically required for Notch signaling in the context of intra-lineage decisions is also consistent with this view. Thus, targeting both receptors and ligands along the newly formed interface during cytokinesis provides an elegant mechanism to restrict signaling between sister cells, thereby ensuring that intra-lineage signaling regulates intra-lineage fate decision. Because Notch generates fate diversity within neural lineages in both vertebrates and invertebrates, this mechanism of intra-lineage signaling may be conserved (Trylinski, 2017).

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1

miR-1> is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. This study shows that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4. Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, it was found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart (Zhu, 2017).

Unexpectedly, overexpression of miR-1 in the anterior-posterior (AP) organizer of the wing disc results in a dose-dependent loss of L3 vein structures, consistent with de-repression of Notch or weakening of a regulatory mechanism that dampens the Notch signal. Using genetic techniques, it was determined that the loss of the distal aspect of L3 could be phenocopied by reducing the gene dose of Notch co-repressors or Nedd4; in the case of Nedd4, the regulation by miR-1 was direct. An expanded model is proposed in which miR-1 expression in the AP organizer has complex effects on Notch signaling owing to its regulation of ligand availability and receptor trafficking. As lower levels of miR-1 expression (18°C) caused wing-vein thickening and tortuosity, and higher levels (22°C) caused vein loss, Delta and Nedd4 may be differentially sensitive to miR-1 regulation, although these studies were not designed to address this issue. It is also possible that indirect effects, such as reductions in Nedd4-mediated ubiquitylation of positive effectors of the Notch receptor (e.g. Deltex) or perturbations in Delta-mediated cis-inhibition, contributed to the de-repression of Notch in the wing-based assay system (Zhu, 2017).

The findings in the mammalian heart indicate that the genetic and biochemical interaction between miR-1 and Nedd4l is physiologically relevant and may provide developmental or tissue-specific regulation of Nedd4l in the myocardium. It is speculated that the additional bands observed on western blots of heart lysates using an anti-Nedd4L antibody might result from post-translational modifications, because Nedd4L can autoregulate its stability through ubiquitylation of its HECT domain. Alternatively, they might represent heart-specific splice variants, because tissue-specific isoforms of Nedd4L have been found in the heart and the liver (Zhu, 2017).

Importantly, although miR-1-mediated reductions in Nedd4 activity caused wing-vein phenotypes induced by Notch, miR-1-mediated dysregulation of Nedd4L in the heart likely affects proteins outside the Notch pathway. Indeed, protein microarrays comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion channels, whereas Nedd4 targets are enriched for signaling pathways. Thus, in the heart, where miR-1 and murine Nedd4L are both expressed, their genetic and biochemical interaction might influence the excitability and connectivity of cardiomyocytes. Indeed, susceptibility to cardiac arrhythmias and sudden death in humans is associated with six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, KCNE1, KCNE2 and RYR2). Murine Nedd4L regulates the cell-surface densities of the sodium channel, the voltage-gated type V alpha subunit (Scn5a), the potassium voltage-gated channel, KQT-like subfamily member 1 (Kcnq1) and the human Ether-a-go-go-related (KCNH2, previously hERG) channel. Furthermore, miR-1 directly regulates human KCNJ2, a channel that maintains cardiac resting potential. These findings suggest that the regulation of murine Nedd4l by miR-1 contributes to some of the electrophysiological abnormalities seen in miR-1 null mice. It would be interesting to determine whether Nedd4L is dysregulated in the heart after an infarction or under ischemic conditions, when miR-1 is upregulated and fatal cardiac dysrhythmias are common (Zhu, 2017).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

During asymmetric division, fate assignation in daughter cells is mediated by the partition of determinants from the mother. In the fly sensory organ precursor cell, Notch signalling partitions into the pIIa daughter. Notch and its ligand Delta are endocytosed into Sara endosomes in the mother cell and they are first targeted to the central spindle, where they get distributed asymmetrically to finally be dispatched to pIIa. While the processes of endosomal targeting and asymmetry are starting to be understood, the machineries implicated in the final dispatch to pIIa are unknown. This study shows that Sara binds the PP1c phosphatase and its regulator Sds22. Sara phosphorylation on three specific sites functions as a switch for the dispatch: if not phosphorylated, endosomes are targeted to the spindle and upon phosphorylation of Sara, endosomes detach from the spindle during pIIa targeting (Loubery, 2017).

Asymmetric cell division plays many roles in development. In particular, stem cells divide asymmetrically to self-renew while also forming differentiated cells. Asymmetric cell division involves the specific partitioning of cell fate determinants (RNA, proteins or organelles) in one of the two sibling daughter cells. The Sensory Organ Precursor cells (SOPs) of the Drosophila notum are a model system of choice to unravel the molecular mechanisms of asymmetric cell division (Loubery, 2017).

The division of each SOP gives rise to a pIIa and a pIIb daughter cell and, after two more rounds of asymmetric cell divisions, to the four cells of the sensory organ: the outer cells (shaft and socket) are progeny of the pIIa, while the pIIb forms the inner cells (sheath and neuron) and a glial cell that rapidly undergoes apoptosis. The Notch signalling pathway controls cell fate determination in this system: a signalling bias between the pIIa-pIIb sibling cells is essential to obtain a correct lineage (Loubery, 2017).

The asymmetric dispatch of cell fate determinants during SOP division is governed by the polarity of the dividing cell. The Par complex (composed by the aPKC, Par-3 and Par-6 proteins) is the master regulator of the establishment of this polarity. Downstream the Par complex, Notch signalling is regulated by endocytosis and endosomal trafficking through four independent mechanisms: (1) The E3 Ubiquitin ligase Neuralized is segregated to the pIIb cell, where it induces the endocytosis and thereby the activation of the Notch ligand Delta; (2) Recycling endosomes accumulate in the perinuclear region of the pIIb cell, in which they enhance the recycling and activation of Delta; (3) The endocytic proteins α-adaptin and Numb are segregated to the pIIb cell, where they inhibit the Notch activator Sanpodo; (4) During SOP mitosis, Sara endosomes transport a signalling pool of Notch and Delta to the pIIa cell, where Notch can be activated. Asymmetric Sara endosomes have also been shown to operate in the larval neural stem cells (Coumailleau, 2009) as well as in the adult intestinal stem cells in flies, where they also play a role during asymmetric Notch signalling. In fish, Sara endosomes mediate asymmetric cell fate assignation mediated by Notch during the mitosis of neural precursor of the spinal cord (Loubery, 2017).

Sara endosomes are a subpopulation of Rab5-positive early endosomes characterised by the presence of the endocytic protein Sara. Sara directly binds the lipid phosphatidyl-inositol-3-phosphate and both molecules are found at the surface of these endosomes. A pulse-chase antibody uptake assay has been established to monitor the trafficking of endogenous internalised Notch and Delta and showed that both Notch and Delta traffic through Sara endosomes. Furthermore, it was shown that Sara endosomes are specifically targeted to the pIIa cell during SOP division, mediating thus the transport of a pool of Notch and Delta that contribute to the activation of Notch in the pIIa. The Notch cargo and its Uninflatable binding partner are required for this asymmetric dispatch. Targeting of Sara endosomes to the central spindle is mediated by a plus-end-directed kinesin, Klp98A. The asymmetric distribution of endosomes at the central spindle results from a higher density of microtubules in pIIb with their plus ends pointed towards pIIa15 (Loubery, 2017).

This study shows that the Sara protein itself controls both the targeting and the final dispatch of Sara endosomes to the pIIa daughter cell. Sara binds and is a target of the PP1 phosphatase complex. The phosphorylation state of Sara functions as a switch that enables the targeting of Sara endosomes to the central spindle of the dividing SOP, and their subsequent detachment from the central spindle, which is necessary to allow their movement to the pIIa daughter cell (Loubery, 2017).

Previous work has shown that a subpopulation of Rab5 early endosomes positive for Sara are asymmetrically dispatched into the pIIa daughter cell during cytokinesis of the SOP. This was monitored by following in vivo either GFP-Sara or internalized Delta or Notch, which reach the Sara endosomes 20 min after their endocytosis in the mother cell. These vesicles were termed iDelta^20' endosomes. In contrast, the pools of Notch in endosomal populations upstream or downstream of the Sara endosomes (that is, the Rab5 early endosomes with low Sara levels and the Rab7 late endosomes, respectively) were segregated symmetrically. Rab5 endosomes show different levels of Sara signal: by a progressive targeting of Sara to the Rab5 endosomes, Rab5 early endosomes mature into Sara endosomes. This prompts the question whether the levels of Sara in endosomes correlate indeed with their asymmetric behaviour (Loubery, 2017).

To study the relationship between the levels of Sara in endosomes and their targeting to the spindle, Matlab codes were written to perform automatic 3D-tracking of the Sara endosomes. Sara endosomes were detected by monitoring a GFP-Sara fusion, which was overexpressed through the UAS/Gal4 system. This way, the position of the endosomes, their displacement towards and away from the central spindle was monitored as well as the levels of Sara. In addition, the position was detected automatically of the Pon cortical crescent, which forecasts the side of the cell that will become the pIIb cell (Loubery, 2017).

The localization of endosomes was studied with respect to a 2 μm-wide box centered in the central spindle during SOP mitosis. The enrichment was measured of endosomes in this central spindle as a function of time. Two phases were observed in the movement of the endosomes during mitosis: (1) targeting to the central spindle and (2) departure into the pIIa cell. The endosomes are progressively accumulating in the central spindle area from the end of metaphase (~450 s before abscission) through anaphase and during cytokinesis until they are enriched at the central spindle by about 10-fold at 250 s before abscission (Loubery, 2017).

Subsequently, the endosomes depart from the central spindle area into the pIIa cell. By fitting an exponential decay to the profile of abundance of the endosomes at the central spindle, the characteristic residence time of the endosomes at the central spindle was measured after the recruitment phase: after recruitment, endosomes remain at the central spindle 98±9.8 s before they depart into one of the daughter cells, preferentially the pIIa cell (Loubery, 2017).

To address a potential role of Sara on central spindle targeting and asymmetric segregation, the behaviour was tracked and quantified of the endosomes in a Sara loss of function mutant (Sara¹²) and in conditions of Sara overexpression in the SOP (Neur-Gal4; UAS-GFP-Sara). In Sara¹² SOPs, targeting of iDelta^20' endosomes to the cleavage plane is severely impaired. Consistent with the fact that the asymmetric dispatch of endosomes to pIIa requires first their targeting to the central spindle as previously shown, in Sara¹² SOPs the dispatch to the pIIa daughter is strongly affected. A slight bias (60% pIIa targeting) is, however, retained in the mutant, consistent with a previous report (Loubery, 2017).

Conversely, overexpression of Sara increases targeting to the central spindle. In these conditions, Sara is found not only in Rab5 endosomes, but also in Rab7 late endosomes as well as in the Rab4 recycling endosomes. Correlating with this, Rab4, Rab5 and Rab7 endosomes, which are not all recruited to the central spindle in wild-type conditions, are now targeted to the central spindle upon Sara overexpression and are asymmetrically targeted (Loubery, 2017).

Furthermore, consistent with the correlation that is observed between the levels of Sara at the endosomes and their displacement towards the cleavage plane, quantification of central spindle targeting of the Sara endosomes upon its overexpression shows that targeting of the endosomes to the cleavage plane is increased by a factor of 2.5 in these conditions. These observations indicate that Sara plays a crucial role on the targeting of the endosomes to the spindle and the subsequent dispatch of the Notch/Delta containing endosomes to pIIa. Does this play a role during Notch-dependent asymmetric cell fate assignation? (Loubery, 2017).

Sara function contributes to cell fate assignation through asymmetric Notch signalling, but this activity is redundantly covered by Neuralized. Neuralized E3 Ubiquitin ligase does play an essential role during the endocytosis and activation of the Notch ligand Delta. Therefore, during larval development, Neuralized is essential for Notch-mediated lateral inhibition in the proneural clusters, which leads to the singling-out of SOP cells from the proneural clusters. Later, during pupal development, Neuralized appears as a cortical crescent in the pIIb side of the dividing SOPs, thereby biasing Delta activation in the pIIb cell and asymmetric activation of Notch in pIIa6 (Loubery, 2017).

Consistently, a partial loss of function of Neuralized by RNAi interference in the centre of the notum (Pnr>Neur^RNAi Control) showed lateral inhibition defects in the proneural clusters, causing the appearance of supernumerary SOPs as well as asymmetric Notch signalling defects in the SOP lineage, leading to supernumerary neurons and loss of the external shaft/socket cells in the lineage. The remaining Neuralized activity in this partial loss of function condition allows many sensory organs (more than forty in the centre of the notum) to perform asymmetric cell fate assignation and to develop, as in wild type, into structures containing at least the two external cells (Loubery, 2017).

In Pnr>Neur^RNAi, Sara¹²/Df(2R)48 transheterozygote mutants, the number of supernumerary SOPs is increased by 35% with respect to the Pnr>Neur^RNAi controls (668±38 versus 498±52). This indicates that during lateral inhibition, Sara endosomes contributes to Notch signalling. This general role of Sara is uncovered when the Neuralized activity during Notch signalling is compromised (Loubery, 2017).

In the case of Neuralized, its localization to the anterior cortex biases Notch signalling to be elicited in the pIIa cell. This is the same in the case of Sara endosomes: asymmetric dispatch of Sara endosomes also biases Notch signalling to pIIa10. Indeed, in Pnr>Neur^RNAi, Sara¹²/Df(2R)48 transheterozygote mutants, the number of bristles (external shaft/socket cells) in the notum is strongly reduced at the expense of supernumerary neurons compared to the Pnr>Neur^RNAi controls. This indicates that Notch-dependent asymmetric cell fate assignation in the SOP lineage is synergistically affected in the Sara/Neuralized mutant. This implies that the SOP lineages which still could generate bristles with lower levels of Neuralized function in Pnr>Neur^RNAi need Sara function to perform asymmetric cell fate assignation: in Pnr>Neur^RNAi, Sara¹²/Df(2R)48 and Pnr>Neur^RNAi, Sara¹²/Sara1 transheterozygote mutants, these lineages failed to perform asymmetric signalling, causing the notum to be largely bald. Therefore, Sara contributes to Notch signalling and asymmetric cell fate assignation, as observed in conditions in which other redundant systems for asymmetric Notch signalling are compromised (Loubery, 2017).

Both Neuralized and Sara play general roles in Notch signalling: they are both involved in lateral inhibition at early stages and, at later stages, in asymmetric cell fate assignation. Indeed, both Neuralized and Sara mutants show early defects in lateral inhibition and, accordingly, they show supernumerary SOPs. In addition, Neuralized and Sara mutant conditions also show defective Notch signalling during cell fate assignation in the SOP lineage and therefore cause the transformation of the cells in the lineage into neurons. In this later step, Notch signalling is asymmetric. The possibility that both Sara and Neuralized play key roles in ensuring the asymmetric nature of this signalling event is only correlative: in the case of Neuralized, it is enriched in the anterior cortex of the cell, which will give rise to pIIb; in the case of Sara, (1) both Delta and Notch are cargo of these endosomes, (2) cleaved Notch is seen in the pIIa endosomes and (3) Sara endosomes are dispatched asymmetrically to pIIa10. It is tantalizing to conclude that the asymmetric localization of these two proteins mediate the asymmetric nature of Notch signalling in the SOP lineage, but further assays will be necessary to unambiguously address this issue. Clonal analysis is unfortunately a too slow assay to sort out the specific requirement of these cytosolic factors (Sara and Neuralized) in the pIIa versus the pIIb cell (Loubery, 2017).

Sara mediates the targeting of Notch/Delta containing endosomes to the central spindle and could contributes to Notch-mediated asymmetric signalling in the SOP lineage. What machinery controls in turn the Sara-dependent targeting of endosomes to the central spindle? Previous proteomic studies uncovered bona fide Sara-binding factors, including the Activin pathway R-Smad, Smox17 and the beta subunit of the PP1c serine-threonine phosphatase (PP1β(9C)). In an IP/Mass Spectrometry approach, those interactions were confirmed and in addition to PP1β(9C), two of the other three Drosophila isoforms of PP1c: PP1α(87B) and PP1α(96A) were found. Furthermore, the PP1c regulatory subunit Sds22 was found, suggesting that Sara binds the full serine-threonine PP1 phosphatase complex. The interaction with Sds22 was confirmed by immunoprecipitation of overexpressed Sds22-GFP and western blot detection of endogenous Sara in the immunoprecipitate (Loubery, 2017).

Prompted by these results, whether the PP1 complex plays a role in the asymmetric targeting of the Sara endosomes was explored by manipulating the activity of Sds22, the common regulatory unit in all the complexes containing the different PP1 isoforms. Sds22 was overexpressed specifically during SOP mitosis, by driving Sds22-GFP under the Neur-Gal4 driver with temporal control by the Gal80ts system. In SOPs where PP1-dependent dephosphorylation is enhanced by overexpressing Sds22, the Sara endosomes fail to be dispatched asymmetrically toward the pIIa daughter cell (Loubery, 2017).

The role of PP1-dependent dephosphorylation in the SOP was examined by knocking down Sds22 (through a validated Sds22-RNAi). Loss of function Sds22 did also affect the asymmetric targeting of endosomes. These data uncover a key role for phosphorylation and PP1-dependent dephosphorylation as a switch that contributes to the asymmetric targeting of Sara during asymmetric cell division (Loubery, 2017).

The observations raise the question of which is the step in the asymmetric dispatch of the endosomes that is controlled by the levels of phosphorylation: central spindle targeting, central spindle detachment or targeting to the pIIa cell? PP1/Sds22-dependent dephosphorylation controls a plethora of mitotic events, including mitotic spindle morphogenesis, cortical relaxation in anaphase, epithelial polarity and cell shape, Aurora B activity and kinetochore-microtubule interactions as well as metabolism, protein synthesis, ion pumps and channels. Therefore, to establish the specific event during the asymmetric dispatch of Sara endosomes that is controlled by PP1/Sds22 dephosphorylation, focus was placed on the phosphorylation state of Sara itself and its previously identified phosphorylation sites. This allowed specific interference with this phosphorylation event and thereby untangle it from other cellular events also affected by dephosphorylation (Loubery, 2017).

