Regulation of membrane localization of Sanpodo by Lethal giant larvae and Neuralized in asymmetrically dividing cells of Drosophila sensory organs

In Drosophila, asymmetric division occurs during proliferation of neural precursors of the central and peripheral nervous system (PNS), where a membrane-associated protein, Numb, is asymmetrically localized during cell division and is segregated to one of the two daughter cells (the pIIb cell) following mitosis. numb has been shown genetically to function as an antagonist of Notch signaling, and also as a negative regulator of the membrane localization of Sanpodo, a four-pass transmembrane protein required for Notch signaling during asymmetric cell division in the central nervous system (CNS). lethal giant larvae (lgl) is required for Numb-mediated inhibition of Notch in the adult PNS. Sanpodo is expressed in asymmetrically dividing precursor cells of the PNS and Sanpodo internalization in the pIIb cell is dependent cytoskeletally-associated Lgl. Lgl specifically regulates internalization of Sanpodo, likely through endocytosis, but is not required for the endocytosis Delta, which is a required step in the Notch-mediated cell fate decision during asymmetric cell division. Conversely, the E3 ubiquitin ligase Neuralized is required for both Delta endocytosis and the internalization of Sanpodo. This study identifies a hitherto unreported role for Lgl as a regulator of Sanpodo during asymmetric cell division in the adult PNS (Roegiers, 2005).

This analysis of Sanpodo function in the adult PNS suggests that, as in the embryo, Sanpodo is expressed only in asymmetrically dividing precursor cells and is required for cell fates dependant on high levels of Notch signaling, perhaps through the direct interaction between Sanpodo and the full length Notch receptor. Sanpodo also interacts directly with Numb in vivo, and in both the embryonic CNS and the adult PNS, numb inhibits plasma membrane association of Sanpodo. Therefore, it appears that Sanpodo plays a similar role in asymmetrically dividing precursor cells in both the CNS and PNS in Drosophila (Roegiers, 2005).

Although there are many similarities between the mechanisms of asymmetric cell divisions in embryonic neuroblasts and adult sensory organ precursor cells, one difference involves the role of lgl. In neuroblasts, lgl is required along with another cortical tumor suppressor, dlg, to target Numb to a basal crescent during mitosis, whereas in pI cells, only dlg is required for Numb crescent formation. While lgl is dispensable for segregation of Numb to the pIIb cell following pI cell mitosis, lgl is required for the inhibition of Notch signaling in the pIIb cell. Based on the current study, it is proposed that Lgl functions with Numb to remove Sanpodo from the membrane, leading to down regulation of the Notch signaling pathway in the pIIb cell. Through what mechanism might Lgl regulate Sanpodo localization? Studies in Drosophila, yeast, and vertebrate cells have implicated Lgl as both a regulator of exocytosis, through its interaction with t-SNARES, and as cytoskeletal effector. In this study, no phenotypes suggesting gross defects in exocytosis were detected; in fact, increased accumulation of the membrane protein Sanpodo at the plasma membrane is seen in lgl mutants. Accumulation of Sanpodo at the plasma membrane in lgl mutants resembles the phenotype of three endocytic proteins Numb, alpha-Adaptin, and Shibire, suggested that lgl may have a broader role in vesicle traffic. Although a potential role for Lgl in endocytosis is observed, this role appears to be specific to Sanpodo, since endocytosis of Delta occurs normally in lgl mutants, suggesting that Lgl is not required for bulk endocytosis. Increasingly, selective endocytosis is being implicated as an important regulator of signaling pathways. Two recent studies demonstrate that Liquid facets, an endocytic epsin participates in the Neuralized-mediated Delta endocytosis, apparently by targeting mono-ubiquitinated Delta to a specific, activating, endocytic compartment. The Notch receptor is also subjected to an ubiquitin-mediated endocytic step required for activation via the E3 ubiquitin ligase Deltex, which targets Notch to the late endosome. However, the roles of Liquid facets and Deltex have not been explored in asymmetrically dividing neural precursors. One possible function for Lgl could be to direct Sanpodo toward a specific endocytic compartment. Alternatively, Lgl may be involved indirectly, by targeting molecules required for Sanpodo endocytosis to the membrane region. This scenario would be more consistent with Lgl's role as an exocytic regulator. An alternative hypothesis may be that Lgl regulates Sanpodo localization through its interaction with the cytoskeleton. Lgl functions as an inhibitor of non-muscle myosin II function in both Drosophila and yeast. The data suggests that cytoskeletal association of Lgl is required for regulating Sanpodo localization, because phosphorylation of Lgl by aPKC, which causes an autoinhibitory conformational change in Lgl that disrupts the association with the cytoskeleton, causes membrane accumulation of Sanpodo. It remains to be determined if Sanpodo endocytosis requires inhibition of myosin II activity (Roegiers, 2005).

Previously, Numb and Neuralized had been implicated in two complementary, and possibly independent, mechanisms to determine cell fate in PNS precursor cells. Numb functions to inhibit Notch autonomously by internalizing Sanpodo in the pIIb cell: while Neuralized acts on Delta in the pIIb cell to induce Notch signaling non-autonomously in the pIIa cell. Both neuralized-dependant uptake of Delta and Sanpodo internalization require dynamin function, suggesting that these steps rely on endocytosis. Unexpectedly, it was found that loss of neuralized function affects both Delta internalization and Sanpodo internalization. Failure to internalize Delta into the pIIb cell causes a cell fate transformation of the pIIa cell into a pIIb cell in neuralized mutants, and this transformation occurs despite the accumulation of Sanpodo at the membrane, suggesting that accumulation of Sanpodo at the membrane is not sufficient to induce Notch signaling in the pIIb cell in the absence of neuralized. It is unclear whether membrane accumulation of Sanpodo in neuralized mutants is due to a direct interaction between Neuralized and Sanpodo, perhaps through ubiquitination of Sanpodo, or through an indirect mechanism. Regardless, the data show that regulation of Sanpodo membrane localization is not completely independent of neuralized function. In summary, this study suggests that Sanpodo is regulated by both neuralized and lgl, while Delta is regulated by neuralized independently of lgl. In addition, this study shows that lgl appears to contribute to the endocytosis of Sanpodo, which suggests a broader role for lgl in vesicle trafficking, which may have important implications for its role as a tumor suppressor. Could the regulation of Notch signaling by Sanpodo, Lgl and Numb be conserved across species? Sequence analysis did not reveal any homologues of Sanpodo beyond other insect species. However, loss of function studies of the mouse homologues of Drosophila numb and lgl in the developing brain show strikingly similar phenotypes. Targeted numb/numblike knockouts in dorsal forebrain and Lgl1 knockouts cause profound disorganization of the layered regions of the cortex and striatum and formation of rosettelike accumulations of neurons. These phenotypes may indicate that Numb and Lgl function together to regulate Notch signaling in mouse neurogenesis as well as in Drosophila PNS development, but a functional homologue sanpodo has yet to be identified in the mouse (Roegiers, 2005).

Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. These results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development (Hutterer, 2005; full text of article).

This analysis shows that Sanpodo regulates Notch signalling during Drosophila ES organ development. In the pIIa cell, Sanpodo is localized at the plasma membrane and is required for Notch activation. In the pIIb cell, Sanpodo is removed from the plasma membrane by Numb- and alpha-Adaptin-dependent endocytosis. This correlates with the inability of this daughter cell to activate Notch signalling, suggesting that it is the plasma-membrane-localized Sanpodo protein that activates the Notch receptor. Previous epistasis experiments have suggested that Sanpodo acts during the intramembranous (S3) cleavage of the Notch receptor. Assuming that this cleavage occurs at the plasma membrane, it is possible that Notch needs to bind to Sanpodo to become a substrate for the protease Presenilin, which carries out the S3 cleavage (Hutterer, 2005).

Although this model is attractive, it does not explain why Sanpodo colocalizes with Notch in endocytic vesicles and why these vesicles are found in both pIIa and pIIb cells. Furthermore, it was found that ectopic expression of Sanpodo during neurogenesis (where Numb is expressed but not asymmetric) causes a neurogenic phenotype. Thus, Sanpodo can both activate and inhibit Notch signalling depending on the absence or presence of Numb. These observations are more consistent with an alternative model in which Sanpodo regulates the endocytosis of Notch. It was recently shown that ubiquitination and subsequent endocytosis can downregulate Notch. Conversely, endocytosis can also positively influence Notch signalling and was shown to be required for Notch activation in vertebrates. It is speculated that Sanpodo might have a general role in Notch endocytosis. In the absence of Numb, endocytosis could be required for Notch signalling, whereas in its presence, the inhibitory endocytic pathway could prevail. Although this model is speculative, it would also explain why expression of Numb in tissues that do not express Sanpodo has little or no influence on Notch signalling (Hutterer, 2005).

AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors

In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. This study shows that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, these data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).

The dorsal thorax of Drosophila pupae, the notum, is a single-layered neuroepithelium that produces epidermal and sensory organ (SO) cells. Each adult SO is composed of four cell types and is derived from a single cell, the sensory organ precursor (SOP, also called the pI cell). Notch regulates binary cell fate decisions in the SO lineage. Each SOP undergoes asymmetric cell division to generate two distinct daughter cells; Notch is activated in the SOP daughter cell that adopts the pIIa fate and is inhibited in the other cell, which becomes a pIIb cell. The pIIa cell divides to generate the external cells of the SO, the shaft and socket cells. The pIIb cell undergoes two rounds of asymmetric cell division to generate the internal cells of the SO, the neuron, the sheath cell, and a glial cell. Although Notch-mediated binary cell fate decision in the SO lineage is tightly controlled by intracellular trafficking, the exact subcellular location of where Notch ligand and receptor interact to produce a signal is subject to debate (Benhra, 2011).

To identify new regulators of Notch signaling involved in intracellular trafficking, a double-stranded RNA (dsRNA) screen was carried out for genes affecting SO development and the clathrin adaptor AP-1 complex was identified. AP-1 is an evolutionarily conserved heterotetrameric complex. Drosophila AP-1 complex is composed of AP-1γ (CG9113), β-adaptin (CG12532), AP-1μ1 (encoded by AP-47, CG9388), and AP-1σ (CG5864) subunits. Although mammalian AP-1 is involved in lysosome-related organelle (LRO) biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 remains largely unknown. Each wild-type SO contains only one socket cell. In contrast, tissue-specific gene silencing of any of the three AP-1 specific subunits, AP-47, AP-1γ, or AP-1σ, gives rise to a Notch gain-of-function phenotype that results in a pIIb-to-pIIa cell fate and/or a shaft-to-socket cell transformation, leading to an excess of socket cells. Following knockdown of AP-1 subunits, 4% to 17% of SO show more than one socket cell. To confirm and extend these dsRNA-induced results, classical mutants were analyzed. Two mutations in AP-47, AP-47SHE11, and AP-47SAE10 were previously recovered as genetic modifiers of presenilin hypomorphic mutations. This stud characterized the AP-47SHE11 allele as a genetic null, whereas the second allele, AP-47SAE10, is hypomorphic. AP-47SHE11/Df(3R)Excel 6264 transheterozygotes die at early first-instar larvae stage, indicating that, as in worms, zebrafish, and mice, AP-47 is essential for viability. To assess the AP-47 loss-of-function phenotype in SO, AP-47 mutant mitotic clones were generated and analyzed in the notum. The same two categories of transformed mutant organs were observed as in the dsRNA experiments. Cell fate transformation was seen in 11% of the mutant organs and in 17% following AP-47dsRNA. The difference could be due to protein perdurance in the mutant clones induced during development. The incomplete penetrance suggests that a compensatory mechanism could bypass the requirement for AP-1. In any case, the results suggest a requirement for the AP-1 complex in Notch-dependent binary cell fate acquisition (Benhra, 2011).

Excess Notch signaling can arise from either disruption of epithelial cell polarity or defects in partitioning of cell fate determinants at mitosis. Because cell polarity relies on the proper apicobasal sorting of membrane proteins, a process requiring both clathrin activity in mammals, this study has analyzed the localization of various polarity markers in AP-47 mutant clones. The Notch gain-of-function phenotype observed in the absence of AP-1 activity cannot be explained by a disruption of epithelial cell polarity, nor by a defect in the partitioning of the cell fate determinants Numb and Neuralized (Neur) at mitosis. Thus, AP-1 activity may be required after unequal segregation of cell fate determinants, possibly at the pIIa/pIIb cell stage to control Notch signaling (Benhra, 2011).

Defects in the endolysosomal degradation, such as in vps25 and erupted mutant cells, result in a Notch gain-of-function phenotype that is caused by ligand-independent mechanisms. Because AP-1 is involved in the biogenesis of LROs in mammals, genetic interaction tests were devised to determine whether excess signaling caused by loss of AP-47 requires the activity of the Notch ligands Delta and Serrate (Ser). Loss of Delta and Ser signaling causes Notch loss-of-function phenotypes, a lateral inhibition defect and a pIIa-to-pIIb cell fate transformation that results in generation of extra neurons, the opposite phenotype to what was observed in AP-47 mutant clones. Loss of external sensory cells accompanied by an excess of neurons is observed in AP-47 Delta Ser triple mutant clones, a phenotype indistinguishable from that of Delta Ser double mutant clones. The reversal of pIIb-to-pIIa transformation phenotype of AP-47 in AP-47 Delta Ser triple mutant clones demonstrates that Delta and Ser are epistatic to AP-47. This finding indicates that the AP-47 mutant phenotype is ligand dependent (Benhra, 2011).

The activity of Delta in the SO lineage is controlled by Neur-dependent endocytosis. Following endocytosis, Delta is recycled, and its trafficking toward apical microvilli requires Arp2/3 and WASp. Mutations in WASp prevent Notch signaling, resulting in a pIIa-to-pIIb cell fate transformation. Excess Notch signaling is observed in AP-47 WASp clones, as in AP-47 clones. These data demonstrate that AP-47 is required for SO formation even in the absence of WASp. These findings suggest that AP-1 is unlikely to act by preventing Delta recycling and raise the possibility that AP-1 acts on Notch receptor signaling (Benhra, 2011).

Sanpodo (Spdo) is a four-pass transmembrane protein required for Notch signaling in asymmetrically dividing cells. Because mutations in spdo result in reduced Notch signaling, the opposite phenotype to what was observed in AP-47 mutant clones, it could be that AP-1 normally represses Spdo activity. To test this hypothesis, AP-47 spdo double mutant clones were generated and a phenotype was observed that is indistinguishable from that of spdo mutant clones. The reversal of the pIIb-to-pIIa transformation phenotype of AP-47 in AP-47 spdo double mutant clones indicates that AP-1 requires the activity of Spdo to control Notch signaling and suggests that AP-1 might control Spdo trafficking and/or localization (Benhra, 2011).

To test for a role of AP-1 in Spdo localization, the subcellular distribution of Spdo was compared in wild-type and AP-47 SO lineages. In the wild-type SOP, Spdo is found in intracellular compartments. After division, Spdo-positive vesicles remain localized in the pIIb cell as a consequence of the unequal inheritance of Numb during SOP mitosis, whereas Spdo localizes preferentially at the plasma membrane of the posterior pIIa cell. Spdo is also detected at the apical cortex of SOP and pIIa/pIIb cells, albeit at a low level. In contrast, in AP-47 mutant SO cells, Spdo accumulates apically, as well as at the interface between the AP-47 SOP daughter cells, where DE-Cad is present. It is concluded that loss of AP-1 results in the specific accumulation of Spdo at the apical plasma membrane in SO cells, as well as at the level of adherens junction in SOP daughters. It is suggested that this defect in Spdo trafficking could explain the excess Notch signaling (Benhra, 2011).

Because AP-1 is required for proper localization of Spdo, an anti-AP-1γ antibody was generated to investigate the subcellular distribution of AP-1 relative to Spdo. AP-1γ is closely juxtaposed to the trans Golgi network (TGN) marker GalT::RFP and colocalizes partially with Liquid facet related (LqfR; CG42250), the Drosophila ortholog of Epsin related (Epsin-R), recently reported to localize at the TGN. AP-1γ also partially colocalizes with Rab11-positive recycling endosomes (RE). Thus, in epithelial cells of the notum, AP-1 is found on two membrane-bound compartments, the TGN and RE, as previously reported in tissue culture cells. In SOPs, Spdo was previously shown to partially colocalize with Notch, Hrs, and Rab5. This study reports that Spdo also colocalizes with AP-1γ and Rab11-positive endosomes, suggesting that Spdo traffics within the TGN and RE (Benhra, 2011).