PP1/Sds22 was shown to bind Sara. It has previously been shown that mammalian Sara itself is phosphorylated at multiple sites and that the level of this Sara phosphorylation is independent on the level of TGF-beta signalling. Three phosphorylation sites have been identified at position S636, at position S709, and at position S774 in Sara protein and these sites were confirmed by Mass Spectrometry of larval tissue expressing GFP-Sara. Phosphorylation of Sara had been previously reported to be implicated in BMP signalling during wing development. However, the role of these three phosphorylation sites during asymmetric division are to date unknown (Loubery, 2017).

ProQ-Diamond phospho-staining of immunoprecipitated GFP-Sara confirmed that Sara is phosphorylated. To test whether PP1/Sds22 controls the phosphorylation state of Sara, ProQ-Diamond stainings of GFP-Sara were performed with and without down-regulation of Sds22. Downregulating Sds22 induced a 40%-increase in the normalized quantity of phosphorylated Sara, showing that PP1/Sds22 does control the phosphorylation state of Sara (Loubery, 2017).

To study the role of Sara phosphorylation during asymmetric targeting of the endosomes, the mitotic behaviour of the endosomes was analyzed in conditions of overexpression of mutant versions of Sara where (1) the three phosphorylated Serines (at position S636, S709, and S774) were substituted by Alanine (phosphorylation defective: GFP-Sara3A) or (2) the PP1 interaction was abolished by an F678A missense mutation in the PP1 binding domain (hyper-phosphorylated: GFP-SaraF678A). Neither mutation affects the general levels of abundance of the Sara protein in SOPs, the targeting of Sara itself to the endosomes, nor the residence time of Sara in endosomes as determined by FRAP experiments. Also, the targeting dynamics of internalized Delta to endosomes are not affected in these mutants (Loubery, 2017).

Upon overexpression of GFP-Sara3A in SOPs, the rate of targeting of the endosomes to the central spindle is greatly increased. In addition, GFP-Sara3A shows impaired departure from the spindle: while the residence time of Sara endosomes at the central spindle after their recruitment is around 100 s in wild type, GFP-Sara3A endosomes stay at the spindle significantly longer (151±21 s). In GFP-Sara3A endosomes, impaired departure leads to defective asymmetric targeting to the pIIa cell while, in wild type, departure from the central spindle occurs well before abscission, in the GFP-Sara3A condition, endosomes that did not depart are caught at the spindle while abscission occurs. These data indicate that the endosomal targeting to the central spindle is greatly favoured when these three sites in Sara are dephosphorylated and suggest that the departure from the microtubules of the central spindle requires that the endosomes are disengaged by phosphorylation of Sara (Loubery, 2017).

Loss of Sara phosphorylation in these sites impairs disengagement from the central spindle. Conversely, impairing Sara binding to the PP1 phosphatase results in defective targeting to the central spindle. Indeed, when binding of Sara to the PP1/Sds22 phosphatase is impaired in the GFP-SaraF678A overexpressing SOP mutants, Sara endosomes fail to be targeted to the spindle. Mistargeted away from the central spindle, the GFP-SaraF678A endosomes fail thereby to be asymmetrically targeted to the pIIa cell. Loss and gain of function phenotypes of the Phosphatase regulator Sds22 during endosomal spindle targeting support the role of Sara phosphorylation during targeting to the central spindle microtubules suggested by the GFP-Sara3A and GFP-SaraF678A experiments (Loubery, 2017).

What are the functional consequences on signalling of impaired phosphorylation/dephosphorylation in Sara mutants? The presence of Sara in endosomes is itself essential for Notch signalling. Sara loss of function mutants show a phenotype in SOP specification (supernumerary SOPs) as well as during fate determination within the SOP lineage (all cells in the lineage acquire a neural fate). In addition, this study showed that Sara is also essential for the targeting of endosomes to the spindle: in the absence of Sara, endosomes fail to move to the spindle in the SOP. They are therefore dispatched symmetrically, but those endosomes do not mediate Notch signalling. As a consequence, both daughters fail to perform Notch signalling in sensitized conditions in which Neuralized is compromised. The result is a Notch loss of function phenotype: the whole lineage differentiates into neurons (Loubery, 2017).

In both Sara3A and SaraF678A mutants, because of reasons that are different in the two cases (either they do not go to the spindle or their departure from the spindle is impaired), functional Sara endosomes are dispatched symmetrically (Fig. 6a,b,e). In contrast to the situation in the Sara loss of function mutant, those endosomes are functional Sara signalling endosomes, which can mediate Notch signalling in both cells. Therefore, these mutations are consistently shown to cause a gain of function Sara signalling phenotype: supernumerary sockets are seen in the lineages (88% of the lineages for Sara3A and 82% of the lineages for SaraF678A). A milder version of this phenotype can be also seen by overexpressing wild-type Sara (34% of the lineages) consistent again with some gain of function Notch signalling phenotype when Sara concentrations are elevated. In summary, this implies that the 3A and F678A mutations impair the phosphorylation state of Sara (with consequences in targeting), but not its function in Notch signalling (Loubery, 2017).

These results indicate that Sara itself plays a key, rate limiting role on the asymmetric targeting of the endosomes by controlling the targeting to the spindle and its departure. Maturation of the early endosomes by accumulating PI(3)P leads to accumulation of the PI(3)P-binding protein Sara to this vesicular compartment. At the endosome, the phosphorylation state of Sara indeed determines central spindle targeting and departure: in its default, dephosphorylated state, Sara is essential to engage the endosomes with the mitotic spindle. Phosphorylation of Sara disengages the endosomes from the central spindle allowing the asymmetric departure into the pIIa cell (Loubery, 2017).

The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating notch receptor trafficking

The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. This study shows that the retromer complex (see Drosophila Vps35) directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7(+) enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. These results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation (Li, 2018).

Unidirectional Notch signaling is a widely used strategy for initiating and maintaining binary cell fates. However, the molecular mechanisms establishing the unidirectionality of Notch signaling in stem cell lineages remain unclear. This study shows that, while asymmetric partition of Numb leads to a biased internalization of the Notch receptor and hence asymmetric dampening of Notch signaling in neural progenitors, it meanwhile poses a high risk of non-canonical endosomal activation of Notch. The retromer complex was found to be the key protein trafficking machinery that resolves this crisis through a timely retrieval of the Notch receptor from its endosomal activation compartments. Upon retromer dysfunction, neural progenitors dedifferentiate into neural stem cell-like status and result in the formation of transplantable tumors. Therefore, retromer acts as a tumor suppressor in Drosophila larval brains. Importantly, mammalian Vps35 physically interacts with Notch, colocalizes with Notch in neural progenitors, and its neuroblast-lineage-specific expression fully rescues neural progenitor-derived brain tumor phenotype in vps35 mutants. Thus, the brain tumor suppressor function of retromer is likely to be conserved in mammals. Intriguingly, downregulation of the retromer complex components has been reported in various human cancers, including glioblastoma. These studies thus provide a new mechanistic link between the retromer complex and carcinogenesis (Li, 2018).

Why the E3 ubiquitin ligase system promoting Notch receptor polyubiquitination and degradation is inherently inefficient in neuroblast lineages? It is speculated that Notch is probably not the only substrate of Su(dx) and Ndfip in neuroblasts or neural progenitors. Therefore, high levels and/or activity of this E3 ubiquitin ligase system above certain threshold may potentially cause imbalanced homeostasis of its critical substrates and hence perturbed neuroblast lineages. Indeed, co-overexpression of Su(dx) and Ndfip led to drastically reduced number of neuroblast lineages and severe tissue atrophy. In this case, a relatively general yet inefficient ubiquitination-degradation system coupled with a highly efficient and selective cargo retrieving system provides a customized regulation of the Notch receptor, ensuring sufficient dampening of Notch signaling in neural progenitors without devastating side effects (Li, 2018).

Intriguingly, previous studies posited that retromer dysfunction causes increased levels of APP (β-amyloid precursor protein) to reside in the endosomes for longer duration than normal, resulting in accelerated processing of APP into amyloid-β, a neurotoxic fragment implicated AD pathogenesis. Furthermore, retromer maintains the integrity of photoreceptors by avoiding persistent accumulation of rhodopsin in endolysosomal compartments that stresses photoreceptors and causes their degeneration. Taken together with this study, these findings indicate that retromer serves as bomb squad to retrieve and disarm harmful or toxic protein fragments from endosomes in a timely manner and thereby safeguard the integrity and fitness of the neuronal lineages (Li, 2018).

How is the Notch receptor ectopically activated in retromer mutants? The idea is favored that Notch is activated in MVBs in a ligand-dependent, cell-autonomous manner, distinct from the majority of non-canonical Notch activation mechanisms. Most of the endosomal Notch activation events identified before, including ectopic Notch signaling activation in ESCRT mutants, BLOS2 mutants, or Rme8 and Vps26 double knockdown background, as well as Hif-alpha-dependent activation of Notch signaling implicated in crystal cell maintenance and survival, are all ligand-independent. It has been proposed that the proteases within the acidifying environment of MVB lumen are sufficient to remove the extracellular domain of Notch, leading to the S3 cleavage of Notch at the limiting membrane. Strongly supporting this notion, blocking the entry of Notch into the ESCRT pathway but not ligand inactivation potently inhibited ectopic Notch activation induced by ESCRT mutations. In sharp contrast to these previously-revealed mechanisms, attenuating ligand activity but not preventing Notch from entering the ESCRT pathway effectively rescues Notch overactivation phenotype caused by retromer dysfunction. Then how Notch signaling is ectopically activated in a ligand-dependent manner in retromer mutants? It is speculated that, upon retromer dysfunction, both Notch and Delta are entrapped in MVBs, where Notch and Delta are presented by limiting membrane and intravesicular membrane respectively and result in ligand-dependent Notch processing and activation, resembling the scenario presented for ligand-dependent Notch signaling activation in Sara endosome. The detailed regulatory mechanisms underlying Notch overactivation in retromer mutants warrants future investigation (Li, 2018).

The ability of vps35 mutant neoplastic neuroblasts to metastasize upon transplantation is intriguing. Metastasis of brain tumor cells derived from neuroblast lineages has never been observed in the developing fly larval brains, likely because the limited time window of fly larval development precludes tumor progression and metastasis. Transplantation assay, however, provides the ectopic microenvironment and allows cancer progression in a much longer time scale (months, or even years upon retransplantation). Importantly, mutations that caused metastasis of fly brain tumor cells upon transplantation have also been implicated in various human cancers. Future studies on the transcriptional profiling of the distal metastatic colonies and stepwise characterization of this long-range metastatic process promise to provide fresh mechanistic insights into the enormously complex process of cancer metastasis (Li, 2018).

Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. This study characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define photoreceptor subtype identity. This model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning (Mishra, 2018).

In the fruit fly Drosophila melanogaster, all parts of the visual system develop from an optic placode, which forms in the dorsolateral region of the embryonic head ectoderm. During embryogenesis, neuroepithelial cells of the optic placode are patterned to form two subdomains. The ventroposterior domain gives rise to the primordium of the larval eye and consists of two photoreceptor (PR) precursor types (primary and secondary precursors), whereas the dorsal domain harbors neuroepithelial precursors that generate the optic lobe of the adult visual system. The basic helix-loop-helix transcription factor Atonal (Ato) promotes PR precursor cell fate in the larval eye primordium. The orphan nuclear receptor Tailless (Tll) is confined to the optic lobe primordium and maintains non-PR cell fate. Hedgehog (Hh) and Notch (N) signaling are critical during the early phase of optic lobe patterning. The secreted Hh protein is required for the specification of various neuronal and non-neuronal cell types, while Notch acts as neurogenic factor preventing ectodermal cells from becoming neuronal precursors by a process termed lateral inhibition. In the optic placode Ato expression is promoted by Hh and the retinal determination genes sine oculis (so) and eyes absent (eya). Notch delimits the number of PR precursors and maintains a pool of non-PR precursors. Ato is initially expressed in all PR precursors in the placode and its expression gets progressively restricted to primary precursors. In a second step, primary precursors recruit secondary precursors via EGFR signaling: primary precursors express the EGFR ligand Spitz, which is required in secondary precursors to promote their survival. After this initial specification of primary and secondary PR precursors, the transcription factors Senseless (Sens), Spalt (Sal), Seven-up (Svp) and Orthodenticle (Otd) coordinate PR subtype specification. Sens and Spalt are expressed in primary PR precursors, while Svp contributes to the differentiation of secondary PR precursors. By the end of embryogenesis, primary PR precursors have fully differentiated into blue-tuned Rhodopsin5 PRs (Rh5), while secondary PR precursors have differentiated into green-tuned Rhodopsin6 PRs (Rh6). While the functional genetic interactions of transcription factors controlling PR subtype specification has been thoroughly studied, it remains unknown how the placode is initially patterned by the interplay of Hh and Notch signaling pathways. Similarly, the mechanisms of how ato and tll-expressing domains are set up to ensure the correct number of primary and secondary PR precursors as well as non-PR precursors of the optic lobe primordium remain unknown (Mishra, 2018).

This study describes the genetic mechanism of neuroepithelial patterning and acquisition of PR versus non-PR cell fate in the embryonic optic placode and provide the link to subsequent PR subtype identity specification. The non-overlapping expression patterns of ato and tll in the optic placode specifically mark domains giving rise to the larval eye precursors (marked by Ato) and the optic lobe primordium (marked by Tll). ato expression in the larval eye primordium is temporally dynamic and can be subdivided into an early ato expression domain, including all presumptive PR precursors and a late ato domain, restricted to presumptive primary PR precursors. The ato expression domain directly forms a boundary adjacent to tll expressing precursors of the optic lobe primordium. tll is both necessary and sufficient to delimit primary PR precursors by regulating ato expression. Hh signaling regulates the cell number in the optic placode and controls PR subtype specification in an ato- and sens-dependent manner. Finally, this study also shows that Notch has two temporally distinct roles in larval eye development. Initially, Notch represses ato expression by promoting tll expression and later, Notch controls the binary cell fate decision of primary versus secondary PR precursors by repressing sens expression. In summary, this study has identified a network of genetic interactions between cell-intrinsic and cell-extrinsic developmental cues patterning neuroepithelial cells of the optic placode and ensuring the timely specification of neuronal subtypes during development (Mishra, 2018).

Neurogenic placodes are transient structures that are formed by epithelial thickenings of the embryonic ectoderm and give rise to most neurons and other components of the sensory nervous system. In vertebrates, cranial placodes form essential components of the sensory organs and generate neuronal diversity in the peripheral nervous system. How neuronal diversity is generated varies from system to system, and different gene regulatory networks have been proposed for each particular type of neuron. Interestingly, some transcription factors, like Atonal, play an evolutionary conserved role during neurogenesis both in Drosophila and in vertebrates (Mishra, 2018).

Neuroepithelial patterning of the Drosophila optic placode exhibits unique segregation of larval eye and optic lobe precursors during embryogenesis. This study has identified genetic mechanisms that control early and late steps in specifying PR versus non-PR cell fate that ensure the expression of precursor cell fate determinants. During germband extension at stage 10, transcriptional regulators (so, eya, ato and tll) show complex and partially overlapping expression patterns in the optic placode. Their interactions with the Notch and Hh signaling pathways define distinct PR and non-PR domains of the larval eye and optic lobe primordium. Intriguingly, the results show a spatial organization of distinct precursor domains, supporting a new model of how the subdivision of precursor domains emerges. In agreement with previous studies initially the entire posterior ventral tip expresses Ato, defining the population of cells that give rise to PR precursors, while neuroepithelial precursors for the presumptive optic lobe are defined by Tll-expression in the anterior domain of the optic placode. Subsequently, Ato expression ceases in the ventral most cells and thus gets restricted to about four primary PR precursors that are located directly adjacent to the Tll expression domain. Hence, a few cell rows are between the primary PR precursors and the ventral most edge of the optic placode. This is in agreement with a recent observation on the transcriptional regulation of ato during larval eye formation. Thus, primary PR precursors are directly adjacent to the Tll-expressing cells while the Ato and Tll negative domain of secondary PR precursors is located at the posterior ventral most tip of the optic placode. Setting the Tll-Ato boundary is critical to define the number of putative secondary PR precursors, which can be recruited into the larval eye, probably via EGFR signaling. A model is proposed during which coordinated action of Hh, Notch and Tll restricts the initially broad expression of Ato to primary PR precursors (see Ato to primary PR precursors). Lack of Tll results in a de-repression of Ato and results in an increased number of primary PR precursors, which in turn recruit secondary PR precursors. Interestingly, while tll mutants show an increase in both primary and secondary PR precursors, the ratio between both subtypes is maintained. This notion further displays similarities of ommatidal formation in the adult eye-antennal imaginal disc, where Ato expressing R8-precursors recruit R1-R6. In the eye-antennal disc, specification of R8-precursors determines the total number of ommatida and therefore also the total number of PRs, the ratio of R8 to outer PRs however always remains the same. Thus, the initial specification of primary PR precursors defines the total number of PRs in the larval eye similarly to R8 PRs, and the ratio of founder versus recruited cells remains constant. Interestingly, the maintenance of primary versus secondary PR precursor ratio is also maintained in ptc mutants further supporting this model (Mishra, 2018).

During photoreceptor development in the eye-antennal imaginal disc hh is expressed in the posterior margin and is required for the initiation and progression of the morphogenetic furrow as well as the regulation of ato expression. During embryogenesis the loss of hh results in a complete loss of the larval eye, while increasing Hh signaling (by means of mutating ptc) generates supernumerary PRs in the larval eye. During early stages, an increase of Ato expression was found in ptc mutants suggesting that similarly to the eye-antennal disc Hh positively regulates ato expression. The observed increase of Ato-expressing cells is not due to a reduction of Tll but is likely due to increased cell proliferation in ptc mutants. Hh also controls proliferation during the formation of the Drosophila compound eye (Mishra, 2018).

During embryonic nervous system development Notch dependent lateral inhibition selects individual neuroectodermal cells to become neuroblasts. Notch represses neuroblast cell fate and promotes ectodermal cell fate. During compound eye development, Notch regulates Ato expression and acts through lateral inhibition to select Ato expressing R8 PR precursors. Similarly, during Drosophila larval eye development, Notch is required for regulating PR cell number by maintaining epithelial cell fate of the optic lobe primordium. Inhibiting Notch signaling leads to a complete transformation of the optic placode to PRs of the larval eye. In the absence of Notch signaling, Ato expression is expanded in the optic placode and as a result the total number of PRs is increased. Despite the increase of the overall PR-number the number of secondary PR precursors is significantly decreased or lost in the absence of Notch activity. In the compound eye Notch promotes R7 cell fate by repressing the R8-specific transcription factor Sens. It was also proposed that genetic interaction between Notch and Sens is required for sensory organ precursor (SOP) selection in the proneural field in a spatio-temporal manner. This study found that during PR subtype specification Notch represses Sens expression, thereby controlling the binary cell fate decision of primary versus secondary PR precursors. Therefore, in the absence of Notch signaling, Sens expression represses the secondary PR precursor fate. As a result, all PR precursors are transformed and acquire primary PR precursor identity. In conclusion, this study observed that Notch is essential for two aspects during optic placode patterning. First, Notch activity is critical for balancing neuroepithelial versus PR cell fate mediated through Tll-regulated Ato expression. Second, Notch regulates the binary cell fate decision of primary versus secondary PR precursor cell fate through the regulation of Sens expression (Mishra, 2018).

Activation of the Notch signaling pathway in vivo elicits changes in CSL nuclear dynamics

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, this study developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or 'assisted' loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which is proposed to confer sensitivity to small input changes and facilitate timely shut-down (Gomez-Lamarca, 2018).

Until recently, most existing models have portrayed CSL as a molecule with long DNA residence that serves as a static platform for exchange between NICD and co-repressors. This analysis, using a combination of FRAP and single-molecule tracking (SMT) to measure Su(H) dynamics, reveals a very different story and highlights two important characteristics. First, in Notch-OFF conditions, Su(H) normally undergoes very transient DNA residency, despite the fact that it is important for repression of the target loci. This implies that prolonged binding is not a prerequisite for repression. It also argues against a model where co-factors are exchanged while CSL remains bound to DNA. Second, in Notch-ON conditions, there is a striking enrichment of Su(H) at E(spl)-C, its primary target locus, where its dwell time is significantly increased. These changes in CSL-binding dynamics, can enable a sensitive and accurate response to NICD at its target sites (Gomez-Lamarca, 2018).

This study has found that NICD enhances both Su(H) recruitment and residence time at its target locus E(spl)-C, via a combination of mechanisms. One key step is that NICD-Su(H) complexes induce local changes in chromatin, which requires Trr (MLL3/4), a long-range co-activator that can contribute to chromatin opening. Notably, the consequence of NICD-induced chromatin opening is that it renders the target enhancers more accessible for additional complexes, regardless of whether they contain NICD or Hairless. Since binding of Hairless and NICD to Su(H) are mutually exclusive, it is likely that these represent discrete activator (Su(H)-NICD) and repressor (Su(H)-Hairless) complexes, although this study has not formally shown that Hairless recruitment relies on Su(H). This enhanced recruitment by NICD resembles that described for the glucocorticoid receptor and other factors, referred to as 'assisted loading,' whereby the binding of one protein complex helps the binding of another. It is proposed that the localized chromatin remodeling brought about by Su(H)-NICD reduces obstacles (e.g., moves nucleosomes) to facilitate DNA binding, i.e., effectively increasing K_ON. Such indirect cooperativity would render the response very sensitive to signal levels (Gomez-Lamarca, 2018).

A second aspect helps explain how the transiently bound Su(H)-NICD complexes can successfully activate transcription. Although at genomic locations with paired binding motifs the dimerization of NICD could enhance binding, the data argue that the presence of Mam itself confers a longer dwell time to the activator complex, most likely by favoring contacts with additional chromatin-associated factors, such as Mediator complex. One candidate to mediate these effects was CBP, a histone acetyltransferase that interacts with Mam and is necessary for its ability to stimulate transcription. However, inhibiting CBP or depleting the Mediator subunit Med7 only slightly modified the Su(H) dynamics, suggesting that each makes at best a modest contribution to the change in its behavior. As neither manipulation fully replicated the effects of Mam inhibition/depletion, despite preventing transcriptional activation, it is likely that they also act at a later step in the initiation process. Thus Mam is likely to exert its early effects on Su(H) recruitment through a combination of other chromatin factors besides CBP. The interaction of the tripartite Su(H)-NICD-Mam complex with these chromatin factors, although still transient, could confer a probabilistic switch between an inactive state and an active state, by leaving a longer-lasting modification or reorganization of the chromatin template or initiation complex (Gomez-Lamarca, 2018).

The fact that the Su(H)-NICD activator complex also enhances recruitment of Hairless co-repressor complexes was entirely unexpected based on prevailing models, and has several important consequences. First, it will bring opposing enzymatic activities (e.g., both histone acetyl-transferases and histone deacetylases), which could create a covalent modification cycle with switch-like properties, potentially further sensitizing responses to Notch. Second, enhanced recruitment of Hairless would ensure that genes are rapidly turned off after the signal decays, the switch operating in the converse direction when NICD levels decrease. Such 'facilitated repression,' where transcriptional activators promote global chromatin decondensation to facilitate loading of repressors, has also been described during circadian gene regulation where it operates as an amplitude rheostat (Gomez-Lamarca, 2018).

In conclusion, in vivo analysis of the mechanisms underlying the transcriptional response to Notch signaling reveal the fundamental importance of changes in DNA-binding dynamics and highlight how different mechanisms combine to enhance Su(H) recruitment and dwell time at E(spl)-C in Notch-ON cells. Whether both mechanisms operate at all Notch-regulated loci remains to be established, but they will likely be relevant for most genes where CSL occupancy was found to increase in Notch-ON conditions. Furthermore, this new insight into Notch signaling leads to a proposal that similar changes in the dynamics of nuclear effectors may also operate to deliver proper transcriptional outputs of other key signaling pathways (Gomez-Lamarca, 2018).

Drosophila neuroblast selection is gated by Notch, Snail, SoxB, and EMT gene interplay

In the developing Drosophila central nervous system (CNS), neural progenitor (neuroblast [NB]) selection is gated by lateral inhibition, controlled by Notch signaling and proneural genes. However, proneural mutants still generate many NBs, indicating the existence of additional proneural genes. Moreover, recent studies reveal involvement of key epithelial-mesenchymal transition (EMT) genes in NB selection, but the regulatory interplay between Notch signaling and the EMT machinery is unclear. This study finds that SoxNeuro (SoxB family) and worniu (Snail family) are integrated with the Notch pathway, and constitute the missing proneural genes. Notch signaling, the proneural, SoxNeuro, and worniu genes regulate key EMT genes to orchestrate the NB selection process. Hence, this study has uncovered an expanded lateral inhibition network for NB selection and demonstrate its link to key players in the EMT machinery. The evolutionary conservation of the genes involved suggests that the Notch-SoxB-Snail-EMT network may control neural progenitor selection in many other systems (Arefin, 2019).

Based upon previous studies, and the findings in this study, SoxN is necessary for NB generation. Regarding the Snail family, previous studies did not find apparent reductions of NBs numbers in sna, esg, and wor triple mutants. However, these studies focused on early stages of neurogenesis and may not have covered the complete span of NB formation. Analyzing embryos at Stage 11, after most, if not all, NBs have formed, this study found that wor mutants simultaneously removing one gene copy of sna and esg do indeed display a significant reduction in NB numbers. In addition, while AS-C and wor mutants both show a partial loss of NBs, this study found that AS-C;wor double mutants display significantly more severe loss than either single mutant alone. Moreover, misexpression of SoxN or wor, from optimized transgenes, reveals sufficiency for these genes in generating ectopic NBs in the ectoderm. Strikingly, SoxN/ wor co-misexpression can generate extensive numbers of ectopic NBs even in a genetic background lacking any AS-C proneural gene activity. Finally, both SoxN and wor are regulated by, and regulate, the Notch pathway. Based upon these findings, it is proposed that SoxN and wor constitute the missing proneural genes (Arefin, 2019).

What are the connections between the Notch pathway, SoxN, and wor? Logically, in NICD and m8 misexpression, which result in fewer NBs, a reduction was observed in SoxN and wor expression. However, counterintuitively, Notch and neur mutants, which display more NBs, also displayed reductions of SoxN and wor expression. One reason for this effect may pertain to the Notch signaling being important for Crb expression, and that crb is important for both SoxN and wor expression. The reciprocal connection, i.e., between SoxN/wor and the Notch pathway, as measured by m8-GFP expression, is also partly dichotomous. Specifically, SoxN and wor mutants display fewer NBs, a NotchON effect, but reduced m8-GFP expression, a NotchOFF effect. Similarly, misexpressions of SoxN^D and wor^D trigger more NBs, a NotchOFF effect, but increased m8-GFP expression, a NotchON effect. However, these findings mirror the activation of E(spl) genes by the proneural genes and point to a balancing act between the proneural, SoxN, and wor genes, as well as the Notch pathway (Arefin, 2019).

Previous studies found that the proneural genes, i.e., ac, sc, and l'sc, are upstream of wor. In line with this, in AS-C mutants loss of wor cells (NBs) and reduction of wor expression levels were observed in the NBs still generated. Similarly, it was previously found that ase misexpression increased wor expression in NBs. Previous studies revealed that SoxN mutants show reduced Ase expression in NBs, and that misexpression of SoxN could activate wor expression, whereas neither wor nor ase GoF or LoF affected SoxN expression in NBs (Bahrampour et al., 2017). In addition, SoxN mutants were found to display loss of ac, l'sc, and wor expression. This indicates that SoxN, which is expressed in the entire early neuroectoderm, acts upstream of the proneural genes, while proneural genes act upstream of wor. However, this SoxN->proneural->wor regulatory flow is complex: while both SoxN and wor misexpression can trigger NB generation, l'sc does not have this potency. Moreover, while SoxN expression may occur first, both AS-C and wor appear to be important for maintained and elevated SoxN expression in NBs. Hence, SoxN, AS-C, and wor appear to be involved in a mutually reinforcing interplay, which ensures robust NB selection once the Notch pathway balanced is tipped (Arefin, 2019).

DNA-binding studies for the factors studied herein, and analysis of related family members (D, Sna, esg, and Ase), suggest that the elaborate transcriptional interplay between all of the aforementioned TDs/co-factors, i.e., NICD/Su(H)/Mam, E(spl), proneural, SoxN, wor, and their respective genes, may result from direct transcriptional regulation (Arefin, 2019).

In Drosophila, crb and sdt control the epithelial polarity in a number of tissues. Recent studies revealed that Crb stabilizes Notch, and accordingly crb mutants show more NBs. This study furthermore found that crb overexpression results in fewer NBs and, interestingly, that crb overexpression can partly rescue Notch mutants. The reduction of NB numbers in crb mutants is logically accompanied by reduced m8-GFP expression, while, surprisingly, crb misexpression also triggered reduced m8-GFP expression. Notch signaling was found to activate Crb expression, evident by downregulation of Crb in Notch and neur mutants, and upregulation of Crb in NICD and m8 misexpression. Hence, with the exception of crb misexpression on m8-GFP, a clear-cut interplay between canonical Notch signaling and crb/Crb emerges. However, this interplay would constitute a runaway loop, with Notch activating crb, and Crb supporting Notch activation. Perhaps the finding that crb overexpression reduces m8-GFP points to a nebulous brake pedal in this loop (Arefin, 2019).

Regarding the connection between crb with SoxN, wor, and the proneural genes, clear interplay, albeit with a reverse logic was found for mutants versus misexpression. Specifically, misexpression of SoxN, wor, or proneural genes, which generates more NBs, with encompassing delamination, also results in reduced Crb expression. However, surprisingly, SoxN, wor, and proneural mutants, which display fewer NBs, also show reduced Crb expression, underscoring the balancing act of these gene regulations. It is envisioned that this may reflect a role for SoxN, wor, and AS-C in the proneural clusters (prepattern), where they may act to ensure Crb expression in the equivalence regions, thereby ensuring an efficient lateral inhibition process (Arefin, 2019).

In addition, further complexity regarding the role of crb stems from recent findings revealing that Neur, an E3 ligase critical for Dl endocytosis, also controls the stability of Sdt, and thereby affects Crb protein levels. It is tempting to speculate that NotchOFF cells (NBs), which maintain proneural gene expression and hence activate neur expression, will have increased Neur, and hence increased endocytosis of Sdt, and thereby decreased Crb levels, leading to reduced Notch receptor activation. Because Neur also increases endocytosis of Dl, high Neur levels would help drive Notch activation in the neighboring cells (epidermal cells). Moreover, since Notch (NICD and m8^CK2) activates Crb expression and represses neur expression, this should ensure more Crb in the NotchON cells (epidermal cells), thereby further supporting Notch activation. By these mechanisms, the transcriptional regulation of crb/neur gene expression and the stability/endocytosis control of Crb/Sdt/Dl levels and localization, and thereby Notch activation, act as a hitherto undiscovered loop providing additional thrust to the lateral inhibition decision (Arefin, 2019).

Similar to the TF interplay described above, the gene-specific and/or genome-wide DNA-binding studies indicate that the gene regulation of crb and neur may be mediated by direct transcriptional regulation of the Notch pathway TFs (NICD/Su(H)/Mam, E(spl), proneural), as well as the SoxB and Snail family TFs (Arefin, 2019).

EMT has been extensively studied in mammals and has revealed roles for the Crb, Scribble (Scrib), and Par complexes, as well as for Notch signaling and the Snail and SoxB families. Previous studies, and the current findings, demonstrate that the majority of these genes also play key roles during Drosophila NB selection and delamination. This supports the notion that NB selection and delamination could be viewed as an EMT-like process. However, the NB-type EMT differs from canonical EMT in several aspects. In canonical EMT, all apical polarity complexes (Crb, Par, and Scrib complexes) and Cadherins are turned off, and there is no activation of asymmetry genes. Hence, delamination is followed by symmetric cell division and cell migration. In contrast, in 'NB-type EMT,' while, similarly, the Crb complex and Cadherins are turned off, the Par complex (baz/par-3, par-6, and aPKC) and the Scribble complex (scrib, dlg, and lgl) remain expressed, and asymmetric genes, e.g., mira, insc, and pros, are turned on. In addition, within NBs, SoxN, wor, and ase activate key cell-cycle driver genes, i.e., Cyclin E and stg, and repress expression of the cell-cycle inhibitor dacapo. These gene expression changes result in NB delamination, but retain apico-basal polarity in the NB, and ensure repetitive rounds of asymmetric cell divisions, generating the unique features of CNS lineages (Arefin, 2019).

Based upon these findings and those previously published, a model emerges wherein SoxB acts early to govern neuroectodermal competence, intersecting with the early transient wave of proneural gene expression in the proneural clusters. SoxN and proneural genes engage in interplay with the Notch-mediated lateral inhibition process, which is also gated by Crb-Sdt-Neur membrane-localized control of Notch receptor activity and Dl ligand endocytosis. The outcome of these interactions is that early NBs become NotchOFF and elevate their SoxN and proneural expressions, as well as activate wor and Ase expression. This results in the downregulation of a subset of EMT genes (i.e., Crb complex and Cadherins), while the Scrib and Par complexes are maintained. The combined action of SoxN, proneural, wor, and Ase triggers activation of asymmetric cell division genes and cell-cycle driver genes, the outcome of which is NB delamination, followed by asymmetric cell divisions and lineage generation. In contrast, the surrounding NotchON cells continue expressing E(spl) genes, downregulating the SoxN, proneural, neur, and Dl genes. This results in the continued expression of the Crb, Scrib, and Par complexes, as well as failure to activate wor, ase, asymmetric, and cell-cycle genes. The combined effect of these regulatory decisions is that these cells remain in the ectoderm and do not divide (Arefin, 2019).

The process of NB selection bears many similarities to the process of peripheral SOP selection. However, while one Snail family gene, esg, is indeed important also for PNS precursor development, there is no study linking sna or wor, nor the SoxB genes SoxN and D, to SOP selection. Hence, while both SOP and NB formation requires AS-C and esg, NB formation additionally requires Sna, wor, SoxN, and Dichaete. It is tempting to speculate that this may relate to two clear differences between SOPs and NBs: EMT and proliferation. Specifically, while NBs undergo a complete EMT-like process, SOPs remain associated with the ectoderm. Moreover, NBs can divide up to 20 times, making 40-cell lineages, while most, if not all, SOPs make 5-cell lineages. The connections between the SoxB and Snail families with NB and GMC proliferation and the EMT pathway (this paper as well as Bahrampour et al., 2017, 2019) suggest that both of these NB-specific properties are driven by the expanded TF code specific to NBs (Arefin, 2019).

In mammals, the neuroepithelial-to-radial glia cell (NE-RGC) transition is in many aspects analogous to the NB selection and delamination process in Drosophila. Intriguingly, recent studies suggest that NE-RGC can perhaps also be viewed as an EMT-like process, although in this case the process has been modified even further, and the RGC retains an apical connection throughout neurogenesis and undergoes interkinetic nuclear migrations. Strikingly, in two recent studies the NE-RGC transition was found to involve the mouse Snail and Scratch factors, both of which are members of the Snail family. Other players in the NB-selection program outlined above also play key roles in the early development of the mammalian CNS and in the NE-RGC transition, although the direct comparison of gene function and cell behavior becomes nebulous. Hence, it would appear that the neuroectoderm->neural progenitor selection and delamination process has undergone several evolutionary modifications, perhaps becoming less and less akin to a canonical EMT process in more derived animals. Nevertheless, it is tempting to speculate that several of the basic principles of EMT are utilized in the mammalian NE-RGC process and that viewing it as such may be helpful for future studies (Arefin, 2019).

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

Information from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2-MCP system to visualize nascent transcripts in single cells in Drosophila embryos, it was revealed how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers, two key principles were uncovered. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context (Falo-Sanjuan, 2019).