Together with the above genetic data, colocalization of AP-1 with Spdo raises the interesting possibility that AP-1 could control the sorting and transport of Spdo. Furthermore, Spdo contains a conserved N-terminal YTNPAF motif that falls into the Y/FxNPxY/F-consensus sorting signal of the LDL receptor whose localization is regulated by clathrin adaptors. If Spdo is an AP-1 cargo, deletion of the sorting motif of Spdo should prevent its interaction with AP-1. To test this prediction, the localization of AP-47-VenusFP (VFP) was analyzed relative to that of Spdo-mChFP versus Spdo-mChFP deleted of its 18 first amino acids containing the YTNPAF motif (SpdoΔ18-mChFP) in the SOP lineage. On average at the two-cell stage, 69% of the AP-47-VFP-positive vesicles are also positive for Spdo-mChFP, whereas only 14% of AP-47-VFP vesicles are positive for SpdoΔ18-mChFP. Thus, the first 18 amino acids of Spdo may be required for its AP-1-mediated sorting. Nonetheless, SpdoΔ18-mChFP does not accumulate at the apical cortex, suggesting that additional sorting motifs or interacting proteins such as Numb, also interacting with Spdo via the YTNPAF motif, contribute to Spdo apical localization. These data reveal that in addition to AP-2, a second clathrin adaptor complex, AP-1, controls the localization of Spdo and regulates Notch signaling. AP-2 and Numb prevent Spdo accumulation at the plasma membrane, whereas AP-1 prevents Spdo accumulation at the apical plasma membrane. Whether AP-1 binds directly to the YTNPAF motif or indirectly via a yet-to-be-discovered clathrin-associated sorting protein (CLASP) like Numb remains unknown. By analogy to Numb and AP-2, the hypothetical CLASP would function together with AP-1 to sort Spdo at the TGN and/or RE (Benhra, 2011).

Based on its localization at the TGN and the RE, AP-1 may ensure sorting of Spdo from the TGN and/or RE. To test whether AP-1 has a role at RE, a functional Spdo construct was generated in which mChFP is inserted in the second extracellular loop of Spdo (SpdoL2::mChFP) and used in a pulse-chase internalization assay with an anti-RFP that recognizes the extracellularly accessible mChFP tag in epithelial cells of the notum. In the control, following a 45 min chase, the anti-RFP has been efficiently internalized and resides primarily in apically localized endosomes. A small pool of anti-RFP is also detected at the level of adherens junctions labeled with DE-cadherin, suggesting that Spdo can be recycled back to adherens junctions, albeit with low efficiency. In cells depleted of AP-1, anti-RFP internalized from the basolateral membrane is efficiently recycled to the adherens junctions, suggesting that AP-1 acts in RE to limit recycling of Spdo toward adherens junctions. In contrast, when AP-2-dependent endocytosis is prevented, anti-RFP remains mostly localized at the basolateral plasma membrane, even after a chase of 45 min, as predicted for a requirement of AP-2 in the internalization of Spdo. Therefore, the data indicate that AP-1 does not regulate endocytosis of Spdo from the basolateral membrane. To test whether AP-1 could regulate apical endocytosis of SpdoL2::mChFP, a pulse-chase internalization assay was conducted in epithelial cells of the wing imaginal discs, a tissue that, in contrast to the pupal notum, allows for access of anti-RFP at the apical plasma membrane. In cells depleted of AP-47, anti-RFP resides predominantly at the apical side at the level of adherens junction at t = 0 and is internalized with similar kinetics as in the control situation. It is concluded that AP-1 does not regulate SpdoL2::mChFP apical internalization. Altogether, these results indicate that AP-1 acts at the RE to prevent or limit apical recycling of Spdo, giving a rationale for why endogenous Spdo accumulates apically in SO mutant for AP-47 (Benhra, 2011).

Does apical accumulation of Spdo cause the Notch gain-of-function phenotype seen in AP-1 mutant SO? Spdo was previously reported to partially colocalize with Notch in large intracellular structures and at the plasma membrane. In wild-type, Notch localizes at the apical membrane of epidermal cells, SOP cells, and SOP daughter cells. Shortly after SOP division, Notch extracellular domain (NECD) is detected apically together with Spdo at the DE-Cad interface between pIIa and pIIb. This specific localization is transient, because NECD and Spdo are detected at the interface of daughter cells in one-third of the cases and are no longer detectable at the pIIa/pIIb interface when the remodeling of the apical cortex of pIIa/pIIb cells takes place. In AP-47 mutant cells, NECD is stabilized with Spdo at the interface of SOP daughter cells, even at a time when control organs have undergone apical cortex remodeling. Similarly, Notch intracellular domain (NICD) is accumulated at the level of adherens junctions in AP-47 mutant cells, whereas it is detected at the interface of wild-type SOP daughters in only half of the cases. To determine whether the stabilization of Notch at the SOP daughter cell interface is caused specifically by AP-47 loss of function, NECD localization was compared in AP-47 versus spdo or AP-47 spdo double mutant clones. Although NECD is enriched at the apical surface in these three mutant situations compared to control cells, stabilization of NECD at the interface of SOP daughter cells occurs in AP-47 single and AP-47 spdo double clones, but not in spdo single clones. These data indicate that, upon loss of AP-47, Spdo is not required for NECD to accumulate at the junction between SOP daughter cells, which raises the interesting possibility that Notch itself may be an AP-1 cargo. Because Spdo and Notch are transiently detected at the interface of wild-type SOP daughter cells, it is proposed that sustained elevated levels of Spdo and Notch at the interface cause the excess signaling observed in AP-47 mutants. These effects of AP-1 appear to be specific to Spdo and Notch, because Delta is transiently detected in punctuated structures at the level of junctions together with Spdo in a similar manner in both control and AP-47 SOP daughter cells. Furthermore, endocytosis of Delta is unaffected by the loss of AP-1. It is thus concluded that AP-1 regulates the amount of Notch and Spdo at this junctional domain, which could serve as a launching pad from which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).

These data have uncover a novel function for AP-1 complex during development. The observations suggest that AP-1 participates in the polarized sorting of Spdo and Notch from the TGN and/or RE toward the plasma membrane. The correlation between the Notch gain-of-function phenotype and the stabilization of Notch and Spdo at the junctions suggests that adherens junctions may be particularly important for Notch activation. Because the effect of loss of AP-1 on Spdo and Notch localization is completely penetrant, it is proposed that a threshold of Spdo and Notch localized at the junctional domain has to be reached in order to cause the cell fate transformation, explaining why only 10% to 20% exhibit the Notch gain-of-function phenotype (Benhra, 2011).

Previous reports have suggested that trafficking of endocytosed Delta to the apical membrane in the pIIb cell is required for its ability to activate Notch that localizes at the apical side in the pIIa cell. Recently, it was reported that most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Based on phalloidin staining, the ARS appears to be unaffected by the loss of AP-47. Notch and Spdo are stabilized at the junctional domain that is included within the ARS and are therefore poised to receive the Delta signal. This would place this domain of the ARS as an essential site for Delta-Notch interaction, leading to productive ligand-dependent Notch signaling (Benhra, 2011).

Could this novel function for AP-1 be conserved in mammals? Spdo is specifically expressed in Dipterans, and no functional ortholog has been described so far, raising the question of the role of AP-1 in Notch signaling in mammals. Nonetheless, Notch is also mislocalized in AP-1 mutant cells even when Spdo activity is missing. Notch also contains evolutionarily conserved tyrosine-based sorting signals, and it cannot be excluded at present that Notch is itself an AP-1 cargo. Finally, the facts that Notch controls several early steps of T cell development and that mice heterozygous for γ-adaptin exhibit impaired T cell development raise the interesting possibility that Notch-dependent decisions in mammals also required AP-1 function (Benhra, 2011).

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. A forward genetic screen identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. It is proposed that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells (Giagtzoglou, 2012).

This study describes the identification of dEHBP1 as a novel, positive regulator of Notch signaling in asymmetrically dividing cells in the ESO lineage in Drosophila. In the absence of dEHBP1, external cell types, such as socket and shaft cells, are transformed into internal cell types, i.e., neuron and sheath cells, one of the hallmarks of loss of Notch signaling. EHBP1 has been previously studied in mammalian cell culture systems and in vivo in C. elegans. In mammalian adipocytes, EHBP1 affects endocytosis and recycling of the glucose transporter GLUT4 in the context of insulin signaling, depending on its interaction via the NPF motifs present in its N-terminal region with EHD2 or EHD1, respectively. However, the fly and worm EHBP1 lack the NPF motifs, suggesting that the EHD-EHBP1 interaction may have emerged later in evolution. In C. elegans, EHBP1 was shown to impair rab10-mediated endocytic recycling of clathrin-independent endocytosed cargoes, such GLR-1 glutamate receptor. This study shows that dEHBP1 is required in the exocytosis and recycling of Delta, a ligand of the Notch receptor. Notch signaling defects were not reported in C. elegans ehbp1 mutants. Therefore, it would be interesting to investigate whether EHBP1 and its homologues play an evolutionarily conserved role of EHBP1 in Notch signaling (Giagtzoglou, 2012).

dEHBP1 is a ubiquitous protein that is associated with the plasma membrane, enriched at the lateral and basal surface of pII cells, where it colocalizes with F-actin. Live imaging with mCherry-dEHBP1 and immunofluorescent stainings with anti-dEHBP1 antisera also reveal dEHBP1-positive, punctate, intracellular structures within ESO lineages. An extensive analysis with a diverse array of intracellular markers revealed that these punctae colocalize with Rab8, indicating their exocytic nature. Importantly, in C. elegans, EHBP1 physically interacts and colocalizes with Rab8 and Rab10, and controls the recruitment of Rab10 in recycling endosomal structures. However, in the current studies, overexpression of dominant-negative forms of Rab10 or Rab8 in the ESO lineages as well as thoracic clones of a newly identified Rab8 loss-of-function allele do not confer any cell fate phenotypes. Furthermore, no interaction was detected between dEHBP1 and Rab8 or Rab10 in a yeast two-hybrid analysis. Therefore, it is believed that loss of either Rab8 or Rab10 function does not underlie the dEHBP1 mutant phenotypes that are describe (Giagtzoglou, 2012).