Developmental signaling pathways have widespread roles, but currently relatively little is known about how signaling information is decoded to generate the right transcriptional outcomes. This study set out to investigate principles that govern how Notch activity is read by target enhancers in the living animal, using the MS2-MCP system to visualize nascent transcripts in Drosophila embryos and focusing on two enhancers that respond to Notch activity in the mesectoderm (MSE). Three striking characteristics emerge. First, MSE enhancers are sensitive to changes in the levels of NICD, which modulate the transcriptional burst size rather than increasing burst frequency. Second, the activation of both mesectoderm (MSE) enhancers is highly synchronous. Indeed, within one nucleus the two enhancers become activated within few minutes of one another. Third, both MSE enhancers confer a sustained response in the wild-type context. This synchronized and persistent activity of the MSE enhancers contrasts with the stochastic and bursty profiles that are characteristics of most other enhancers that have been analyzed and relies on the MSE enhancers being 'primed' by regional transcription factors Twist and Dorsal. It is proposed that such priming mechanisms are likely to be of general importance for rendering enhancers sensitive to signals so that a rapid and robust transcriptional response is generated (Falo-Sanjuan, 2019).

Transcription of most genes occurs in bursts interspersed with refractory periods of varying lengths that are thought to reflect the kinetic interactions of the enhancer and promoter. However, the MSE enhancers appear to sustain transcription for 40-60 min, without detectable periods of inactivity, though very short off periods might not have been resolved by the assays used in this study. Calculation of the autocorrelation function (ACF) in traces from these nuclei suggest very slow transcriptional dynamics, consistent with one long period of activity rather than overlapping short bursts. This fits with a model where promoters can exist in a permissive active state, during which many 'convoys' of polymerase can be fired without reverting to a fully inactive condition (Tantale, 2016). The rapid successions of initiation events are thought to require Mediator complex (Tantale, 2016), which was also found to play a role in the NICD-mediated increase in residence time of CSL complexes (Gomez-Lamarca, 2018). It is proposed that sustained transcription from m5/m8 and sim reflects a switch into a promoter permissive state, in which general transcription factors like Mediator remain associated with the promoter so long as sufficient NICD is present, allowing repeated re-initiation (Falo-Sanjuan, 2019).

However, the ability to drive fast and sustained activation is not a property of NICD itself. For example, when ectopic NICD was supplied, cells in many regions of the embryo responded asynchronously and underwent short bursts of activity. Furthermore, variable and less sustained cell-by-cell profiles were generated in the MSE region when the binding motifs for Twist and Dorsal in m5/m8 were mutated. The presence of these regional factors appears to sensitize the enhancers to NICD, a process referred to as enhancer priming. This has two consequences. First, it enables nuclei to rapidly initiate transcription in a highly coordinated manner once NICD reaches a threshold level. Second, it creates an effective 'state transition' so that the presence of NICD can produce sustained activity. A priming mechanism, rather than classic cooperativity, is proposed because Twist and Dorsal alone are insufficient to drive enhancer activity. Furthermore, since m5/m8 and sim rapidly achieve sustained activity when NICD is produced, it is likely that Twist and Dorsal are required prior to NICD recruitment, although both may continue to play a role after transcription is initiated, as suggested by the lower mean levels when Twist or Dorsal motifs are mutated. Another contributory factor may be recruitment of the co-repressor complex containing CSL and Hairless, whose presence at primed enhancers could poise for activation and set the threshold (Falo-Sanjuan, 2019).

It is suggested that the synchronous activation of the MSE enhancers reflects their requirements for a critical concentration of NICD is borne out by their responses when levels of NICD are increased. Notably, while sim and m5/m8 had almost identical dynamics in wild-type embryos, their response to ectopic NICD differed, suggesting that they detect different thresholds. Indeed, doubling the dose of ectopic NICD further accelerated the onset time of sim in agreement with the model that the enhancers detect NICD levels. Threshold detection does not appear to rely on the arrangement of CSL motifs, as onset times of sim or m5/m8 were unaffected by changes in the spacing of CSL paired sites. In contrast, mutating Twist- or Dorsal-binding motifs in m5/m8 delayed the onset, arguing that these factors normally sensitize the enhancer to NICD, enabling responses at lower thresholds (Falo-Sanjuan, 2019).

It is proposed that enhancer priming will be widely deployed in contexts where a rapid and consistent transcriptional response to signaling is important, as in the MSE where a stripe of cells with a specific identity is established in a short time window. In other processes where responses to Notch are more stochastic, as during lateral inhibition, individual enhancers could be preset to confer different transcription dynamics. This appears to be the case for a second enhancer from E(spl)-C (m8NE), which generates a stochastic response in the MSE cells, similar to that seen for the MSE enhancers when Twist and Dorsal sites are mutated. This illustrates that the presence or absence of other factors can toggle an enhancer between conferring a stochastic or deterministic response to signaling (Falo-Sanjuan, 2019).

Manipulating NICD levels revealed that Notch-responsive enhancers act as analog devices that can measure and broadcast variations in levels. Increased NICD levels have a consistent effect on enhancer activity irrespective of the priming state of the enhancer: an increase in burst size. Transcriptional bursting has been formalized as a two-state model where the promoter toggles between ON and OFF states, conferring a transcription initiation rate when ON. Changes in duration or frequency of bursts lead to an overall increase in transcription. Most commonly, differences in enhancer activity have been attributed to changes in switching-on probability (K_on), leading to changes in burst frequency. It was therefore surprising to find that higher doses of NICD did not increase burst frequency. Instead, they produced bigger bursts, both by increasing bursting amplitude, equivalent to the rate of transcription initiation, and bursting length, indicative of the total time the enhancer stays in the ON state. Modifications to the CSL motifs also impacted on the same parameters. Thus, enhancers with paired motifs (SPS) produced larger transcription bursts than those where the motifs are further apart. This suggests that paired motifs can 'use' the NICD present more efficiently, potentially because optimally configured sites increase the likelihood that at least one NICD will be bound at any time. Interestingly, even though m5/m8 and sim contain different arrangements and numbers of CSL motifs they have converged to produce the same mean levels of transcription in wild-type embryos (Falo-Sanjuan, 2019).

Two models would be compatible with the observations that effective NICD levels alter the burst size. In the first model, increasing the concentration of NICD when the enhancer is activated would create larger Pol II clusters. This is based on the observation that low-complexity activation domains in transcription factors can form local regions of high concentration of transcription factors, so-called 'hubs,' which in turn are able to recruit Pol II. As the lifetime of Pol II clusters appears to correlate with transcriptional output, the formation of larger Pol II clusters would in turn drive larger bursts. In the second model, NICD would be required to keep the enhancer in the ON state, for example, by nucleating recruitment of Mediator and/or stabilizing a loop between enhancer and promoter, which would in turn recruit Pol II in a more stochastic manner. General factors such as Mediator have been shown to coalesce into phase-separated condensates that compartmentalize the transcription apparatus (Cho, 2018, Sabari, 2018, Boija, 2018), and these could form in a NICD-dependent manner. Whichever the mechanism, persistence of the clusters and/or ON state requires NICD yet must be compatible with NICD having a short-lived interaction with its target enhancers (Gomez-Lamarca, 2018). Furthermore, the fact that the activity of m5/m8 saturates with one eve2 directed NICD expression construct and cannot be enhanced by providing a more active promoter suggests that there is a limit to the size or valency of the clusters that can form (Falo-Sanjuan, 2019).

Although unexpected, the ability to increase burst size appears to be a conserved property of NICD. Live imaging of transcription in response to the Notch homolog, GLP-1, in the C. elegans gonad also shows a change in burst size depending on the signaling levels (Lee, 2019). As the capability to modulate burst size is likely to rely on the additional factors recruited, the similarities between the effects in fly and worm argue that a common set of core players will be deployed by NICD to bring about the concentration-dependent bursting properties (Falo-Sanjuan, 2019).

TM2D genes regulate Notch signaling and neuronal function in Drosophila

TM2 domain containing (TM2D) proteins are conserved in metazoans and encoded by three separate genes in each model organism species that has been sequenced. Rare variants in TM2D3 are associated with Alzheimer's disease (AD) and its fly ortholog almondex is required for embryonic Notch signaling. However, the functions of this gene family remain elusive. This study knocked-out all three TM2D genes (almondex, CG11103/amaretto, CG10795/biscotti) in Drosophila and found that they share the same maternal-effect neurogenic defect. Triple null animals are not phenotypically worse than single nulls, suggesting these genes function together. Overexpression of the most conserved region of the TM2D proteins acts as a potent inhibitor of Notch signaling at the gamma-secretase cleavage step. Lastly, Almondex is detected in the brain and its loss causes shortened lifespan accompanied by progressive motor and electrophysiological defects. The functional links between all three TM2D genes are likely to be evolutionarily conserved, suggesting that this entire gene family may be involved in AD (Salazar, 2021).

Alzheimer's disease (AD) is the most common neurodegenerative disease affecting the aging population and accounts for the large majority of age-related cases of dementia. AD is pathologically characterized by histological signs of neurodegeneration that are accompanied by formation of extracellular plaques and intra-neuronal tangles. Numerous studies have identified genetic factors that contribute to AD risk and pathogenesis. Rare hereditary forms of AD are caused by dominant pathogenic variants in APP (Amyloid Precursor Protein), PSEN1 (Presenilin 1) or PSEN2 (Presenilin 2). These three genes have been extensively studied using variety of experimental systems, and the resultant knowledge has led to greater understanding of how they contribute to the formation of extracellular plaques found in both familial and sporadic AD brains. PSEN1 and PSEN2 are paralogous genes that encode the catalytic subunit of the γ-secretase, a membrane-bound intramembrane protease complex. γ-secretase substrates include many type-I transmembrane proteins including APP as well as Notch receptors that play various roles in development and physiology (Salazar, 2021).

While studies of genes that cause familial AD have been critical in providing a framework to study pathogenic mechanisms of this disorder, pathogenic variants in APP and PSEN1/2 are responsible for only a small fraction of AD cases. Familial AD can be distinguished from more common forms of AD because most patients with pathogenic APP or PSEN1/2 variants develop AD before the age of 65 [early-onset AD (EOAD)]. The majority (>95%) of AD cases are late-onset (LOAD, develops after 65 years of age), sporadic and idiopathic in nature. In these patients, it is thought that multiple genetic and environmental factors collaborate to cause damage to the nervous system that converges on a pathway that is affected by APP and PSEN1/2. To reveal common genetic factors with relatively small effect sizes, multiple genome-wide association studies (GWAS) have been performed and identified over 40 loci throughout the genome that confer increase risk to developing AD. The most notable risk-factors are variant alleles in Apolipoprotein E (APOE). Although the precise molecular mechanism by which different alleles of APOE increase or decrease the risk of AD has been extensively debated, a number of studies have proposed that this gene is involved in the clearance of toxic Aβ peptides. A recent meta-analysis has also identified ADAM10 (encoding a β-secretase enzyme that cleaves APP and Notch) as an AD associated locus, suggesting that genes involved in familial EOAD and sporadic LOAD may converge on the same molecular pathway. Functional studies of these and other newly identified risk factors for AD are critical to fully understand the etiology of this complicated disease that lack effective treatments or preventions (Salazar, 2021).

A rare missense variant (rs139709573, NP_510883.2:p.P155L) in TM2D3 (TM2 domain containing 3) is significantly associated with increased risk of developing LOAD through an exome-wide association study in collaboration with the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) consortium. This variant was also associated with earlier age-at-onset that corresponds to up to 10 years of difference with a hazard ratio of 5.3 after adjusting for the common ε4 allele of APOE. Although the function of this gene in vertebrates was unknown and this missense variant was not predicted to be pathogenic based on multiple variant pathogenicity prediction algorithms including SIFT, PolyPhen and CADD, it was experimentally demonstrated that p.P155L has deleterious consequences on TM2D3 function based on an assay established using Drosophila embryos. The Drosophila ortholog of TM2D3, almondex (amx), was initially identified based on an X-linked female sterile mutant allele (amx¹) generated through random mutagenesis. Although homozygous or hemizygous (over a deficiency) amx¹ mutant females and hemizygous (over Y chromosome) males are viable with no morphological phenotypes, all embryos laid by amx¹ hemi/homozygous mothers exhibit severe developmental abnormalities including expansion of the nervous system at the expense of the epidermis (Shannon, 1973; Lehmann, 1983). This 'neurogenic' phenotype results when Notch signaling mediated lateral inhibition is disrupted during cell-fate decisions in the developing ectoderm . By taking advantage of this scorable phenotype, this study showed that the maternal-effect neurogenic phenotype of amx¹ hemizygous females can be significantly suppressed by introducing the reference human TM2D3 expressed under the regulatory elements of fly amx, but TM2D3p.P155L expressed in the same manner fails to do so (Jakobsdottir, 2016). This showed that the function of TM2D3 is evolutionarily conserved between flies and humans, and the molecular function of TM2D3 that is relevant to LOAD may also be related to Notch signaling. More recently, another rare missense variant (p.P69L) in this gene has been reported in a proband that fit the diagnostic criteria of EOAD or frontotemporal dementia, indicating that other TM2D3 variants may be involved in dementia beyond LOAD (Salazar, 2021).

TM2D3 is one of three highly conserved TM2 domain containing (TM2D) proteins encoded in the human genome. The two other TM2 domain-containing proteins, TM2D1 and TM2D2, share a similar protein domain structure with TM2D3, and each protein is encoded by a highly conserved orthologous gene in Drosophila that has not been functionally characterized. All TM2D proteins have a predicted N-terminal signal sequence and two transmembrane domains that are connected through a short intracellular loop that are found close to the C-terminus. Within this loop, there is an evolutionarily conserved DRF (aspartate-arginine-phenylalanine) motif, a sequence found in some G-protein coupled receptors that mediates their conformational change upon ligand binding. The extracellular region between the signal sequence and first transmembrane domain is divergent in different species as well as among the three TM2D containing proteins. In contrast, the sequences of the two transmembrane domains as well as the intracellular loop is highly conserved throughout evolution as well as between the three TM2 domain containing proteins. The three proteins also have short C-terminal extracellular tails that are evolutionarily conserved within orthologs but vary among the three proteins (e.g. TM2D1 has a slightly longer C'-tail than TM2D2 and TM2D3). The molecular functions of these conserved and non-conserved domains of TM2D proteins are unknown (Salazar, 2021).

Amx has been proposed to function at the γ-secretase cleavage step of Notch activation based on a genetic epistasis experiment performed previously (Michellod, 2008). Notch signaling activation is initiated by the binding of the Notch receptor to its ligands (Delta or Serrate in Drosophila). This induces a conformational change of Notch to reveal a cleavage site that is recognized by ADAM10 [encoded by kuzbanian (kuz) in Drosophila]]. Notch receptor that has undergone ADAM10 cleavage (S2 cleavage) is referred to as NEXT (Notch extracellular truncation) and becomes a substrate for γ-secretase. NEXT that is cleaved by γ-secretase (S3 cleavage) releases its intracellular domain (NICD), which then translocates to the nucleus and regulates transcription of downstream target genes. To determine how amx regulates Notch signaling, Michellod and Randsholt (2008) attempted to suppress the embryonic neurogenic phenotype of embryos produced from amx¹ mutant mothers by zygotically overexpressing different forms of Notch using a heat-shock promoter. While NICD was able to weakly suppress the neurogenic defect, NEXT was not able to do so, suggesting that Amx somehow modulates the cleavage step that involves the γ-secretase complex. However, because the phenotypic suppression observed by NICD in this study was very mild and since the authors used Notch transgenes that were inserted into different regions of the genome (thus NICD and NEXT are likely to be expressed at different levels and cannot be directly compared), additional data is required to fully support this conclusion (Salazar, 2021).

In this study, clean null alleles of all three Drosophila TM2D genes were generated using CRISPR/Cas9-mediated homology directed repair (HDR), and their functions assessed in vivo. Surprisingly, CG10795 (TM2D1) and CG11103 (TM2D2) knockout flies are phenotypically indistinguishable from amx (TM2D3) null animals, displaying severe maternal-effect neurogenic phenotypes. Double- and triple-knockout animals were assessed to determine whether these three genes have redundant functions in other Notch signaling dependent contexts during development. The triple-knockout of all TM2D genes did not exhibit any obvious morphological phenotypes but shared the same maternal-effect neurogenic phenotype similar to the single null mutants, suggesting these three genes function together. Further evidence is provided that Amx functions on γ-secretase to modulate Notch signaling in vivo, and a previously unknown role of this gene in the maintenance of neural function in adults was uncovered (Salazar, 2021).

This study functionally characterized TM2D genes through gene knockout and over-expression strategies in Drosophila to gain biological knowledge on this understudied but evolutionarily conserved gene family that has been implicated in AD. It was first shown that the knockout allele of amx (Drosophila homolog of TM2D3) generated by CRISPR is phenotypically indistingusiable from the classic amx¹ allele and displays female sterility and a maternal-effect neurogenic defect. Recently, it was reported that this allele also shows a maternal-effect inductive signaling defect to specify the mesoectoderm during embryogenesis which is another Notch-dependent event (Das, 2020), demonstrating that amx is maternally required for multiple Notch signaling dependent processes during embryogenesis. In addition, the first knockout alleles of amrt (Drosophila ortholog of TM2D2) and bisc (Drosophila ortholog of TM2D1) were generated ,and each null allele that was phenotypically documented mimics the loss of amx. Furthermore, it was revealed that the triple knockout of all three TM2D genes in Drosophila show identical maternal-effect neurogenic phenotypes without exhibiting other obvious Notch signaling-related developmental defects. Moreover, although the over-expression of the full-length Amx do not cause any scorable defects, it was serendipitously found that expression of a truncated form of Amx that lacks the majority of the extracellular domain can strongly inhibit Notch signaling in the developing wing imaginal disc, a tissue in which all three fly TM2D genes are expressed endogenously. Through genetic epistatic experiments using newly generated UAS-Notch transgenic lines, this inhibitory effect was mapped to the γ-secretase cleavage step of Notch activation. Subsequently, it was found that amx null animals have a shortened lifespan, a phenotype that can be rescued by reintroduction of Amx in neurons. This shortening of lifespan phenotype was also seen in amrt and bisc null flies, suggesting that these three genes may also function together in the aging brain. Finally, through assement of climibing behavior and electrophysiological recordings of the giant fiber system, amx null flies were shown to have age-dependent decline in neural function. In summary, it was demonstrated that all three TM2D genes play critical roles in embryonic Notch signaling to inhibit the epithelial-to-neuron cell fate transformation as maternal-effect genes, and that amx is required for neuronal maintenance in the adult nervous system, a function that may be related to the role of human TM2D3 in AD (Salazar, 2021).

Within commonly used genetic model organisms, TM2D genes are found in multicellular animals (both in invertebrates and in vertebrates) but are absent in yeasts (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe) and plants (e.g. Arabidopsis thaliana), suggesting that this family of genes arose early in the metazoan lineage. In humans and flies, there are three TM2 domain-containing genes (TM2D1, TM2D2, TM2D3 in Homo sapiens, and three in Drosophilam, bisc, amrt, amx, each corresponding to a single gene in the other species. Interestingly, this 1:1 ortholog relationship is also seen in mouse (Tm2d1, Tm2d2, Tm2d3), frog (Xenopus tropicalis: tm2d1, tmd2d, tm2d3) zebrafish (Danio rerio: tm2d1, tm2d2, tm2d3) and worm (Caenorhabditis elegans: Y66D12A.21, C02F5.13, C41D11.9). In general, most genes have more paralogous genes in humans compared to flies (for example, one Drosophila Notch gene corresponding to four NOTCH genes in human) as vertebrates underwent two rounds of whole-genome duplication (WGD) events during evolution. Furthermore, teleosts including zebrafish underwent an extra round of WGD, leading to formation of extra duplicates in 25% of all genes (e.g. one NOTCH1 gene in human corresponds to notch1a and notch1b in zebrafish). Hence it is interesting that each of the three TM2D genes remained as single copy genes in various species despite whole genome level evolutional changes, suggesting that there may have been some selective pressure to keep the dosage of these genes consistent and balanced during evolution (Salazar, 2021).

Although the in vivo functions of TM2D1 and TM2D2 have not been studied extensively in any organism, several lines of studies performed in cultured cells suggest that these genes may also play a role in AD pathogenesis. Through an yeast-two hybrid screen to identify proteins that bind to Aβ42, TM2D1 was identified, and this protein was referred to as BBP (beta-amyloid binding protein). TM2D1 was also shown to interact with Aβ40, a non-amyloidogenic form of Aβ, and there is preliminary data that it also binds to APP. The interaction between Aβ peptides and TM2D1 was shown to require the extracellular domain as well as a portion of the first transmembrane domain of TM2D1. Because overexpression of TM2D1 in a human neuroblastoma cell line (SH-SY5Y) increased the sensitivity of these cells to cell death caused by incubation with aggregated Aβ and since the DRF motif was found to be required for this activity, the authors of this original study proposed that TM2D1 may function as a transmembrane receptor that mediates Aβ-toxicity. However, a follow-up study from another group refuted this hypothesis by providing data that TM2D1 is not coupled to G proteins using a heterologous expression system in Xenopus oocytes. Therefore, although the physical link between TM2D1 and Aβ42 is intriguing, the significance of this interaction and its role in AD pathogenesis has been obscure (Salazar, 2021).

Surprisingly, loss of bisc/TM2D1 and amrt/TM2D2 were phenotypically indistinguishable from the loss of amx/TM2D3 in Drosophila. The zygotic loss of each gene did not exhibit any strong developmental defects into adulthood, despite their relatively ubiquitous expression pattern according to large transcriptome datasets Furthermore, the loss of either amrt or bisc caused a reduction of lifespan, again mimicking the loss of amx. More interestingly, the triple null mutants are viable and do not exhibit any morphological defect, suggesting that these genes are not required zygotically during development. In contrast, maternal loss of any single TM2D gene causes a strong embryonic neurogenic defect, which is also seen in embryos laid by triple knockout animals. Neurogenic defect is a classical phenotype in Drosophila that was originally reported in the mid-1930s, and the study of mutants that show this phenotype led to the establishment of the core Notch signaling pathway in the late 1980s and early 1990s. Although the study of neurogenic phenotypes and genes has a long history, this phenotype is a very rare defect that has so far been associated with only 19 genes according to FlyBase, prior to this work. Seven genes show this defect as zygotic mutants [aqz, bib, Dl, E(spl)m8-HLH, mam, N and neur], seven genes are zygotically-required essential genes with large maternal contributions (hence the need to generate maternal-zygotic mutants by generating germline clones to reveal the embryonic neurogenic defect) [Gmd, Gmer, gro, Nct, O-fut1, Psn and Su(H)], one gene has only been investigated by RNAi (Par-1) and four genes including amx are non-essential genes that show maternal-effect neurogenic defects (amx, brn, egh, pcx). Hence, this study has revealed two new genes that are evolutionarily closely linked to amx in this Notch signaling related process (Salazar, 2021).

The similarity of sequences and phenotypes caused by loss of amx, amrt and bisc suggests that the proteins encoded by these genes may function together. The lack of additive or synergistic phenotypes in the double and triple null mutant flies also support this idea. Interestingly, high-throughput proteomics experiments based on co-immunoprecipitation mass spectrometry (co-IP/MS) from human cells have detected physical interactions between TM2D1-TM2D3 and TM2D2-TM2D3, suggesting these proteins may form a protein complex. Further biochemical studies will be required to clarify the functional relationship between the three TM2D proteins. Two additional mammalian datasets further support the hypothesis that these three proteins functions together. First, all three TM2D genes were identified through a large scale cell-based CRISPR-based screen to identify novel regulators of phagocytosis. Individual knockout of TM2D genes in a myeloid cell line was sufficient to cause a similar phagocytic defect based on the parameters the authors screened for (e.g. substrate size, materials to be engulfed). Although the authors of this study did not generate double or triple knockout cell lines to determine whether there were any additive or synergistic effects when multiple TM2D genes were knocked out, this suggests that these three genes may function together in phagocytosis. The authors further note that because these genes are broadly expressed in diverse cell types beyond phagocytic cells in the nervous system, they may play other roles in the brain, consistent with the finding that amx is required neuronally to maintain a normal lifespan. It would be interesting to explore whether age-dependent phenotypes seen in TM2D null mutants can also be suppressed through glial specific rescue experiments to understand the in vivo significance of this group's in vitro findings. Second, preliminary phenotypic data from the International Mouse Phenotyping Consortium indicates that single knockout of mice of Tm2d1, Tm2d2 and Tm2d3 are all recessive embryonic lethal prior to E18.5. Although detailed characterization of these mice will be required and further generation of a triple knockout line is desired, the shared embryonic lethality may indicate that these three genes potentially function together in an essential developmental paradigm during embryogenesis in mice (Salazar, 2021).

Attempts to unravel the function of Amx through overexpression of the full-length protein was uninformative since this manipulation did not cause any scorable phenotype. However, it was serendipitously found that a truncated form of Amx, which only contains the most conserved region of TM2D family proteins, has the capacity to strongly inhibit Notch signaling during wing and notum development. These results were surprising because no wing or bristle defects were seen in the triple TM2D gene family knockout flies, even though all three genes are endogenously expressed in the wing imaginal disc. Through epistasis experiments using a set of new UAS-Notch transgenic lines, AmxΔECD to inhibit Notch signaling at the S3 cleavage step which is mediated by the γ-secretase complex. This data is consistent with earlier epistasis experiments performed on amx¹ in the context of embryonic neurogenesis, further supporting the idea that amx may regulate γ-secretase function in vivo. It was further determined that over-expression of AmxΔECD in the wing imaginal disc causes an accumulation of Notch protein within the cell and at the cell membrane. This phenotype is similar to what is seen upon knockdown of Psn in the wing imaginal disc, which is consistent with earlier findings showing Notch accumulation at the cell membrane in neuroblasts of Psn mutants. In summary, this study showed that ectopic over-expression of a portion of Amx that is conserved among all TM2D proteins causes a strong Notch signaling defect by disrupting a process that realates to γ-secretase, providing additional links between Amx/TM2D3 and AD (Salazar, 2021).

By aging the amx null male flies that are visibly indistinguishable from the control flies (amx null flies with genomic rescue constructs), it was found that loss of amx causes a significant decrease in lifespan, a phenotype that was observed in amrt and bisc null flies. By generating a functional genomic rescue transgene in which Amx is tagged with an epitope tag, this protein was found to be expressed in the adult brain. The short lifespan defect of amx null flies was effectively rescued by reintroduction of Amx in neurons, demostrating the importance of this gene in this cell type. In addition to the lifespan defect, amx null flies also exhibited an age-dependent climing defect, suggesting that their neural function declines with age. By further performing electrophysiological recordings of the giant fiber system, which is a model circuit that is frequently used in neurological and neurodegenerative research in Drosophila, it was found that there is indeed an age-dependent decline in the integrity of this circuit. Through this assay, it was observed that the DLM branch of the giant fiber system begins to show failures earlier than the TTM branch. The DLM is activated by giant fiber neurons that chemically synapse onto PSI (peripherally synapsing interneuron) neurons through cholinergic synapses, which in turn chemically synapse onto motor neurons (DLMn which are glutamatergic) through cholinergic connections. The TTM, in contrast, is activated by giant fiber neurons that electrically synapse onto motor neurons (TTMn which are glutamatergic) through gap junctions, causing a more rapid response. Considering the difference in the sensitivity of the two branches, cholinergic neurons/synapses may be more sensitive to the loss of amx, a neuronal/synaptic subtype that is severely affected in AD in an age-dependent manner (Salazar, 2021).

How does amx maintain neuronal function in aged animals and is this molecular function related to AD? One potential molecular mechanism is through the regulation of γ-secretase in the adult brain. By knocking down subunits of the γ-secretase complex, Psn and Nct, specifically in adult neurons, it was shown that reduction of γ-secretase function decreases lifespan, which was associated with climbing defects as well as histological signs of neurodegeneration. The requirement of γ-secretase components in neuronal integrity has also been reported in mice, suggesting this is an evolutionarily conserved phenomenon. Interestingly, the role of the γ-secretase complex in neuronal maintenance is unlikely to be due to defects in Notch signaling because neurodegeneration has not been observed upon conditional removal of Notch activity in post-developmental brains in flies and in mice. While the precise function of γ-secretase in neuronal maintenance is still unknown, several possibilities including its role in regulating mitochondrial morphology and calcium homeostasis has been proposed based on studies in C. elegans and mice. Investigating whether Amx does indeed regulate γ-secretase in adult neurons and whether it impacts the aforementioned processes will likely facilitate understanding on how this gene regulates neuronal health. Furthermore, considering that TM2D3 and other TM2D genes have been proposed to function in phagocytic cells, and because phagocytosis process plays many roles beyond engulfment of toxic Aβ molecules in the nervous system, Amx may also be playing a role in engulfing unwanted materials that are harmful for the adult brain. For example, loss of the phagocytic receptor Draper in glia cells causes age-dependent neurodegeneration that is accompanied by accumulation of non-engulfed apoptotic neurons throughout the fly brain. Interestingly, a recent study has shown that over-expression of phagocytic receptors can also promote neurodegeneration, indicating the level of phagocytic activity needs to be tightly controlled in vivo. Further studies of amxΔ mutants (as well as amxΔ amrtΔ biscΔ triple mutants) in the context of phagocytosis will likely reveal the precise molecular function of Amx and other TM2D proteins in this process (Salazar, 2021).

Finally, could there be any molecular link between the role of TM2D genes in Notch signaling (proposed based on experiments in Drosophila) and phagocytosis (revealed based on mammalian cell culture based studies), or are they two independent molecular functions of the same proteins? All TM2D proteins have two transmembrane domains connected by a short intracellular loop, making them an integral membrane protein. By tagging the amx genomic rescue construct with a 3xHA tag that does not influence the function of Amx, it was observed that 3xHA::Amx is localized to the plasma membrane as well as intracellular puncta, which likely reflects intracellular vesicles. Interestingly in embryos laid by amxΔ mutant females, a mild and transient but significant alteration was observed in Notch distribution during early embryogenesis. Moreover, a strong accumulation of Notch was observed when AmxΔECD was overexpressed in the developing wing primordium. These data indicate that Amx may affect protein trafficking, which in turn may impact the processing of Notch by the γ-secretase complex. Indeed, Notch signaling is highly regulated by vesicle trafficking and alterations in exocytosis, endocytosis, recycling and degradation all impact the signaling outcome. In fact, multiple studies have proposed that γ-secretase cleavage occurs most effectively in acidified endocytic vesicles. Hence, while amx may be specifically required for the proper assembly or function of the γ-secretase complex, it may alternatively be necessary to bring Notch and other substrates to the proper subcellular location for proteolytic cleavages to occur efficiently. The subcellular localization differences observed between the punctate AmxΔECD, which causes a dramatic Notch signaling defect accompanied by mistrafficking of Notch, and the membranous AmxFL, which does not have this effect, may further support the idea that Amx function as a trafficking module. Similar to Notch signaling, phagocytosis also requires coordination of many cellular trafficking events to expand the plasma membrane to form a phagophore, internalize the particle of interest to generate a phagosome, and fuse the phagosome to lysosomes to degrade its content. By studying the role of TM2D genes and proteins in embryonic Notch signaling, phagocytosis and age-dependent neuronal maintenance, the precise molecular and cellular function of this evolutionarily conserved understudied protein family will likely be understood, which will likely lead to further understanding of molecular pathogenesis of AD and other human diseases. Considering the phenotypic similarities of amrt and bisc to amx in Drosophila embryonic neurogenesis, the similarities between TM2D1-3 in human cells in the context of phagocytosis, and the similarities of Tm2d1-3 knockout mice in the context of embryogenesis, it is proposed that studies of rare genetic variants, epigenetic regulators or proteomic changes in other TM2D genes may reveal novel risk factors or biomarkers in epidemiologic study of AD and other forms of dementia (Salazar, 2021).

Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. This study examined the role of almondex in Notch signaling during early Drosophila embryogenesis. In addition to being required for lateral inhibition in the neuroectoderm, almondex was also found to be partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, it was found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, it is speculated that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis (Das, 2020).

Analysis of amx^N, the first clean null allele of amx, clearly showed that maternal amx is required for Notch signaling during early embryogenesis; this is consistent with previous findings for the amx¹ and amxm alleles. A study using the amxm allele reported that amx is also required zygotically for imaginal disc development (Michellod et al., 2003). However, as with amx¹ zygotic mutants, amxN zygotic mutant flies had no morphological phenotype. Considering that amx^m, is a complex allele with the potential to affect other genes, the zygotic phenotypes reported for the amx^m, allele are likely due to defects in genes other than amx (Das, 2020).

amx^mz embryo phenotypes were examined; amx is required not only for neuroectoderm specification during mid-embryogenesis but also for mesectoderm specification during early embryogenesis. Notch signaling is required for sim expression in mesectodermal cells; in various mutants of core Notch signaling pathway genes, including Notch and Suppressor of Hairless, sim expression is restricted to a very few cells. Interestingly, sim expression was reduced only partially in amx^mz embryos, although these embryos exhibited a strong neurogenic phenotype with full penetration. These results show a partial requirement of amx for activating Notch signaling in mesectodermal cells. This is reminiscent of pcx, another maternal neurogenic gene with a strong neuroblast-segregation defect, since pcx is partially required for activating sim during mesectoderm specification. One interpretation of this differential contribution is that amx and pcx are absolutely required in neuroectodermal cells, but only partially required for mesectoderm specification. However, the molecular mechanisms that would account for such differences remain elusive (Das, 2020).

This analysis demonstrates that the subcellular distribution of the Notch receptor, but not the Dl ligand, depends on a function of maternal amx during early cellularization— specifically in mid-stage 5, when the structure of the apical plasma membrane with its microvilli changes drastically. Immunostaining did not show any noticeable structural changes in F-Actin or the apical plasma membrane in the absence of amx function, and the ER, Golgi, and endosome morphology also appeared unaltered. Thus, amx appears to have a specific role in regulating Notch trafficking during this stage (Das, 2020).

In amx^mz embryos at mid-stage 5, Notch accumulated abnormally in a mesh-like subcellular structure but without any marked increase in colocalization with markers for the ER, Golgi apparatus, or endosomal compartments. Thus, it is clear that Notch mislocalizes in the absence of amx function, but it is not clear which intracellular compartment(s) it localizes to. It is important to note that Notch mislocalization in amx^mz embryos is not caused by aberrant Notch signaling, since Notch localizes normally in pcx^mz embryos at mid-stage 5. It is tempting to speculate, however, that Notch mislocalization might contribute to defects in Notch signaling in the absence of amx function (Das, 2020).

Vesicular Notch trafficking can activate or inhibit Notch signaling according to the context. Importantly, endocytic Notch trafficking facilitates S3 Notch cleavage, likely because γ-secretase cleavage is a pH-dependent protease that is more active in acidic environments, such as the late endosome. Considering that epistasis analysis of amx and full-length and activated forms of Notch placed amx in the S3 cleavage step of signal transduction, it would be interesting to further explore the relationship between γ-secretase-mediated S3 cleavage and defective Notch trafficking in the absence of amx function. It would also be interesting to investigate exactly how Amx is involved in Notch trafficking through live-imaging analyses of Notch intracellular transportation (Das, 2020).

Notch mediates inter-tissue communication to promote tumorigenesis

Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of Epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, this study shows that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, this study provides evidence that signaling can be mediated by cytonemes from Delta-expressing cells. It is thus proposed that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating (Boukhatmi, 2020).

Normal tissue mesenchymal cells are thought to have important roles in promoting the growth and metastasis of many tumors. To do so, they must be educated by the aberrant cancerous cells to acquire the properties needed to sustain tumorigenesis. Using a Drosophila model of EGFR/Ras-driven tumorigenesis, this study demonstrates that Notch activity in the unmodified mesenchymal cells is essential for tumor growth. Downregulating Notch specifically in mesenchymal cells reduced their proliferation rates, promoted their differentiation, and significantly compromised the size of tumors that developed. Strikingly, the activation of Notch in these supporting cells appears to rely on direct communication from the cancerous epithelial cells, illustrating that this pathway can operate in long-range signaling between tissue layers (Boukhatmi, 2020).