Notably, many key players that affect cell polarity or mark subcellular compartments, including Arm, Rab11, Sec15, and F-actin, are not affected by the loss of dEHBP1. In addition, cell fate determinants Numb and Neuralized are correctly segregated upon asymmetric cell division in dEHBP1 mutant cells. However, loss of dEHBP1 specifically affects the abundance and localization of Spdo, a regulator of Notch signaling in asymmetrically dividing ESO cells, and the exocytosis and trafficking of Delta (Giagtzoglou, 2012).

Spdo facilitates reception of Notch signal at the plasma membrane of the signal-receiving cell. Therefore, accumulation of Spdo in dEHBP1−/− ESO clusters and its presence in the plasma membrane should result in a Notch gain of function, instead of the loss-of-function phenotype that was observed. No effects have been observed of Spdo overexpression upon cell fate acquisition in the ESO lineage. Alternatively, the accumulation of Spdo in the absence of dEHBP1 in these cells may reflect defects in its trafficking and membrane localization, which render the activation of Notch signaling more difficult (Giagtzoglou, 2012).

dEHBP1 mutations cannot suppress the gain of function phenotype of overexpressed ligand-independent, activated Notch intracellular domain. In addition, dEHBP1 does not affect the steady-state levels of Notch protein, as well as its endocytosis. Therefore, it is concluded that dEHBP1 functions at a level upstream of presenilin-mediated S3 cleavage of Notch during reception of the signal. Although it cannot be excluded that dEHBP1 functions in the signal-receiving cell, where it may control the trafficking and localization of Spdo, it is concluded that dEHBP1 also functions in the sending of the signal. This conclusion is based on the fact that dEHBP1 mutations are able to suppress the gain of function of Notch phenotype conferred by the overexpression of DaPKCΔN. Overexpressed constitutively active DaPKCΔN places Spdo at the plasma membrane, enabling the activation of Notch signaling. This study found that upon loss of dEHBP1, Spdo is still found at the plasma membrane under conditions of overexpression of DaPKCΔN. Therefore, the suppression of the overexpression phenotype of DaPKCΔN by loss of dEHBP1 may be because of other defects, such as loss of the ability of Delta to signal. Furthermore, loss of dEHBP1 leads to development of additional neurons despite the concomitant ectopic expression of DeltaR+, a variant of Delta, in clones within pupal nota at 36 h APF. Because the steady-state levels of Delta are not affected in dEHBP1−/− ESO lineages, whether dEHBP1 affects Delta trafficking in the signal-sending cell was examined. Upon loss of dEHBP1, the abundance of Delta at the cell surface is significantly reduced, suggesting that exocytosis is defective. Importantly, most of the remaining extracellular Delta protein localizes at the basal side of the signal-sending cell. This suggests that in addition to affecting exocytosis of Delta, dEHBP1 may also play a role in basal-to-apical trafficking of Delta. This leads to a reduced level of Delta at the signaling interface, which interferes with proper Notch signaling in the cell receiving the signal. Although the results do not exclude a possible role of dEHBP1 in other aspects of Delta trafficking, such as endocytosis, reduced exocytosis of Delta should mask an endocytic defect in the assays. The enrichment of dEHBP1 in the basal and lateral area of the plasma membrane, its colocalization with F-actin at the actin-rich structure at the interface of the pIIa and pIIb cells, the reduction of Delta exocytosis in mutant cells, and the absence of Delta at the interface and the apical surface of the ESO cluster in mutant cells indicate a role of dEHBP1 in the Sec15/Rab11 recycling pathway. Indeed, the colocalization of dEHBP1 and Delta in sec15−/− ESO lineages implies that the exocyst component, Sec15, controls exocytosis of Delta, Spdo, and dEHBP1 to the apical plasma membrane through a common compartment. Because loss of dEHBP1 does not affect the localization of either Rab11 or Sec15, it is concluded that sec15 lies more upstream in the trafficking pathway regulating the localization of multiple components, while dEHBP1 functions during the later stages of intracellular trafficking. Furthermore, the physical interaction between dEHBP1 and Sec15 as well as Rab11 suggest a mechanism how dEHBP1 may regulate the membrane localization of Delta via its interaction with Sec15 and Rab11 at the pII cells interface, even though such interaction was detected under transient overexpression conditions. It is proposed (see Model of dEHBP1 function) that dEHBP1 is an adaptor of the Rab11/Sec15-positive, Delta-bearing vesicles required for exocytosis (Giagtzoglou, 2012).

The identification of dEHBP1 provides further compelling evidence that the exocytosis and recycling pathway of Delta during asymmetric divisions is tightly regulated. The recycling pathway of Delta appears to be context dependent, i.e., it is not required in all cells that use Notch signaling. Still, the discovery of dEHBP1 as a novel player in Notch signaling provides the opportunity to test its role in Notch-related neurobiological behaviors, such as sleep and addiction, as well as in Notch-related diseases, as for example in Wiskott-Aldrich syndrome, an immunodeficiency characterized by abnormal differentiation and function of T cell lineages. Furthermore, because the anthrax toxins lethal factor (LF) and edema factor (EF) inhibit the Sec15/Rab11-dependent Delta-recycling pathway in flies and endothelial cells, it would be interesting to hypothesize whether they target dEHBP1 to mediate their toxicity (Giagtzoglou, 2012).

A fluorescent tagging approach in Drosophila reveals late endosomal trafficking of Notch and Sanpodo

Signaling and endocytosis are highly integrated processes that regulate cell fate. In the Drosophila melanogaster sensory bristle lineages, Numb inhibits the recycling of Notch and its trafficking partner Sanpodo (Spdo) to regulate cell fate after asymmetric cell division. This paper used a dual GFP/Cherry tagging approach to study the distribution and endosomal sorting of Notch and Spdo in living pupae. The specific properties of GFP, i.e., quenching at low pH, and Cherry, i.e., slow maturation time, revealed distinct pools of Notch and Spdo: cargoes exhibiting high GFP/low Cherry fluorescence intensities localized mostly at the plasma membrane and early/sorting endosomes, whereas low GFP/high Cherry cargoes accumulated in late acidic endosomes. These properties were used to show that Spdo is sorted toward late endosomes in a Numb-dependent manner. This dual-tagging approach should be generally applicable to study the trafficking dynamics of membrane proteins in living cells and tissues (Couturier, 2014).



To follow the expression and subcellular localization of Spdo, antibodies specific to two overlapping regions of the predicted cytoplasmic domain of Spdo were generated. Using either antibody, it was found that Spdo is expressed in all NBs, all GMCs, and transiently in most, if not all, neurons in the CNS. In the PNS, Spdo is expressed in all SOPs and their progeny. In the mesoderm, Spdo is expressed in heart and somatic muscle precursors that undergo spdo-dependent asymmetric divisions. Spdo is also expressed in the asymmetrically dividing cells of the posterior midgut. Thus, all embryonic cells known to undergo asymmetric divisions, even those thought to divide asymmetrically in a spdo-independent manner, appear to express Spdo. Consistent with Spdo playing a role to regulate asymmetric NB divisions, a weak, but consistent, duplication of GMC1-1a is observed in spdo mutant embryos (O'Connor-Giles, 2003).

Sec15, a component of the exocyst, promotes Notch signaling during the asymmetric division of Drosophila sensory organ precursors: a proposed role for Spdo in Delta recycling in the signal-sending cell

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, a mutation was isolated in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15 sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, it is proposed that a defect in Delta recycling causes cell fate transformation in sec15 sensory lineages. The data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila (Jafar-Nejad, 2005).