The conclusion that Notch receptors in the mesenchymal cells are activated from ligands presented by nearby epithelial cells is unexpected because most examples of Notch signaling occur between cells within an epithelial cell layer. The fact that the ligands are transmembrane proteins means that direct cell-cell contacts are required to elicit signaling and that signaling usually occurs between neighboring cells. More recently, examples have emerged where signaling occurs across longer distances that appear to involve contacts mediated by cell protusions, such as filopodia or cytonemes. Evidence indicates that a similar mechanism operates in the tumors. Delta is produced in the epithelial cells and can be detected in fine processes that extend through the nearby mesenchymal cells, which is consistent with a recent report describing cytonemes in these EGFR-psqRNAi tumors. In a heterologous system, it was found there was robust activation of a Notch target gene rapidly after ligand-expressing cells made contact through cell processes. Likewise, ectopic patches of Delta in the disc epithelium led to the expression of the Notch-regulated m6-GFP in the underlying mesenchyme. Thus, it is proposed that the widespread upregulation of Delta in the epithelial compartment of the tumorous wing discs, in turn, activates the Notch pathway in the neighboring mesenchymal cells by long cellular processes. As a consequence, the mesenchymal cells become coordinated with the cancer epithelial cells and are maintained in an undifferentiated state (Boukhatmi, 2020).

Although the data demonstrate that Delta-Notch-mediated inter-tissue signaling is important for sustaining tumor growth, it is evident that other signals are also required. First, it was previously shown that Dpp from the cancerous epithelium is essential for these tumors to grow. Because the Dpp pathway was still activated in the mesenchyme when Notch was depleted, it is proposed that Dpp and Notch operate in parallel. This may explain why apicobasal polarity was not fully restored when Notch activity was impaired and highlights the likelihood that several different pathways are coopted to drive tumorigenesis. Second, the fact that tumorigenesis is rescued by perturbing Notch or Dpp signaling in the mesenchyme argues that there must be a reciprocal signal to the epithelium. Notably, the relative growth of the two populations appears highly co-ordinated in the tumors, unlike the wild type where the epithelial growth predominates. A plausible model is that combined inputs from Notch and Dpp are required to produce reciprocal signal(s), and it will be interesting to discover whether the reciprocal signaling also operates through cytonemes, given that the mesenchymal cells emit processes (Boukhatmi, 2020).

One of the key effectors of Notch activity in the tumor mesenchyme is Zfh1/ZEB, which is important for maintaining the muscle progenitors in normal conditions. In a similar manner, its expression is kept high in the tumor mesenchyme, due to Notch activity, where it helps prevent their differentiation. Downregulating zfh1 in mesenchymal cells induces their premature differentiation and prevents tumor growth. The role of Zfh1/ ZEB in promoting progenitors and stem cell proliferation appears to be widespread. Furthermore, ZEB1 is upregulated in many cancers, where it can cause the expansion of cancer stem cells and frequently drives epithelial-to-mesechymal transition to promote metastasis. Whether its activation in these conditions also involves Notch activation and inter-tissue signaling remains to be determined (Boukhatmi, 2020).

Conservation and divergence of related neuronal lineages in the Drosophila central brain

Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, this study mapped 18 Drosophila central brain lineages. While large aggregate differences between lineages were found, shared patterns of morphological diversification were discovered. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms (Lee, 2020).

In order to discern how NBs guide neuronal diversification, It is necessary to appreciate neuronal development at the single-cell level. In other words, individual neurons need to be mapped back to their developmental origins. Achieving this with stochastic clone induction (i.e. labeling GMC offspring as isolated, single-neuron clones and assigning the neurons to specific lineages based on lineage-characteristic morphology) is possible but laborious and can be extremely challenging for lineages that contain similar neurons. Targeted cell-lineage analysis using lineage-restricted genetic drivers is therefore preferred for mapping specific neuronal lineages of interest with single-cell resolution. To date, only three of the about 100 distinct neuronal lineages have been fully mapped at the single-cell level in adult fly brains: the mushroom body (MB), anterodorsal antennal lobe (ALad1) and lateral antennal lobe (ALl1) lineages. These mapped lineages consist of 1) MB Kenyon cells (KC), 2) AL projection neurons (PN), and 3) AL/AMMC PNs and AL local interneurons (LN), respectively. All three lineages produce morphologically distinct neuron types in sequential order, indicating a common temporal cell-fating mechanism. However, the progeny's morphological diversity varies greatly from one lineage to the next. The four identical MB lineages are composed of only three major KC types; moreover, paired KCs from common GMCs show no evidence for binary sister fate determination. By contrast, the two AL NBs produce progeny that rapidly change type (producing upwards of 40 neuron types) and the GMCs generate discrete A/B sister fates. In the ALl1 lineage, differential Notch signaling specifies PNs versus LNs. Notably, the paired PN and LN hemilineages show independent temporal-fate changes, as evidenced by windows with unilateral switches in production of distinct PNs or LNs. Moreover, the ALl1 PN hemilineage alternately yields Notch-dispensable AL and Notch-dependent AMMC PNs. Together, these phenomena demonstrate a great versatility in lineage-guided neuronal diversification (Lee, 2020).

Assembling complex region-specific intricate neural networks for an entire brain requires exquisite cell specificity. In fact, cellular diversity-as characterized by gene expression-is higher during development than in mature brains, signifying that the underpinnings of the connectome can be understood by studying development. Such developmental diversity is reflected by characteristic neurite projection and elaboration patterns. This study therefore aimed to elucidate the roles of NB lineage specification, temporal patterning and binary sister-fate decisions upon neuronal morphology. By doing this, it is hoped to gain insight about how a limited number of NBs can specify such enormous brain complexity. This study mapped a large subset of NB lineages, enough to make generalizations but not so many to confound analysis. To this end, NBs expressing the conserved spatial patterning gene vnd were selected. With single-neuron resolution, 25 hemilineages derived from 18 vnd-expressing NBs were mapped. Hemilineage-dependent morphological diversity were observed at two levels. First, neurons of the same hemilineage may uniformly innervate a common neuropil or differentially target distinct neuropils. Second, neurons show additional structural diversity in terminal elaboration, which depends on neuropil targets rather than lineage origins. Once you factor in the differences of the neuropil targets, hemilineages which seem grossly distinct actually show comparable temporal patterns in the diversification of neuron morphology. Many hemilineages exhibit recurrent production of analogous neuron types and/or cyclic appearance of characteristic morphological features, implicating dynamic fating mechanisms. Moreover, non-sister hemilineages were discovered that make similar or even identical neuron types with common temporal patterns. These observations suggest involvement of conserved lineage-intrinsic cell-fating mechanisms in the derivation of diverse neuronal lineages (Lee, 2020).

Despite having predetermined fates, it is hard to imagine how complex neuronal morphology is controlled. Mapping neuron morphology for 25 hemilineages in this study reveals that primary trajectories and thus neuropil targets are mainly dependent upon both lineage identity and Notch signaling-that is hemilineage identity. Notably, sister hemilineages can vary greatly in the extent of innervation. Hemilineages with excessive coverage areas are consistently associated with the Notch^off (N^off) state. However, the larger coverage area of the B hemilineage could be a result of only a subset of neurons with lengthy projections. At the single-cell level, the average length of the main trajectory (defined as the total length of the segmented neuron after pruning branches shorter than 50 microns) is significantly greater on the N^off than N^on side in only two (CREa1 and SMPp&v1) of the seven Vnd lineages composed of dual hemilineages. Instead, evidence was found in support of presence of more diverse morphological groups and/or topological classes of B neurons (as opposed to a dominant group/class of A neurons). First, the degree (coefficient) of variation in the length of the main trajectory is significantly higher on the N^off than N^on side. Second, there indeed exist significantly higher numbers of topological neuronal classes in the B than A hemilineages. However, it is unclear if the hemilineage Notch state also affects diversity of neuronal topology in the Drosophila thoracic ganglion with well-defined hemilineages (Lee, 2020).

Notch signaling as a binary switch delivers context-dependent outcomes, including grossly opposite phenotypes. For instance, Notch can promote or suppress neuronal cell death depending on lineage identity. This can lead to unpaired hemilineages, in which only one viable neuron is produced after each GMC division. From the 11 Vnd unpaired hemilineages, seven are N^on and four are N^off. Given this random association, it is curious that correlation is seen between Notch state and hemilineage complexity. Higher gross diversity is frequently seen on the N^off side. The same applies to the previously mapped ALl1 lineage where the N^on hemilineage consists exclusively of AL local interneurons, whereas its N^off sister hemilineage consists of projection neurons innervating diverse neuropils, including AL, AMMC, LH, PLP, and VLP. However, the striking Notch-dependent LN/PN fate separation appears to be a characteristic of only the ALl1 lineage. This study found instead that neurons of the same hemilineage can adopt various topologies. In fact, both general topology and terminal arborization seem primarily tailored by the targets innervated. Further, unrelated hemilineages with striking similarities (e.g., CREa1A/CREa2A and LALv1A/AOTUv4A) consistently have the same Notch state. Such resemblance across non-sister hemilineages could simply reflect their evolutionary relatedness at the lineage level. Nonetheless, Notch may directly regulate neuropil targeting, as implicated by the complete segregation of the N^on and N^off neuronal processes observed in six of the seven (not LALv1) dual lineages. Further, Notch can promote cell adhesion, either by acting as a cell adhesion molecule or by upregulating canonical cell adhesion molecules such as integrins. It is speculated that Notch may strengthen neurite-neurite affinity, as higher affinity in A hemilineages could suppress neurite defasciculation resulting in more uniform trajectory, and facilitate extension of long neurite fascicles. By contrast, reduced affinity in B hemilineages could promote gross diversity through serial defasciculation of primary projections. Further, reduced affinity across sister branches could enhance neurite elaboration within targeted neuropils. Nonetheless, additional factors (e.g., neuropil-characteristic topographic maps) might modulate the gross manifestation of Notch's morphogenetic effects (Lee, 2020).

The orderly derivation of morphologically distinct neuron types within a given hemilineage is indicative of temporal fating. However, the final neuron morphology depends not only on temporal fate, but also on lineage identity and Notch binary sister fate, as well as the anatomy of target neuropils. Despite the complexities, similar temporal features are observable across diverse hemilineages. First, it is common to see beginning neurons with uniquely elaborate projections and ending neurons with reduced morphology (see Common themes of neuronal lineage temporal patterning). Second, there are temporally ordered neuropil targets characteristic of each hemilineage. Although rarely restricted to a single window, most morphological groups show select windows of production. These phenomena indicate long-range temporal patterning. However, recurrent neuropil targeting is also common. Moreover, a comparable series of related neuron types or progressive morphological changes can appear multiple times in a hemilineage. These recurrences suggest repetition of dynamic factors. Taken together, the temporal changes in neuron morphology and targeting indicate the combination of both long-range temporal patterning and reiteration of temporal windows (Lee, 2020).

As to underlying molecular mechanisms, the observed birth order-dependent neuronal morphogenesis is unlikely due to the environmental differences over the course of larval neurogenesis, since the final neuronal targeting and innervation occur in a rather synchronized manner at the early pupal stage. Further, some of the temporal patterning phenomena may be explained by the intrinsic temporal factors that have been previously described in the literature. The Cas and Svp embryonic temporal transcription factors are expressed in NBs during early postembryonic neurogenesis and are thus candidates to promote the uniquely exuberant neurite projections in first-born neurons. Opposing temporal gradients of Imp and Syp RNA binding proteins in cycling NBs have been shown to control neuronal temporal fate in MB, AL, and complex type II lineages as well as global NB termination. Imp/Syp are likely to govern long-range temporal patterning of most, if not all, neuronal lineages. Imp and Syp gradients shape the descending protein gradient of Chinmo. The hierarchical temporal gradients of Imp/Syp and Chinmo could define serial temporal windows with expression of various terminal selector genes. For instance, Mamo (a temporally patterned terminal selector gene) is selectively expressed in the window defined by both weak Chinmo and abundant Syp in MB and AL lineages (Liu, 2019; Lee, 2020 and references therein).

However, it is not known exactly how the opposite Imp/Syp protein gradients can define ~30 serial temporal fates in a protracted neuronal lineage. Interestingly, recurrent production of related neuron types has emerged as a dominant theme in the temporal patterning of Vnd lineages. Dynamic Notch signaling may underlie some alternating temporal fates, as Notch has been shown to control alternate production of AL and AMMC projection neurons in the lateral AL lineage. However, it is unlikely that Notch alone can mediate multiple recurring features as seen in most Vnd hemilineages. Therefore involvement of parallel recurring factors is proposed to elicit unsynchronized repetition of distinct features. In sum, there likely exist multiple temporal fating mechanisms that act in concert to expand neuron diversity, thus resulting in complex temporal patterns (Lee, 2020).

Given the need for large neuronal diversity, it was surprising to see the production of two identical, long series of dopaminergic neuron types by CREa1A and CREa2A. Strikingly, the NB homology extends throughout postembryonic neurogenesis. The FB neurons born prior to dopaminergic neurons in CREa1A are also morphologically indistinguishable from those in CREa2A. The only difference is the selective loss of the first larval-born CREa2A neuron. Contrasting the almost identical CREa1A and CREa2A hemilineages, their paired sister hemilineages (CREa1B and CREa2B) are easily distinguishable, as only CREa1B neurons cross the midline. These phenomena implicate that neighboring CREa1 and CREa2 lineages may have arisen from NB duplication followed by a change in midline crossing. Thus, it is believed that one way for brain complexity to increase is through lineage duplication and subsequent divergence (Lee, 2020).

In conclusion, this high-resolution, comprehensive analysis of Vnd lineages reveals how a complex brain can be reliably built from differentially fated neural stem cells. This seminal groundwork lays an essential foundation for unraveling brain development from genome to connectome (Lee, 2020).

A developmental framework linking neurogenesis and circuit formation in the Drosophila CNS

The mechanisms specifying neuronal diversity are well-characterized, yet it remains unclear how or if these mechanisms regulate neural circuit assembly. To address this, the developmental origin was mapped of 160 interneurons from seven bilateral neural progenitors (neuroblasts), and they were identified in a synapse-scale TEM reconstruction of the Drosophila larval CNS. Lineages were found to concurrently build the sensory and motor neuropils by generating sensory and motor hemilineages in a Notch-dependent manner. Neurons in a hemilineage share common synaptic targeting within the neuropil, which is further refined based on neuronal temporal identity. Connectome analysis shows that hemilineage-temporal cohorts share common connectivity. Finally, this study showed that proximity alone cannot explain the observed connectivity structure, suggesting hemilineage/temporal identity confers an added layer of specificity. Thus, this study demonstrated that the mechanisms specifying neuronal diversity also govern circuit formation and function, and that these principles are broadly applicable throughout the nervous system (Mark, 2021).

Tremendous progress has been made in understanding the molecular mechanisms generating neuronal diversity in both vertebrate and invertebrate model systems. In mammals, spatial cues generate distinct pools of progenitors, which generate neuronal diversity in each spatial domain. The same process occurs in invertebrates like Drosophila, but with a smaller number of cells, and this process is particularly well understood. The first step occurs when spatial patterning genes act combinatorially to establish single, unique progenitor (neuroblast) identities. These patterning genes endow each neuroblast with a unique spatial identity (Mark, 2021).

The second step is temporal patterning -- the specification of neuronal identity based on birth-order, an evolutionarily conserved mechanism for generating neuronal diversity. This study focused on Drosophila embryonic neuroblasts, which undergo a cascade of temporal transcription factors: Hunchback (Hb), Krüppel (Kr), Pdm, and Castor (Cas). Each temporal transcription factor is inherited by ganglion mother cells (GMCs) born during each expression window. The combination of spatial and temporal factors endows each GMC with a unique identity (Mark, 2021).

The third step is hemilineage specification, which was initially characterized in Drosophila larval and adult neurogenesis, and may also be used in vertebrate neurogenesis. Hemilineages are formed by GMC asymmetric division into a pair of post-mitotic neurons; during this division, the Notch inhibitor Numb (Nb) is partitioned into one neuron (NotchOFF neuron), whereas the other sibling neuron receives active Notch signaling (NotchON neuron), thereby establishing NotchON and NotchOFF hemilineages. In summary, three mechanisms generate neuronal diversity within the embryonic central nervous system (CNS): neuroblast spatial identity, GMC temporal identity, and neuronal hemilineage identity (Mark, 2021).

A great deal of progress has also been made in understanding neural circuit formation in both vertebrates and invertebrate model systems, revealing a multi-step mechanism. Neurons initially target their axons to broad regions (e.g., thalamus/cortex), followed by targeting to a neuropil domain (glomeruli/layer), and finally forming highly specific synapses within the targeted domain (Mark, 2021).

Despite the progress in understanding the generation of neuronal diversity and the mechanisms governing axon guidance and neuropil targeting, how these two developmental processes are coordinated remains largely unknown. While it is accepted that the identity of a neuron is linked to its connectivity, the developmental mechanisms involved are unclear. For example, do clonally related neurons target similar regions of the neuropil due to the expression of similar guidance cues? Do temporal cohorts born at similar times show preferential connectivity? This study addressed the question of whether any of the three developmental mechanisms (spatial, temporal, hemilineage identity) are correlated with any of the three circuit-wiring mechanisms (neurite targeting, synapse localization, connectivity). This study mapped the developmental origin for 80 bilateral pairs of interneurons in abdominal segment 1 (A1) by identifying and reconstructing these neurons within a full CNS TEM volume -- this is over a quarter of the ~300 neurons per hemisegment. The unexpected observation was made that hemilineage identity determines neuronal projection to sensory or motor neuropils; thus, neuroblast lineages coordinately produce sensory and motor circuitry. In addition, it was shown that neurons with shared hemilineage-temporal identity target pre- and post-synapse localization to similar positions in the neuropil, and that hemilineage-temporal cohorts share more common synaptic partners than that produced by neuropil proximity alone. Thus, temporal and hemilineage identity plays essential roles in establishing neuronal connectivity (Mark, 2021).

This study determined the relationship between developmental mechanisms (spatial, temporal, and hemilineage identity) and circuit assembly mechanisms (projections, synapse localization, and connectivity). To do this, both developmental and circuit features were mapped for 160 neuronal progeny of 14 neuroblast lineages in a serial section TEM reconstruction - this allows characterization neurons that share a developmental feature at single synapse resolution. It is important to note that the seven neuroblasts in this study were chosen based on successful clone generation and availability of single neuroblast Gal4 lines, and thus there should be no bias towards a particular pattern of neurite projections, synapse localization, or connectivity. The results show that individual neuroblast lineages have unique but broad axon and dendrite projections to both motor and sensory neuropil; hemilineages restrict projections and synapse localization to either motor or sensory neuropil; and distinct temporal identities within hemilineages provide additional specificity in synapse localization and connectivity. Thus, all three developmental mechanisms act combinatorially to progressively refine neurite projections, synapse localization, and connectivity (Mark, 2021).

In mammals, clonally related neurons often have a similar location, morphology, and connectivity. In contrast, this study found that clonally related neurons project widely in the neuropil, to both sensory and motor domains, and thus lack shared morphology. Perhaps as brain size expands to contain an increasing number of progenitors, each clone takes on a more uniform structure and function. Yet the observation that each neuroblast clone had highly stereotyped projections suggests that neuroblast identity (determined by the spatial position of the neuroblast) determines neuroblast-specific projection patterns. Testing this functionally would require manipulating spatial patterning cues to duplicate a neuroblast and assay both duplicate lineages for similar projections and connectivity (Mark, 2021).

This study found that hemilineages produce sensory and motor processing units via a Notch-dependent mechanism. Pioneering work on Drosophila third instar larval neuroblast lineages showed that each neuroblast lineage is composed of two hemilineages with different projection patterns and neurotransmitter expression. These studies were extended to embryonic neuroblasts and showed that Notch signaling determines motor versus sensory neuropil projections in all lineages examined. Surprisingly, the NotchON hemilineage always projected to the dorsal/motor neuropil, whereas the NotchOFF hemilineage always projected to the ventral/sensory neuropil. The relationship between the NotchON hemilineage projecting to the motor neuropil may be a common feature of all 30 segmental neuroblasts or it could be that the NotchON/NotchOFF provides a switch to allow each hemilineage to respond differently to dorsoventral guidance cues, with some projecting dorsally and some projecting ventrally. Analysis of additional neuroblast lineages will resolve this question. Another point to consider is the potential role of Notch in post-mitotic neurons as these experiments generated Notchintra misexpression in both newborn sibling neurons as well as mature post-mitotic neurons. Future work manipulating Notch levels specifically in mature post-mitotic neurons undergoing process outgrowth will be needed to identify the role of Notch in mature neurons, if any (Mark, 2021).

Elegant work has identified neuropil gradients of Slit and Netrin along the mediolateral axis, Semaphorins along the dorsoventral axis, and Wnt5 along the anteroposterior axis. The finding that neurons in a hemilineage project to a common region of the neuropil strongly suggests that all neurons within a hemilineage respond in the same way to these global pathfinding cues. Conversely, the finding that neurons in different hemilineages target distinct regions of the neuropil suggests that each hemilineage expresses a different palette of guidance receptors, which enable them to respond differentially to the same global cues. For example, neurons in ventral hemilineages may express Plexin receptors to repel them from high Semaphorins in the dorsal neuropil (Mark, 2021).

Hemilineages have not been well described in vertebrate neurogenesis. Notch signaling within the Vsx1 + V2 progenitor lineage generates NotchOFF V2a excitatory interneurons and NotchON V2b inhibitory interneurons, which may be distinct hemilineages. Interestingly, both V2a and V2b putative hemilineages contain molecularly distinct subclasses; this study raises the possibility that these subtypes arise from temporal patterning within the V2 lineage. In addition, NotchON/NotchOFF hemilineages may exist in the pineal photoreceptor lineage, where NotchON and NotchOFF populations specify cell-type identity (Mark, 2021).

Only recently have the role of hemilineages been tested for their functional properties. In adults, activation of each larval hemilineage from NB5-2 showed similar behavioral output, whereas each hemilineage from NB6-1 elicited different behaviors. Previous work showed that the Eve+, Saaghi, and Jaam neurons are part of a proprioceptive circuit (Heckscher, 2015); this study shows that each class of neurons represents a hemilineage-temporal cohort. Note that the Jaam neurons process sensory input and are in a NotchOFF hemilineage, supporting the conclusion that NotchOFF hemilineages are devoted to sensory processing; the Saaghi premotor neurons are in a NotchON hemilineage consistent with their role in motor processing. Interestingly, both input and output neurons in this circuit arise from a common progenitor (NB5-2), which may generate late-born Jaam/Saaghi sibling neurons. In the future, it would be interesting to determine if other sibling hemilineages are in a common circuit to generate a specific behavior (Mark, 2021).

The hemilineage results have several implications. First, the results reveal that sensory and motor processing components of the neuropil are being built in parallel, with one half of every GMC division contributing to either sensory or motor networks. This would be an efficient mechanism to maintain sensory/motor balance as lineage lengths are modified over evolutionary time. Second, the results suggest that looking for molecular or morphological similarities in full neuroblast clones may be misleading due to the full neuroblast clone comprising two different hemilineages. For example, performing bulk RNAseq on all neurons in a neuroblast lineage is unlikely to reveal key regulators of pathfinding or synaptic connectivity due to the mixture of disparate neurons from the two hemilineages (Mark, 2021).

The cortex neurite length of neurons was used as a proxy for birth-order and shared temporal identity. This is thought to be a good approximation, but it clearly does not precisely identify neurons born during each of the Hb, Kr, Pdm, Cas temporal transcription factor windows. Nevertheless, there was sufficient resolution to observe that neurons with the same temporal identity clustered their pre- or postsynapses, rather than localizing them uniformly through the hemilineage neuropil domain. Interestingly, the three-dimensional location of each hemilineage temporal cohort synaptic cluster is identical on the left and right side of A1, ruling out the mechanism of stochastic self-avoidance. Other possible mechanisms include hemilineage-temporal cohorts expressing different levels of the presynapse spacing cue Sequoia or hemilineage-temporal cohorts exhibiting different responses to global patterning cues. Testing the function of temporal identity factors in synaptic tiling will require hemilineage-specific alteration of temporal identity, followed by assaying synapse localization within the neuropil (Mark, 2021).

The results strongly suggest that hemilineage identity and temporal identity act combinatorially to allow small pools of neurons to target pre- and postsynapses to highly precise regions of the neuropil, thereby restricting synaptic partner choice. Yet precise neuropil targeting is not sufficient to explain connectivity as many similarly positioned axons and dendrites fail to form connections. The model is favored that hemilineages direct gross neurite targeting to motor or sensory neuropil, whereas temporal identity acts combinatorially with each hemilineage to direct more precise neurite targeting and synaptic connectivity. Thus, the same temporal cue (e.g., Hb) could promote targeting of one pool of neurons in one hemilineage and another pool of neurons in an adjacent hemilineage. This limits the number of regulatory mechanisms needed to generate precise neuropil targeting and connectivity for all ~600 neurons in a segment of the larval CNS (Mark, 2021).

In conclusion, this study demonstrates how developmental information can be integrated with connectomic data. Lineage information, hemilineage identity, and temporal identity can all be accurately predicted using morphological features (e.g., number of fascicles entering the neuropil for neuroblast clones and radial position for temporal cohorts). This both greatly accelerates the ability to identify neurons in a large EM volume as well as sets up a framework in which to study development using data typically intended for studying connectivity and function. This framework is used to relate developmental mechanism to neuronal projections, synapse localization, and connectivity. Lineage, hemilineage, and temporal identity were found act sequentially to progressively refine neuronal projections, synapse localization, and connectivity, and the data supports a model where hemilineage-temporal cohorts are units of connectivity for assembling motor circuits (Mark, 2021).

Enhancer architecture sensitizes cell-specific responses to Notch gene dose via a bind and discard mechanism

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, there is only a limited mechanistic understanding of phenotype variability due to gene dosage. This study unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, it was shown that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. Mathematical modeling was combined with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration (Kuang, 2020).

Haploinsufficiency, or the inability to complete a cellular process with one functional allele of a given gene, manifests in tissue and organ defects with variable penetrance and severity. For example, Notch (N) haploinsufficiency, which was discovered in Drosophila, causes a variety of tissue-specific defects including wing notching and extra sensory bristle formation that can vary greatly in penetrance and expressivity. Notch pathway haploinsufficiency was subsequently observed in mammals, as Notch1 heterozygous mice have heart valve and endothelium defects, whereas Notch2 heterozygotes have defects in bone, kidney and marginal zone B cells. A single allele of NOTCH2 or the JAG1 ligand can also cause pathological phenotypes in humans, as heterozygosity of either gene can result in a variably penetrant developmental syndrome known as Alagille. Thus, Notch gene dose sensitivity has been observed in a variety of Notch-dependent tissues in both humans and animals. Unfortunately, a mechanistic understanding of what causes some tissues to be highly sensitive to Notch gene dose and what factors impact the variable penetrance and severity of Notch haploinsufficiency phenotypes is lacking (Kuang, 2020).

Molecularly, Notch signaling is initiated by ligand-induced proteolysis of the Notch receptor to release the Notch intracellular domain (NICD) from the membrane. NICD subsequently transits into the nucleus, binds to the Cbf1/Su(H)/Lag1 (CSL) transcription factor (TF) and the adaptor protein Mastermind (Mam), and induces gene expression via two types of DNA binding sites: independent CSL sites that bind monomeric NICD/CSL/Mam (NCM) complexes, and Su(H) paired sites (SPS) that are oriented in a head-to-head manner to promote cooperative binding between two NCM complexes. Once bound to an enhancer, the NCM complex activates transcription of associated genes via the P300 co-activator. Thus, the production of NICD is converted into changes in gene expression that ultimately regulate cellular processes during development (Kuang, 2020).

Haploinsufficiency of Notch receptor and ligand encoding genes suggests that decreased gene dosage results in a sufficiently large decrease in NICD production to cause phenotypes in a subset of tissues. There is also growing evidence that genetic changes that reduce NICD degradation can alter signal strength with pathological consequences in specific cell types. In the mammalian blood system, for example, Notch1 mutations that remove an NICD degron sequence have been associated with increased NICD levels and the development of T-cell Acute Lymphoblastic Leukemia (T-ALL) in mice and humans. Intriguingly, NICD turnover via this degron sequence has been directly linked to transcription activation, as the Mam protein interacts with the Cdk8 kinase module (CKM) of the Mediator complex, which can phosphorylate NICD to promote its ubiquitylation by the Fbxw7 E3-ligase and degradation by the proteasome. Accordingly, gene mutations that lower CKM activity have also been associated with increased NICD levels and T-ALL initiation and progression. Thus, perturbations in mechanisms that regulate either NICD production or degradation can induce cell and/or tissue specific phenotypes (Kuang, 2020).

This study used Drosophila genetics, quantitative trait and expression analysis, and mathematical modeling to unravel a unique regulatory mechanism that impacts Notch signal strength in a tissue-specific manner. First, it was unexpectedly found that an enhancer containing as few as 12 Notch dimer binding sites can induce tissue-specific phenotypes via a CKM-dependent mechanism that can be uncoupled from transcription activation. Second, based on quantitative analysis and mathematical modeling, changes in NICD degradation rates are shown to be predicted to preferentially impact long duration Notch-dependent processes, whereas genetic changes in NICD production rates (i.e., Notch haploinsufficiency) affect both short and long duration processes. Collectively, these findings provide new insights into how distinct Notch-dependent cellular processes can be differentially impacted by both enhancer architecture and signal duration to induce tissue-specific Notch defects within a complex animal (Kuang, 2020).

The results show that simply increasing the number of clustered Notch dimer sites (SPS) linked to sites for a pioneer TF can cause a tissue-specific N haploinsufficiency phenotype via a Cdk8-dependent mechanism. These findings have important implications for both enhancer biology and the mechanisms regulating Notch signal strength in specific tissues. First, the proposed Cdk8-dependent mechanism links the rapid degradation of the Notch signal (NICD) with its binding to specific loci (SPSs) in a manner that can be uncoupled from transcription activation. This 'bind and discard' mechanism reveals an unexpected global link between accessible binding sites in the epigenome, such that the collective 'drain' loci can reduce Notch-dependent transcription at other loci in the same nucleus. Moreover, since the binding sites do not have to be coupled with transcription to induce a notched wing phenotype, the findings highlight the possibility that seemingly non-functional genomic binding events could impact TF metabolism in a Cdk8-dependent manner. Given that Cdk8 interacts with many genomic loci and that a previous phospho-proteomic study identified numerous transcriptional regulators are targets of CDK8/19 phosphorylation (CDK19 is a paralog of CDK8), such a mechanism may be quite general and apply to transcription regulators beyond Notch (Kuang, 2020).

While genetic and cell culture data, as well as previous phosphorylation studies, support a direct link between CKM activity and NICD degradation, it should be noted that the CKM also phosphorylates other proteins that could contribute to the differences in wing versus macrochaetae phenotypes. Intriguingly, one of the high confidence CKM targets in mammalian cells was MAML1, indicating that CKM activity may directly regulate Notch output by phosphorylating multiple components of the Notch transcription complex. In addition, CCNC (CycC), MED12, and MED13 were all found to be high confidence CDK8/19 targets, suggesting that CKM activity may directly impact the turnover of key components of its own complex. It's thus not surprising that removing an allele of cycC, kto (med12) or skd (med13) had a much larger impact on the Notch haploinsufficient wing phenotype compared to changing the gene dose of cdk8. Moreover, these genetic data are consistent with prior studies in yeast showing that structural/regulatory components of macromolecular complexes, such as CycC/Med12/Med13, are enriched in haploinsufficiency genes, whereas enzymes are generally under-represented from the list of dose sensitive genes (Kuang, 2020).

Second, the data support the idea that not all Notch binding sites are equally capable of marking NICD for degradation, and that enhancer architecture plays a key role in modulating NICD turnover. For instance, only Notch dimer but not Notch monomer sites are sufficient to generate phenotypes, and even SPS-containing enhancers differ in their ability to induce phenotypes based on the absence/presence of pioneer TF sites. Notably, this study found that enhancers with either synthetic SPS sites designed to limit additional TF input or an endogenous E(spl)m8 SPS with adjacent binding sites capable of providing negative feedback were sufficient to induce notched wing phenotypes when coupled to pioneer TF sites. Since Grh and Zld binding is sufficient to increase chromatin opening, these findings suggest enhancer accessibility alters the rate at which NICD is metabolized by Notch dimer sites. Intriguingly, ChIP-seq data for Grh and Zld reveals extensive binding to the Enhancer of Split (E(spl)) locus that contains numerous SPS-containing Notch regulated enhancers. However, it is important to note that while an SPS-containing enhancer lacking pioneer TF sites failed to induce phenotypes in wild type flies, it did significantly increase wing notching in a sensitized genetic background. These findings suggest that SPS-containing enhancers promote NICD degradation at differing rates based on the presence of nearby TF sites. While the mechanistic basis for how SPS but not CSL sites promote NICD degradation is not known, these data highlight a potential mechanism by which enhancer architecture (i.e., Notch dimer vs monomer sites) and epigenetic 'context' (i.e., accessibility due to pioneer TF binding) can fine tune the global Notch response in different tissues (Kuang, 2020).

The finding that introducing as few as 12 SPS sites into the genome can induce notched wing phenotypes raises the question of how many functional SPS sites exist in the endogenous genome. Recent studies found that about one third of direct Notch target genes (38 of 107 genes) in human T-ALL are regulated by SPS sites, and a mouse mK4 cell line has an estimated 2500 Notch dimer dependent binding sites. These findings suggest that many SPS sites are accessible across the mammalian genome. The estimated number of SPS sites within the Drosophila genome, which is an order of magnitude smaller than most mammalian genomes, remains to be determined. However, of the 154 Notch-responsive genes identified in a Drosophila wing disc-derived cell line, eight encode E(spl) genes that are clustered within a common 40 kb locus and many E(spl) genes contain one or more SPS sites. In comparison to the E(spl) locus, the 6 SPS sites within the G6S-lacZ transgene are found within ~300 bps, and thus it is possible that concentrating SPS sites might provide an avidity impact that increases the probability of a recruited NICD molecule being marked for degradation. In fact, it is estimated that the cumulative effect of the Drosophila genome is equal to ~5 highly accessible, linked SPS-GBE sites. Future studies using endogenous SPS-containing enhancers will be needed to provide a better understanding of both the role of nearby binding sites for other TFs and how concentrating SPS sites in specific loci impacts the wing notching phenotype (Kuang, 2020).

Third, it is proposed that the differential sensitivity of Notch-dependent tissues to changes in NICD degradation (i.e., SPS-GBE sites or CKM heterozygotes) or production rates (N heterozygotes) reflects the temporal requirement for Notch signal duration. An appealing aspect of this Notch signal duration model is that it predicts that any perturbation that alters NICD signal degradation will preferentially affect long-duration processes over short duration processes, whereas perturbations that impact NICD signal production will affect both long and short duration events. Moreover, the differential sensitivity of the Notch duration model to changes in production versus degradation rates may be generalizable to the study of other signaling pathways. However, it's worth noting that additional differences between the wing and macrochaetae besides signal duration may contribute to the magnitude of change in Notch signal strength in each tissue. As an example, cis-inhibition, which determines the fraction of functional Notch receptors on the cell membrane, could play a cell-specific role in modulating NICD production to a larger degree in one tissue over another. Taking this mechanism into consideration, the assumption that Notch heterozygotes reduce NICD production by 50 percent in each tissue may be over-simplified. Thus, further experiments using a system that is amenable to systematic changes in the length of Notch signal induction are needed to thoroughly test the signal duration model in multiple tissues (Kuang, 2020).

Intuitively, the duration model suggests a mechanism underlying cell-specific context that may have implications for both developmental processes and tumorigenesis. For example, mutations in the NICD PEST domain that decouple DNA binding and degradation are common in T-cell acute lymphoblastic leukaemia (T-ALL), and CycC (CCNC) functions as a haploinsufficient tumor suppressor gene in T-ALL, at least in part, by stabilizing NICD. These findings suggest that T-ALL is highly sensitive to alterations in NICD degradation. Indeed, T-ALL cells are 'addicted' to Notch and are thus dependent on a long duration signal. As ~30% of Notch target genes in T-ALL use SPS containing enhancers, the current findings provide insight into how Notch PEST truncations and CCNC heterozygotes could each promote tumorigenesis by slowing CKM-mediated NICD turnover on SPS enhancers. Future studies focused on enhancers that recruit the CKM and other Notch-dependent cellular processes will help reveal how the temporal requirements for nuclear activities contributes to both normal development and disease states (Kuang, 2020).