In a genetic screen designed to identify novel players in Drosophila sensory organ development, a mutation in sec15 was isolated that caused a pIIa to pIIb transformation phenotype. Sec15 is a component of a multiprotein complex called the exocyst or Sec6/8 complex. Mutations in exocyst components were originally isolated in a yeast screen for secretion-defective mutants. Subsequent analysis of the exocyst complex in yeast and mammalian cell culture systems has indicated that it functions in intracellular vesicle transport. In yeast, the exocyst mediates the post-Golgi to membrane targeting of exocytic cargo via an interaction with the Rab GTPase Sec4p. In Madin-Darby canine kidney (MDCK) epithelial cells, the exocyst localizes to areas of cell-cell contact and is involved in basolateral delivery of vesicles. However, none of the studies on the exocyst components have implicated these proteins in cell fate determination. The data suggest that Sec15 mediates highly specific intracellular trafficking events that promote N signaling and thereby ensure proper cell fate specification in Drosophila mechanosensory organs (Jafar-Nejad, 2005).

The various cell types that form an adult sensory organ in Drosophila are generated via asymmetric divisions of a pI and its progeny. Differential activation of the N signaling pathway between the two daughter cells of each division ensures that each sensory organ acquires the proper complement of cell types necessary to function. Sec15, a component of the evolutionarily conserved exocyst complex as reported here, is required for proper cell fate specification of the pI progeny. Studies on sec15 mutations in the eye did not reveal any fate change in the photoreceptors. Loss-of-function mutations in three other exocyst components have been reported previously: sec5 and sec6 in flies and sec8 in mice. sec8 mutant mice die at day E7.5, before the development of specific neuronal populations can be studied. Also, sec5 and sec6 mutations are cell lethal in the Drosophila eye. Therefore, this report is the first to identify a role for an exocyst component in cell fate determination. At this point, it cannot be predicted if sec5 and sec6 also play a role in neuronal cell fate specification. However, given the data obtained from studies of the fly eye, the hypothesis is favored that components of the exocyst may form more than a single functional unit and/or have subunit-specific roles (Jafar-Nejad, 2005).

Live imaging of dividing pI cells indicates that Sec15 is associated with a vesicular compartment that traffics between apical and subapical areas. In sec15 SOPs, an expanded compartment is observed that contains Spdo, N, and Dl. Unlike wt pI and pIIb cells, in which Spdo/N/Dl+ vesicles tend to reside at or above the level of septate junctions, in mutant SOPs these puncta accumulate at the basal side of the cell. Together, these observations suggest that Sec15 is involved in vesicle trafficking to the apical parts of the cell. The defect in the apical trafficking of proteins does not seem to be a general one, since localization of E-Cad and Arm at the adherens junction is not disrupted in mutant tissue. Therefore, the data link a specific vesicle trafficking event to a developmental decision made by sensory precursor cells (Jafar-Nejad, 2005).

Genetic experiments and immunohistochemical stainings strongly suggest that Sec15 and Spdo function in the same pathway in sensory cell fate determination process. It has been proposed, based on studies performed on the asymmetric divisions of Drosophila embryonic neuroblasts, that Spdo promotes N signaling at the membrane of the signal-receiving cell. In contrast, Numb and α-Adaptin in the signal-sending cell might promote endocytosis of Spdo and its removal from the membrane, thereby preventing the reception of signal by this cell. The subcellular distribution of Spdo in pIIa and pIIb cells is similar to its localization in embryonic neuroblast progeny, suggesting that this model might also apply to adult bristle formation. Notably, however, Spdo is observed at or close to the membrane of both pI progeny in sec15 clones. Therefore, while the proposed role for Spdo in promoting N signaling at the membrane of the signal-receiving cell cannot be ruled out, the data suggest a role for Spdo in Dl recycling in the signal-sending cell. It should be noted, though, that these two models are not mutually exclusive. Presence of a significantly higher number of vesicles containing both Dl and N in pIIb compared to the pIIa in wt sensory precursors has been implicated in the ability of the pIIb cell to send the Dl signal. Colocalization of Spdo with Dl in a significant fraction of these vesicles suggests that a defect in Spdo/Dl trafficking in pIIb contributes to the sec15 loss-of-function phenotype (Jafar-Nejad, 2005).

Presence of endocytosed Dl in vesicles that accumulate in sec15 clones implicates these vesicles in the endocytic traffic of Dl. This notion is further supported by the observation that in both wt and sec15 SOPs, the Spdo/Dl/N puncta show a significant colocalization with the endosomal markers Rab5 and HRS. It has recently been proposed that in order to signal, Dl needs to traffic through a specific endocytic compartment, which will lead to recycling of the protein. A defect in Dl recycling is further suggested by the aberrant accumulation of the recycling endosomal marker Rab11 in sec15 clones. The Rab11+ endosomal compartment is thought to be a central trafficking intermediate in both exocytic and endocytic pathways and is shown to control the traffic of cargo from the perinuclear recycling endosomal compartment to the membrane. Interestingly, it has been shown that Sec15 meets the criteria of being an effector for Rab11 in mammalian cell lines: Sec15 physically binds Rab11 in a GTP-dependent manner; Sec15 colocalizes with Rab11 in the perinuclear region of the cells; Sec15 labels structures containing an endocytosed protein in immuno-EM experiments. Similarly, Drosophila Sec15 and Rab11 interact physically and show a high level of colocalization in SOPs. Altogether, these data are compatible with a model in which Sec15 regulates the traffic of a subset of endocytosed Dl to the membrane of the pIIb cell via a Rab11+ recycling endosomal compartment. Sec15 traffics symmetrically in pIIa and pIIb. Therefore, it is proposed that an intrinsic difference between the endocytic traffic of Dl in pIIa and pIIb allows the pIIb cell to employ the Sec15-Rab11 machinery differentially from the pIIa cell and thereby assume the role of signal-sending cell. The most likely mechanisms for the proposed intrinsic difference are unequal segregation of Neur into the pIIb, which promotes Dl endocytosis in this cell, and asymmetric distribution of the Rab11+ recycling endosomes in the pIIb versus pIIa, which is thought to specifically mediate Dl recycling in the pIIb (Jafar-Nejad, 2005).

These data suggest that at least some of the Spdo/N/Dl-containing vesicles that accumulate in the basal areas of sec15 SOPs are of a mixed exo-endocytic nature. This is not unprecedented, since traffic from the TGN to an endosomal compartment has been documented. Accordingly, it has been proposed that some exocytic cargo might pass through the recycling endosome on its way from the TGN to the plasma membrane. Recently, it has been shown that upon exit from the Golgi apparatus, newly synthesized E-Cad fuses with a Rab11+ recycling endosomal compartment before it reaches the plasma membrane. It is interesting to note that members of the exocyst complex have been shown to localize to both the TGN and recycling endosomes in polarizing epithelial cells. Although the recycling endosome has been proposed as an intermediate to transfer the exocytic cargo to the plasma membrane, it is possible that passing through these vesicles somehow enhances the signaling ability of internalized Dl. In other words, presence of Spdo might be part of the specific environment that Dl needs to traffic through. Although Dl endocytosis and recycling are also implicated in N signaling during lateral inhibition, no lateral inhibition defects are observed in sec15 clones. It is proposed that the link to Spdo results in the specificity of the sec15 phenotype to the asymmetric divisions, since loss of spdo similarly does not affect lateral inhibition (Jafar-Nejad, 2005).

In summary, the data indicate that one component of the highly conserved exocyst complex affects the asymmetric division of the sensory precursors in the Drosophila PNS through specific vesicle trafficking events. Components of the exocyst complex are conserved from yeast to human, and several reports have shown parallels between the contribution of asymmetric divisions to Drosophila and vertebrate neurogenesis. Therefore, it is conceivable that Sec15, and perhaps other members of the exocyst complex, are involved in neural cell fate determination in other species (Jafar-Nejad, 2005).

Effects of Mutation or Deletion

The function of the gene sanpodo (spdo) is illustrative of a fundamental process in developmental biology: the specification of cell fate through asymmetric cell division. Before looking at spdo in some detail, a brief outline of cell fate specification in the peripheral nervous system (PNS): the event is mediated in part through asymmetric cell divisions, a process in which one cell divides to give rise to two daughter cells with distinctly different fates. In the external sensory (es) organ lineage, the primary sensory organ precursor (SOPI) divides asymmetrically to produce two daughter cells, SOPIIa and SOPIIb. These two divide, producing (respectively) a hair and socket cell, and a neuron and glial cell. Many genes that play a role in specifying the fate of PNS cells and neurons have been found to be located in the Notch signaling pathway: Notch, Delta, Suppressor of Hairless, Enhancer of split, Mastermind and Kuzbanian. Loss of Delta, Notch, Supressor of Hairless or Kuzbanian leads to an increase in the number of neurons, the neurogenic phenotype. Another component, Numb, has been integrated into the Notch signaling pathway. Numb is a cytoplasmic protein that has been shown to bind to the intracellular domain of Notch and to repress Notch function. Interestingly, in the SOPI of es organs the Numb protein is localized to a crescent at the cell surface. When SOPI divides, the Numb protein segregates into the SOPIIb daughter cell. The resulting daughter-cell-specific repression of Notch signaling is necessary for the SOPIIa and b cells to adopt different fates (Dye, 1998 and references).

Where does sanpodo fit in a discussion of cell fate specification? The gene was identified by Salzberg (1994), as well as in subsequent screens (Salzberg, 1997) for mutations altering the developmental pattern of the peripheral nervous system. All mutants are embryonic lethal. spdo embryos display an approximate doubling of the number of neurons in the PNS when compared to wild-type embryos. This phenotype is the basis for the name Sanpodo, the Korean word for mountain grapes. In spite of the increased number of neurons, PNS neurons remain distributed among the four typical clusters within each segment, but, unlike other neurogenic mutants (e.g. Notch, Delta), the overall morphology of spdo embryos is not affected. In addition, no defects in axon pathfinding were observed in the PNS (Dye, 1998).

An increased number of multiple dendritic (md) or external sensory (es) neurons, as observed in spdo mutants, can be obtained by any of several different mechanisms: an increased recruitment of sensory organ precursors (SOPs), extra divisions of the progeny of SOPs (SOPI and SOPII) or extra divisions of the neuron, or transformation of the es hair, socket or glial cells into neurons. It has been shown that in spdo mutants there are no extra SOPs recruited, and that the total number of cells within the dorsal and lateral clusters of the PNS is not increased significantly. It is therefore concluded that the supernumerary neurons must result from a fate change of cells in the same lineage and it is proposed that the two sibling cells of the SOPIIb (neuron and glia) take on the same fate, i.e. neurons (Salzberg, 1994). Since spdo mutant embryos lack virtually all Prospero and Bar-positive glial support cells in the PNS, it is concluded that the two sibling cells derived from the es SOPIIb take the same neuronal fate in spdo mutants (Dye, 1998).

The dorsal bipolar dendritic (dbd) lineage is the most simple and well-characterized asymmetric cell division in the PNS: the precursor cell divides asymmetrically to give rise to an md neuron and a glial cell. The dbd neuron is often duplicated in spdo embryos. Glial markers show no glia associated with the duplicated dbd neurons. Hence the sibling cell is transformed into an md neuron. This transformation is the opposite of the numb phenotype, which consists of two glial cells instead of two dbd neurons. This suggests that in spdo mutants, for those md neurons that have a lineage-related sibling, the non-neuronal cells as well as the neuronal cell adopt the neural fate. Staining with an md-specific marker reveals another feature of spdo mutants. Es neurons are programmed to become md neurons in spdo mutants. This observation is particularly interesting because the same phenotype occurs in Notch mutants, suggesting that Notch signaling is impaired in spdo mutants. These observations suggest that spdo is a neurogenic gene and when mutated results in too many neurons, the hallmark of the neurogenic phenotype (Dye, 1998).

Spdo functions downstream of Numb, a protein known to be involved in the Notch pathway. Within the es lineage, numb function is required in both the SOPIIb and its daughter cells to correctly specify cell fate. spdo function is also required within the es lineage, but in a manner just the opposite of that in numb. To place spdo in the Numb/Notch pathway, an analysis of numb;spdo double mutants was undertaken. spdo function is likely downstream of numb, because numb;spdo embryos have many more neurons than those that only lack numb. Alternatively, Spdo functions as an antagonist of Numb. Based on the epistatic interaction studies, it is proposed that the transformation of SOPIIb into SOPIIa brought about by loss of numb is often blocked in the absence of spdo. Because loss of Numb in the SOPIIb leads to increased Notch signaling, Notch activation and/or signaling may require Spdo function. A role for Spdo in Notch signaling is supported by the finding that reducing Notch protein function at the time of SOPIIa division decreases the number of es glial cells and increases the number of es neurons. The es neurons express md-specific markers in spdo mutants, as is observed in Notch mutants. It is therefore unlikely that Spdo functions in an independent pathway, although at the present time this possibility can not be formally excluded (Dye, 1998).

sanpodo mutation reveals an additonal role for Sanpodo in external sensory organ hair differentiation. Examination of cuticle preparations of pharate first instar larvae reveal that the three hairs of Keilin organs, a cluster of external sensory organs in the thoracic segments, are almost always affected in spdo mutants. When compared to wild type, the hairs are most often reduced in size or absent. The morphology of the socket cells is often abnormal: in approximately 10% of the mutant embryos there appear to be extra hair cells and a corresponding lack of socket cells, suggesting that the socket cells are transformed into hair cells. The typical structures corresponding to the ventral Kolbchen do not appear to be present; the Kolbchen are refractive club-like sensory structures found in the thoracic segments. Hairs in the abdominal segments are often present, albeit reduced in size. It has been concluded that the differentiation of the external structures of many PNS organs are affected in spdo mutants and that these defects resemble those associated with mutations in genes encoding actin binding proteins. Staining with phalloidin reveals a severe decrease in F-actin staining in Keilin organs and Kolbchen organs in spdo mutants when compared to wild type. This decrease in F-actin staining is observed for the external structures of the entire PNS in mutants (Dye, 1998).

Although most glial CNS cell lineages are not characterized, it can be demonstrated that CNS glia are affected by mutations in sanpodo. Staining with anti-RK2/REPO, a glial marker, reveals a severe loss of glial cells in the CNS. Analysis of the expression of another glial marker, glial cells missing (gcm) , shows that lack of spdo causes a severe decrease in the number of Gcm-positive cells in stage 15 embryos. Because gcm loss-of-function mutations transform glial cells into neurons and overexpression of gcm transforms neurons into glia, gcm is considered to be a glial identity gene. Thus, the absence of gcm expression in spdo mutants suggests that a subset of CNS glia have not adopted a glial fate. The lineages of most CNS glia are not well defined and it is not known if these cells have siblings. As a result, it could not be determine if the absence of gcm expression in spdo mutants results from a cell fate switch, as is occurring in the PNS, or from a failure to maintain gcm expression (Dye, 1998).

Staining of axons in the CNS with monoclonal antibodies mAb BP102 and mAb 1D4 shows that mutations in spdo cause very noticeable disruptions of the longitudinal tracts. The anterior and posterior commissures form and separate properly, but they appear thicker and less condensed than normal. The phenotype in the ventral nerve cord in spdo mutants is strikingly similar to the phenotype reported for gcm loss-of-function mutations. These observations suggest that loss of spdo alters cellular identity in the CNS as well (Dye, 1998).

sanpodo mutation has been shown to disrupt external sensory organ hair differentiation. The hairs of external sensory organs have been shown to be reduced in size and altered in morphology in singed and chickadee mutants. These genes encode, respectively, an actin-bundling protein (Fascin), and G-actin binding protein (Profilin). A determination was made whether lack of spdo caused any defects in the morphology of the extra sensory (es) organ hairs of first instar larvae. Examination of cuticle preparations of pharate first instar larvae reveals that the three hairs of Keilin organs, a cluster of external sensory organs in the thoracic segments, are almost always affected in spdo mutants. When compared to wild type, the hairs are most often reduced in size or absent. In addition, the morphology of the socket cells is often abnormal. In approximately 10% of the mutant embryos extra hair cells are observed and a corresponding lack of socket cells, suggesting that the socket cells are transformed into hair cells. Absent as well from the thoracic segments are the typical structures corresponding to the ventral Kolbchen, a refractive club-like sensory structure that appear as round structures in the tops of wild-type embryos. Hairs in the abdominal segments are often present, albeit reduced in size. It is therefore concluded that the differentiation of the external structures of many PNS organs are affected in spdo mutants and that these defects resemble those associated with mutations in genes encoding actin binding proteins. Staining with phalloidin in spdo mutants reveals a severe decrease in F-actin staining in Keilin organs and Kolbchen organs, when compared to wild type. This decrease in F-actin staining is observed for the external structures of the entire PNS in such mutants (Dye, 1998).

It is thought that in numb mutant embryos there are increased levels of Notch signaling, resulting in a transformation of SOPIIb cells into SOPIIa cells. Consequently, loss of numb reduces the number of PNS neurons. Because this phenotype is the opposite of that in spdo mutants, the epistatic relationship between numb and spdo was determined. numb;spdo double homozygous embryos were analyzed with both neuronal and glial markers to determine if the phenotype caused by loss of numb requires the presence of Spdo or vice versa. When compared to wild-type, spdo mutants exhibit roughly twice the normal amount of neurons; embryos that lack only numb function have 3-8 neurons per hemisegment (compared to approximately 35 in wild-type). Double mutants never exhibit the numb phenotype; rather, the number of neurons is more similar to that seen in spdo embryos, showing that loss of spdo partially suppresses the numb phenotype. Analysis of the epistatic interactions between numb and spdo within a single PNS lineage also demonstrates that spdo is epistatic to numb. The dbd lineage of the PNS consists of one neuron and one glial cell derived from a single precursor cell. In the absence of numb, two dbd glial cells are seen; in spdo mutants, the dbd glial cell is absent. In double mutants, the dbd glial cell is missing, as is seen in spdo embryos. These observations suggest that spdo functions downstream of, or antagonistically to, numbto specify cell fate in this cell lineage (Dye, 1998).