The Notch pathway regulates the Second Mitotic Wave cell cycle independently of bHLH proteins

Notch regulates both neurogenesis and cell cycle activity to coordinate precursor cell generation in the differentiating Drosophila eye. Mosaic analysis with mitotic clones mutant for Notch components was used to identify the pathway of Notch signaling that regulates the cell cycle in the Second Mitotic Wave. Although S phase entry depends on Notch signaling and on the transcription factor Su(H), the transcriptional co-activator Mam and the bHLH repressor genes of the E(spl)-Complex were not essential, although these are Su(H) coactivators and targets during the regulation of neurogenesis. The Second Mitotic Wave showed little dependence on ubiquitin ligases neuralized or mindbomb, and although the ligand Delta is required non-autonomously, partial cell cycle activity occurred in the absence of known Notch ligands. This study found that myc was not essential for the Second Mitotic Wave. The Second Mitotic Wave did not require the HLH protein Extra macrochaetae, and the bHLH protein Daughterless was required only cell-nonautonomously. Similar cell cycle phenotypes for Daughterless and Atonal were consistent with requirement for neuronal differentiation to stimulate Delta expression, affecting Notch activity in the Second Mitotic Wave indirectly. Therefore Notch signaling acts to regulate the Second Mitotic Wave without activating bHLH gene targets (Bhattacharya, 2017).

Differences were observed between how Notch signaling regulates the SMW compared to how Notch regulates photoreceptor differentiation (see Model of the cell cycle and cell fate specification in the morphogenetic furrow). The Notch pathway suppresses the specification of photoreceptor cells in a manner similar to regulation of neurogenesis by Notch in many other tissues. That is, activation of the transmembrane ligand Delta by the ubiquitin ligase Neuralized leads allows Delta to activate Notch, leading to release of the Notch intracellular domain, which then acts in a nuclear complex with Su(H) and Mastermind to induce the transcription of the E(spl)-C family of transcriptional repressors and prevent neural fate specification and differentiation. By contrast to the pathway regulating neural differentiation, cell cycle entry in the SMW occurred in the absence of the E(spl)-C. This suggested that a distinct transcriptional target of Su(H) is involved, but unusually, this Su(H) function did not seem to require the co-activator Mam. Although most studies find that Mam is required for transcriptional activation by Su(H), mutations in mam often have weaker phenotypes than other neurogenic genes. It is possible that Mam protein might exhibit exceptionally strong perdurance, but there is also in vitro evidence for mam-independent transcriptional activation by Su(H) (Bhattacharya, 2017).

SMW entry also occurred in the absence of neur. In the case of neurogenesis, cells lacking neur show a weaker neurogenic phenotype than cells lacking other components of the pathway, both in the embryonic CNS and in the eye, which is thought to reflect the activities of two other ubiquitin ligases, Mindbomb and Mindbomb2. No effect was found on the SMW of mutated mib1, but neither mib2 nor cells lacking two or more of these possibly redundant ubiquitin ligases were examined (Bhattacharya, 2017).

Eoles of ligands were examined in more detail. It has been reported that SMW entry depended on Delta, which is also the ligand necessary for regulation of neurogenesis in the eye as in other parts of the nervous system, but although their figures show an obvious reduction in cell cycle entry in the clones of cells lacking Dl, some cell cycle entry still seems to occur. Those experiments were repeated using cells lacking both Dl and the other known ligand Ser, with similar results, ie, cell cycle entry was obviously disrupted in clones lacking both Notch ligands, nevertheless some cells still entered the cell cycle. A potential complicating factor is that reducing Notch function typically results in excessive neurogenesis, raising the question of whether some Dl Ser mutant cells were prevented from entering the cell cycle by differentiation as photoreceptors. The excess neurogenesis of such clones was partially suppressed by expression of Argos or Ras-DN. Although there is clearly a considerable disruption of the SMW in the absence of N ligands, there is also clear evidence of cell cycle entry, occurring at approximately the same time as the normal SMW (Bhattacharya, 2017).

There are at least three possible explanations for these findings. First, it possible that another ligand besides Dl or Ser activates N in the SMW. Secondly, it has been reported that Dl can signal across several cell diameters using filopodia. During neurogenesis, rescue of Dl mutant clones appears to extend beyond cells immediately adjacent to wild type cells. Signaling across many cell diameters would be required to account for the cell cycle progression seen in Dl Ser mutant clones. Thirdly, Notch can be activated independently of ligands if delivered to certain cellular compartments. Recently it has been suggested that such ligand-independent activation is suppressed by cis-inactivation, in which ligands inhibit N signaling in the same cells, so that ligand-independent activation can be revealed in cells lacking Dl and Ser. Cis-inactivation is potentially an important contributor to the patterning of Notch-mediated lateral inhibition during neurogenesis, but direct evidence of such a role has not yet been obtained. If ligand-independent signaling is occurring in Dl Ser clones, some other pathway must be active near the SMW to explain why ligand-independent signaling cell cycle entry was limited to cells at the same stage posterior to the furrow as those that undergo the normal, ligand-dependent SMW (Bhattacharya, 2017).

The cell cycle targets of Notch are of some interest. Many Drosophila cell cycles depend on regulated transcription of Cyclin E. Cyclin E is clearly required for the SMW suggested that Cyclin E might not be limiting for SMW cell cycle entry, however, because Cyclin E was seen to accumulate more in the differentiating photoreceptor precursors that do not enter the SMW. Alternatively, the lack of Cyclin E accumulation in SMW cells might reflect Cyclin E protein instability in S phase, with accumulation only in differentiating cells that can't enter the cell cycle. A Cyclin E transcriptional reporter appears to accumulate in the same cells as the Cyclin E protein, however, which is not indicative of post-translational regulation (Bhattacharya, 2017).

It has been suggested that Cyclin A was part of the Notch dependent machinery promoting cell cycle entry in the SMW, although it was concluded that it could not be the only important Notch target. What was observed, however, was dependence of Cyclin A protein levels on Notch signaling that exactly paralleled the dependence of S-phase entry on Notch signaling in various genotypes. Like Cyclin B, Cyclin A protein is degraded by APC/Cyclosome activity during G1, so that Cyclin A protein cannot accumulate until the G1/S transition. Therefore these results are also expected if Cyclin A simply accumulates whenever the G1/S transition has occurred, and is not a direct target of Notch signaling (Bhattacharya, 2017).

In mammalian cells, c-Myc appears to be an important Notch target. Indeed, human Notch was first identified as a proto-oncogene that causes leukemia through c-Myc. c-Myc is an activator of the nucleolar protein fibrillarin, which transiently increases during the SMW. Although Dmyc is important for growth in Drosophila, so that clones of myc null mutant cells in the eye disc are small, they nevertheless enter the SMW at the normal time, indicating that myc is unlikely to be the critical Notch target in the SMW (Bhattacharya, 2017).

Emc is a transcriptional target of N signaling in the eye as in other tissues. Even though Emc protein levels seem to be less affected by N signaling than is emc RNA, it has been suggested that N regulates the eye cell cycle by upregulating emc, thereby inhibiting Da, which is proposed to inhibit cell cycle entry. Contrary to this model, previous studies have reported that Da is in fact required for the SMW. We investigated this potential role for emc directly, finding no evidence that emc was required for the SMW. These studies confirmed previous findings that da is required for S phase entry into the SMW, although not apparently for cell cycle activity anterior to the morphogenetic furrow (Bhattacharya, 2017).

The cell-autonomy of da requirement in the cell cycle had not been determined previously. da mutant cells sometimes enter the SMW at the posterior boundaries of da clones, where they were adjacent to wild type cells. This suggested that da was required for the production of a short-range non-autonomous signal that is required for SMW entry, and could be provided to da mutant cells that were near to wild type cells (Bhattacharya, 2017).

It is instructive to compare the effect of da on the cell cycle to that of ato. Ato is the heterodimer partner of Da that is required for R8 specification. Da is also required independently of ato to specify R2,R3,R4 and R5. Interestingly, this requirement in R2-5 seems to be incomplete, since some da mutant R2-5 cells do differentiate at reduced frequencies. Because of this requirement for da outside R8, R1-7 photoreceptor cells can differentiate near borders of ato mutant clones, but fewer photoreceptors are expected near the borders of da mutant clones, and mostly of the R1, R6 and R7 cell types. Proneural genes often promote Notch ligand expression, and accordingly ato is required for normal Dl expression during these stages of eye development. Reduced Dl expression is a plausible explanation for the absence of the SMW within ato clones, and the more extensive requirement for da in photoreceptor differentiation and Dl expression may explain why non-autonomous rescue of the SMW is more pronounced near the borders of ato clones than near the borders of da clones (Bhattacharya, 2017).

A model is present for SMW. Unpatterned proliferation of the eye imaginal disc is terminated ahead of the morphogenetic furrow by Hh and Dpp signaling. Hh and Dpp affect many aspects of eye development including expression of master regulators of retinal determination such as Eyeless, Teashirt, Dachshund, and Sine Oculis, as well as Homothorax. Cells posterior to the furrow can still respond to growth, because extra cell cycles are driven in unspecified cells by overexpression of CycD/cdk4, Inr, or myc, as well as by Cyclin E. It is possible that Notch regulates cellular growth in the SMW, since there appears to be a small but discernible increase in nucleolar size at this stage, and progenitor cells are not noticeably smaller after dividing, indicating that growth accompanies the SMW cell division. The SMW, however, does not depend on myc. It appears to depend on a transcriptional target of Su(H) outside of the E(spl)-C of bHLH genes and that can be transcribed in the absence of Mam. The possibility of a function of Su(H)/Nicd other than transcription cannot be ruled out. The SMW clearly depends on CycE, but CycE may not be the N-dependent gene required for SMW entry, because it is not obviously elevated in SMW cells. Contrary to a recent proposal, the SMW does not depend on the HLH gene emc and is not inhibited by the bHLH protein Da. Instead da is required for entry into the SMW. The positive requirement for da is cell-nonautonomous, as is the requirement for its heterodimer partner ato, and consistent with the role of ato and da in promoting Dl expression during retinal differentiation, which is likely to account for the cell cycle defect. Surprisingly, the requirement for Dl in the SMW cell cycle was not absolute, either because an unidentified ligand exists, long-range cell-nonautonomy occurs, or Dl (and Ser) may contribute to cis-inactivation of N, so that some ligand-independent Notch signaling could occur in the unphysiological circumstance that Notch ligands are completely absent (Bhattacharya, 2017).

Early Treatment of Notch in The Interactive Fly

Notch is a surface receptor. It transmits signals received from outside the cell to the cell's interior. Notch ligands, such as Delta, Serrate and Scabrous interact with epidermal growth factor repeats contained in Notch's extracellular domain.

The intracellular domain of Notch binds Suppressor of Hairless, a multifunction transcription factor that acts as a signal transducing molecule shuttling between the cytoplasm and the nucleus. The intracellular domain of Notch might also have a nuclear function, as first suggested by Lieber, 1993. A nuclear function has been documented for the mammalian Notch homolog (Lu, 1996), and has now been documented for Drosophila as well (Struhl, 1998).

When Notch is bound by a ligand, a signal is passed across the cell membrane releasing the Suppressor of Hairless protein, freeing this protein to enter the nucleus and assume its role in activating transcription of Enhancer of split complex genes. E(spl)-C proteins act in turn to repress the adoption of neural and other differentiated states. Deltex, an intracellular docking protein, replaces Suppressor of Hairless as Su(H) leaves the site of interaction with the intracellular tail of Notch.

The Notch receptor is function is called neurogenic, but this confusing nomenclature refers to the phenotype established in the absence of functional Notch. Notch's function is to repress the adoption of differentiation by cells that carry the Notch protein. A look at the principle ligand of Notch (Delta) and its function makes the anti-neural function of Notch more easily understood.

Delta is not secreted, but is cell bound. The Delta-Notch interaction serves a cell adhesive function between ligand and receptor bearing cells. The receptor bearing cell is inhibited in assuming a differentitated state, while the ligand bearing cell is free to do so. During neurogenesis, this latter cell delaminates, that is, it migrates out of the epithelial cell layer in which it formerly resided, and assumes the differentiated state of a neuroblast in its new physical location within the developing nervous system. Thus Notch is involved in neurogenesis with respect to cells that bears the ligands for Notch: Delta, Serrate and Scabrous.

Lateral inhibition is a process whereby a single cell is fated to differentiate through the interaction of Notch-Delta, while other cells simultaneously retain their undifferentiated state. A state of competition is imposed upon a cluster of cells. Perhaps the single cell, seemingly selected at random, is the one with the highest density of ligand. However, very little is left to chance. Three other proteins are involved in fate determination of the selected cell. Inscuteable, Numb, Prospero assure a neural fate for the ligand bearing cell The selected cell proceeds along a neural differentiation pathway, synthesizing higher levels of the proneural proteins, Achaete and Scute.

Lateral inhibition is one of the major themes of development. The process of lateral inhibition and cell selection is repeated hundreds of times in Drosophila, with differentiation that takes place in nearly every kind of tissue. For example, Notch is required to limit the number of neural precursors, limit the number of muscle precursors, limit the growth of Malpighian tubules, and regulate the growth of the ovary. Notch also functions as receptor for both Serrate and Delta in organizing the dorsal-ventral boundary of the wing. One important target of Serrate and Notch in this context is wingless (Diaz-Benjumea, 1995).

Two extreme models can be envisioned for lateral inhibition. The first implicates the Notch pathway in the choice of a single precursor via a negative feedback loop. This process could be random in some cases. The second model postulates that the precursor is pre-determined by some mechanism other than Notch signaling, and that Notch signaling then serves only to mediate mutual, uniform repression of other cells and ensure development of a single precursor. Studies concerning the physical spacing of precursors for the microchaetes of the peripheral nervous system suggest the existence of a regulatory loop under transcriptional control between Notch and its ligand Delta. Activation of Notch leads to repression of the achaete-scute genes, which are themselves known to regulate transcription of Delta; this regulation may perhaps be direct (Seugnet, 1997a).

Neuroblast segregation was studied in embryos lacking both the maternal and the zygotic forms of either Notch or Delta. A seven-up-LacZ marker was used to follow neuralization of 5-2 and 7-4 neuroblast groups. In the absence of Notch signaling, the cells with an equivalence group do not enter the neural differentiation pathway simultaneously. Neuralization within a group is progressive with two or three cells segregating early and several more later. This suggests that neural potential is not evenly distributed among these cells. A requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos was tested by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression, suggesting that transcriptional regulation of Notch is not necessary for many aspects of development of the larval CNS and PNS. In particular, it is dispensable both before and after neuroblast segregation, implying that it is not a necessary component of neuroblast segregation, per se. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases; in the remaining 20%, more than one neuroblast segregates from a single group of cells. Thus transcriptional regulation of Delta is largely dispensable, with only a small percentage of multiple neurons segregating in each cluster. The possibility is discussed that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Genes such as achaete, scute, extramacrochaete, and wingless could be responsible for local differences in proneural activity. Notch signaling would enable all cells to mutually repress one another; only a cell with an elevated neural potential could overcome this repression (Seugnet, 1997a).

GENE STRUCTURE

Bases in 5' UTR - 799

Exons - nine

Bases in 3' UTR - 1262

PROTEIN STRUCTURE

Amino Acids - 2703

Structural Domains

There are two regions of high hydrophobicity, an N-terminal signal sequence and a transmembrane segment between residues 1745 and 1767. There is an epidermal growth factor domain repeated 36 times, each domain consisting of approximately 38 amino acids (Kidd, 1986).

The Drosophila Notch (N) gene encodes a conserved single-pass transmembrane receptor that transduces extracellular signals controlling cell fate. Evidence has been found that the intracellular domain of Notch gains access to the nucleus in response to ligand, possibly through a mechanism involving proteolytic cleavage and release from the remainder of the protein. These results suggest that signal transduction by Notch depends on the ability of the intracellular domain, particularly the portion containing the CDC10 repeats, to reach the nucleus and to participate in the transcriptional activation of downstream target genes (Struhl, 1998).

A sensitive approach was used to detect the physical presence of intracellular portions of Notch in the nucleus. The chimeric transcription factor Gal4-VP16 (GV) was inserted at various positions in otherwise wild-type Notch protein and the resulting N+-GV proteins expressed under heatshock control in embryos that also carry a UAS-lacZ transgene. The Gal4-VP16 protein contains the DNA-binding domain of the yeast Gal4 transcription factor coupled to the transcriptional activation domain of the viral VP15 protein. The UAS-lacZ gene contains copies of the UAS-binding site for Gal4 and is transcribed in response to Gal4 as well as the Gal4-VP16 protein. It was reasoned that expression of the UAS-lacZ gene would provide a sensitive assay for nuclear access of the inserted Gal4-VP16 domain and hence for events that lead to nuclear import of the Notch intracellular domain (Struhl, 1998).

Gal4-VP16 domains inserted in the intracellular domain of Notch do indeed have access to the nucleus, as judged by their ability to activate UAS-lacZ transcription. Access of a specific insertion positioned just C-terminal to the Notch transmembrane domain is ligand-dependent and correlates with Notch signal-transducing activity. A minimal fragment of the Notch intracellular domain was defined containing the CDC10 repeats that have intrinsic transducing activity. This intrinsinc transducing activity depends on its having access to the nucleus. Addition of sequences that permit or target this polypeptide to accumulate in the nucleus retain transducing activity, whereas sequences that target this polypeptide to extranuclear membranes block the activity. The presence of the VP16 activator domain renders the protein constitutively active, provided that it is inserted at a position that allows it to gain access to the nucleus irrespective of ligand. Adding repressor motifs from either Engrailed or Hairy blocks the signal-transducing activity of the resulting Notch proteins. It is suggested that the only limited step in the mechanism of signal transduction by Notch is the proposed ligand-dependent cleavage event that releases the intracellular domain from the membrane and allows it to enter into the nucleus. Notch signal transduction also appears to depend on several proteins that associate physically with the Notch intracellular domain, such as Su(H), Dishevelled, Deltex, and Numb, as well as other proteins such as Mastermind and Hairless (Struhl, 1998).

date revised: 1 March 2024 

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