Eleven alleles of spdo were isolated based on dramatic alterations in even-skipped expression in the CNS. Embryos homozygous for the null spdo ZZ27 allele, subsequently called 'spdo embryos,' show normal Eve + GMCs but an equalization of sibling neuron identity as detected by Eve and other markers. The RP2 motoneuron (derived from GMC 402a) is duplicated at the expense of the RP2sib, as shown by staining for Eve, Zfh-1 and 22C10. The aCC motoneuron (derived from GMC1-1a) is duplicated at the expense of the pCC interneuron, as shown by staining for Zfh-1, 22C10 and by following axonal projections. The Usib (derived from GMC7-1a) fates are duplicated at the expense of the U neurons, as shown by Eve staining. dMP2 is duplicated at the expense of vMP2, as shown by Odd-skipped and 22C10 staining. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation characteristic of Notch mutations. spdo germline clones yield an Eve CNS phenotype identical to embryos that lack only zygotic spdo function. Thus, spdo does not appear to function during Notch-mediated lateral inhibition in the neuroectoderm (Skeath, 1998).

Numb is known to bind to the intracellular domain of Notch and antagonize Notch signaling but, with the exception of the dMP2/vMP2 neurons, it has not been reported to play a role in sibling neuron development in the CNS. However, due to the widespread role of Notch in specifying asymmetric sibling neuron identity, the CNS function of numb was re-investigated. Sibling neuron development was assayed using four different numb alleles and a deficiency that uncovered the numb locus. In embryos homozygous for the strongest numb allele (nb 2 ), an equalization of sibling neuron phenotype is observed for all siblings tested, with the exception of aCC/pCC. RP2 is transformed into RP2sib approximately 50% of the time; three Usibs are transformed into three U neurons; and dMP2 is transformed into vMP2. The numb phenotypes for RP2, Usib and dMP2 neurons are reciprocal to those observed in spdo, Delta, Notch or mam embryos. This is consistent with studies showing that Numb antagonizes Notch function and extends this interaction to a diverse array of CNS sibling neurons. In addition, a strong decrease in the number of Eve + EL neurons is observed in numb mutant embryos. There is clear evidence of maternal numb function during CNS development, which may account for the lack of a fully penetrant numb sibling neuron phenotype. Changing the dose of maternal numb product directly affects CNS development and suggests that numb may have earlier CNS functions in addition to sibling neuron specification (Skeath, 1998).

spdo and numb have opposite sibling neuron phenotypes and so the epistatic relationship between the two genes was determined by examining the phenotype of a numb;spdo double mutant. The numb;spdo double mutant phenotype is identical to embryos lacking spdo alone. Thus, spdo is genetically downstream of numb, just as has been observed for Notch pathway mutations in other lineages. sanpodo and numb exhibit dosage-sensitive interactions, as gene products that act in the same biochemical pathway often do. The sibling neuron phenotype in numb embryos is sensitive to the level of spdo. For example, homozygous nb 2 embryos show a loss of EL and RP2 neurons, but reducing the dosage of spdo by one-half in nb 2 embryos leads to a recovery of Eve + EL and RP2 neurons. Thus, halving the dosage of spdo strongly suppresses the numb CNS phenotype. These results show that the numb phenotype is extremely sensitive to the dosage of spdo, consistent with the two proteins acting in the same biochemical pathway (Skeath, 1998).

In numb embryos, there is a striking decrease in the number of Eve + EL neurons. Notch, Delta, mam and spdo single mutants do not alter the number of Eve + EL neurons. Importantly, numb;spdo double mutant embryos show a complete rescue of Eve + EL neurons , suggesting that Numb acts to prevent Spdo-mediated downregulation of eve expression (i.e. in the absence of Spdo, the loss of Numb is irrelevant). These data are consistent with a model in which Numb blocks Notch/Spdo-mediated downregulation of eve in the neurons of the EL lineage (Skeath, 1998).

Spdo regulates Notch-mediated sibling cell fate decisions but is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles) and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway' genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm. However, mutations in neuralized and E(spl) have no effect on the identity of the sibling neurons that were assayed, despite strong defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. For the most part the basis of asymmetric cell division has been analyzed in neuroblasts in the process by which neuroblast division yields another neuroblast and a secondary precursor cell: the ganglion mother cell (GMC). In the embryonic central nervous system of Drosophila melanogaster, GMCs divide and produce postmitotic neurons that take on different cell fates. The current study analyses the process of binary fate decision of two pairs of sibling neurons that occurs during cell division in GMCs. This process is accomplished through the intrinsic fate determinant, Numb. GMCs have apical-basal polarity; Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. These results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, the data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells (Buescher, 1998).

The focus for the analysis of the roles of insc, numb, and components of the N-signaling pathway in fate specification, was on the only two pairs of GMC-derived neurons for which sibling relationships have been established: the RP2/RP2sib and the aCC/pCC neurons. These neurons are derived from two GMCs that can be identified unambigously by their specific expression of the nuclear protein Even-skipped (Eve). GMC1-1a divides into the aCC/pCC neurons that have approximately equal size and continue to express Eve. However, at later stages of development, aCC is distinguished from pCC by the expression of Zfh-1 and 22C10 (a membrane associated antigen). aCC is a motoneuron and forms an ipsilateral projection that pioneers the intersegmental nerve. GMC4-2a divides to form the sibling neurons RP2/RP2sib that are morphologically distinguishable. In 88% of the hemisegments, the newborn siblings show a significant difference in the size of their nuclei and cell bodies. This asymmetry appears to be initiated during cell division. In GA1019 mutant embryos, in which GMC4-2a fails to complete cytokinesis, cells are formed that contain one large and one small nucleus. This strongly suggests that the difference in size is generated early, prior to the completion of cytokinesis. The larger cell always adopts the RP2 fate, which is characterized by the expression of Eve, Zfh-1, and 22C10. RP2 forms an antero-ipsilateral projection. The smaller sibling always adopts the RP2sib fate, which is characterized by a further decrease in cell and nuclear size and the loss of Eve immunreactivity. Zfh-1 and 22C10 expression have not been shown in RP2sib. These observations suggest that the cell and nuclear size difference may serve as an early physical marker that will allow one to differentiate between the two progeny of GMC4-2a, irrespective of the molecular markers they express later (Buescher, 1998).

Mutations in mastermind (mam),sanpodo, and Notch equalize aspects of sibling cell fate but retain the difference in cell and nuclear size of sibling neurons. In mam mutant embryos, both progeny of GMC4-2a can adopt the RP2 fate with respect to Eve, Zfh-1, and 22C10 expression. However, despite this apparent change from the RP2sib to the RP2 cell fate, the unequal sizes of the GMC4-2a daughter cells remain; that is, their sizes are unaffected. mam is required for the correct fate specification of RP2sib and pCC but not for that of RP2 and aCC. The requirement for mam suggests that N signaling may be involved in the resolution of distinct sibling neuron cell fate. Mutations in mam and N result in similar defects and support the notion that N signaling is required for the resolution of sibling neuron fate. In inscuteable mutant embryos, GMC1-1a and GMC4-2a are correctly formed and express normal levels of Eve (and in the case of GMC4-2a, also Pdm-1). However, GMC1-1a divides to form two sibling neurons that both adopt the aCC fate (94%) with respect to marker gene expression. Similarly, GMC4-2a division results in two sibling cells, both of which adopt the RP2 fate (96%) with respect to expression of Eve, Zfh-1, and 22C10, as well as axon morphology. This strongly suggests that in wild-type embryos, the divisions of GMC1-1a and GMC4-2a are asymmetric in an insc-dependent manner and produce sibling cells that are intrinsically different; loss of insc function leads to the generation of sibling neurons with equivalent cellular identities. Moreover, in contrast to mam, sanpodo, and Notch mutant embryos, the duplicated RP2s seen in insc mutants are equal with respect to their cell and nuclear size. These observations are consistent with the idea that the size difference seen in wild-type embryos is generated by an insc-dependent process during the GMC cell division and occurs prior to the events mediated by mam, spdo, and N that presumably act at the level of the postmitotic sibling cells. No size asymmetry between the sibling neurons should be generated in an insc background regardless of whether the other functions (e.g., spdo) are present or not (Buescher, 1998).

In Drosophila, much has been learned about the specification of neuronal cell fates but little is known about the lineage of mesodermal cells with different developmental fates. During development, individual mesodermal precursor cells are initially singled out to become the founder cells for specific muscles. The selection of muscle founder cells is thought to employ a Notch-mediated process of lateral inhibition, similar to what is observed for the specification of neural precursors. These muscle founder cells then seem to fuse with the surrounding, uncommitted myocytes, inducing the formation of muscle fiber syncytia. In contrast, the differentiated progeny of neural precursor cells are usually the result of a fixed pattern of asymmetric cell divisions that are directed, in part, by interactions among Numb (a localized intracellular-receptor protein); Sanpodo, and Notch (a transmembrane receptor protein). The roles of these neural lineage genes have been examined in the cell fate specification of muscle and heart precursors. numb and spdo mutations have opposite effects in the specification of muscle founder cells. In all numb mutant embryos examined, the number of Kruppel and S59/NK1 expressing muscle cells is dramatically reduced or absent in stage 12/13 embryos. In spdo mutant embryos the number of S59 and Kr expressing muscle founders is increased (Park, 1998).

A progenitor cell that generates both a pericardial heart cell and a somatic muscle founder cell was the focus of investigation. The two sibling cells studied were a single dorsal muscle, DA1, and a non-muscle pericardial cell (termed EPC), which is associated with the heart. Both cell types express Eve, however, they can be distinguished from one another morphologically. The precursor for both the EPCs and the putative founders of DA1 muscle emerges from a small cluster of mesodermal Eve-expressing cells in each hemisegment at mid-stage 11. At first, these mesodermal Eve cells are indistinguishable from one another, and they co-express Mef2, which is expressed in the entire early mesoderm and later in all (contractile) muscle types. Subsequently Mef2 expression ceases in the future EPCs as they begin to differentiate as non-muscle, pericardial cells. The putative DA1 founder seems to maintain Mef2 expression and begins fusing with surrounding myocytes. In Mef2 mutants, no fusion occurs but the putative muscle founders maintain expression of their muscle precursor markers, such as Eve. The asymmetric segregation of Numb into one of these daughter cells antagonizes the function of Notch and Spdo by preventing the presumptive muscle founder from assuming the same fate as its cardiac sibling. In numb mutants, most DA1 muscles are physically absent and the remaining ones lack Eve (and Kr) expression; in addition, the number of EPCs is doubled. These data suggest that in numb mutants, the putative DA1 founders are transformed into EPCs, because the Eve progenitor cells that normally give rise to DA1 founders and EPCs now only produce EPCs. Overexpression of numb or loss-of-spdo-function result in a failure to generate EPCs but allow for the formation of DA1 muscles. Similarly, expression of constitutively active Notch leads to a failure of DA1 muscle formation and an increase in the number of EPCs. Studies of double mutants indicate that spdo is epistatic to numb (in numb;spdo double mutant embryos, the spdo phenotype is apparent), suggesting that it acts downstream of numb. These results suggest that asymmetric cell divisions, in addition to the previously-documented inductive mechanisms, play a major role in cardiac and somatic muscle patterning. In addition, the cytoskeleton may have a role in the asymmetrical localization of cell fate determinants (Park, 1998).

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. A new population of heart cells is reported here: the Odd skipped-positive pericardial cells (Odd-pericardial cells). Descriptive, lineage tracing and genetic assays were used to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. Within a hemisegment, Odd-pericardial cells develop from three heart progenitors and these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors, each of which produces two cardioblasts. Furthermore, numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above (Ward, 2000).

Having established a wild-type profile of Odd-pericardial cell development it was of interest to identify the genetic regulatory mechanisms that govern Odd-pericardial cell development. Genes that control asymmetric divisions regulate Eve-pericardial cell development. Thus, whether loss of sanpodo or numb function affect Odd-pericardial cell and cardioblast development was examined. Normally 4.2 Odd-pericardial cells and 6.0 cardioblasts develop within each abdominal hemisegment of late-stage embryos. In numb mutant embryos, 6.0 Odd-pericardial cells and 4.2 cardioblasts were detected per hemisegment. Conversely, 7.6 cardioblasts and 2.7 Odd-pericardial cells per hemisegment were detected in sanpodo mutant embryos. Thus, in numb mutant embryos roughly two extra Odd-pericardial cells and two fewer cardioblasts were detected per hemisegment. Conversely, in sanpodo mutant embryos roughly two fewer Odd-pericardial cells and two additional cardioblasts form per hemisegment (Ward, 2000).

These results demonstrate that sanpodo promotes Odd-pericardial cell development and opposes cardioblast development. Conversely, numb opposes Odd-pericardial cell development and promotes cardioblast development. In addition, they suggest that two cardioblasts and two Odd-pericardial cells arise via the asymmetric divisions of numb/sanpodo dependent heart progenitors. These results are consistent with the known requirement for Notch in pericardial cell development. Loss of numb function disrupts the precise alignment of cardioblasts leading to 'broken rows' of cardioblasts in numb mutant embryos (Ward, 2000).

Multiple models can explain the reciprocal effects of sanpodo and numb on cardioblast and Odd-pericardial cell development. For example, one model predicts that two mixed-lineage heart progenitors each divide to yield one cardioblast and one Odd-pericardial cell. A second model predicts the existence of four progenitors: two would divide with each producing one Odd-pericardial cell and one cell of unknown fate; the other two progenitors would divide each producing one cardioblast and one cell of unknown fate. In these and other models, loss of numb or sanpodo function would equalize all asymmetric divisions and could result in the observed Odd-pericardial cell and cardioblast phenotypes (Ward, 2000).

An enhancer trap in the seven-up gene identifies the two mixed-lineage heart progenitors. Towards the end of the lineage analyses it was discovered fortuitously that an enhancer trap in the gene seven-up labels four heart cells in each abdominal hemisegment. This enhancer trap is referred to as svp-lacZ). Two of these cells reside at the dorsal midline and are cardioblasts since they express Mef2. The other two cells reside just lateral and slightly ventral and anterior to the svp-lacZ cardioblasts. These two cells are Odd-pericardial cells because they express Odd. The relative positioning of the svp-lacZ cardioblasts and Odd-pericardial cells closely resembles that of the sibling cardioblasts and Odd-pericardial cells marked by the mixed lineage heart clones. This suggests that the svp-lacZ heart cells may identify the four progeny of the two mixed lineage heart progenitors that arise in each hemisegment. If the four svp-lacZ heart cells are the progeny of these two progenitors, then loss of sanpodo function should convert all svp-lacZ heart cells to cardioblasts and loss of numb function should convert all svp-lacZ heart cells to Odd-pericardial cells. In sanpodo mutant embryos, all four svp-lacZ cells acquire the cardioblast fate and in numb mutant embryos all four svp-lacZ cells acquire the Odd-pericardial cell fate. The results from these experiments demonstrate that svp-lacZ identifies the progeny of the two mixed lineage heart progenitors and that numb and sanpodo mediate the asymmetric divisions of these mixed-lineage heart progenitors (Ward, 2000).

The regulation of apoptosis by Numb/Notch signaling in the serotonin lineage of Drosophila

Apoptosis is prevalent during development of the central nervous system, yet very little is known about the signals that specify an apoptotic cell fate. The role of Numb/Notch signaling in the development of the serotonin lineage of Drosophila has been studied; it is necessary for regulating apoptosis. When Numb inhibits Notch signaling, cells undergo neuronal differentiation, whereas cells that maintain Notch signaling initiate apoptosis. The apoptosis inhibitor p35 can counteract Notch-mediated apoptosis and rescue cells within the serotonin lineage that normally undergo apoptosis. Furthermore, tumor-like overproliferation of cells is observed in the CNS when Notch signaling is reduced. These data suggest that the distribution of Numb during terminal mitotic divisions of the CNS can distinguish between a neuronal cell fate and programmed cell death (Lundell, 2003).

The segmented Drosophila nerve cord develops from stereotyped division of 30 neuroblasts (NB) in each hemisegment. A pair of serotonergic neurons in each hemisegment arises from NB7-3. The divisions of the NB7-3 lineage have recently been determined using a combination of molecular markers and clonal analysis. NB7-3 produces three GMCs. GMC-1 produces two neurons: GW, a motoneuron, and EW1, the more medial serotonergic neuron. GMC-2 produces EW2, the more lateral serotonergic neuron. GMC-3 produces EW3, a neuron that synthesizes the neuropeptide corazonin. The GW neuron projects an axon ipsilateral and posteriorly, and the three EW interneurons all project axons anterior to the posterior commissure (Lundell, 2003 and references therein).

The results of this study demonstrate that the intercellular Notch signaling pathway can be modulated during terminal divisions of the CNS to direct a choice between neuronal development and programmed cell death. The division of GMC-1 produces two distinct neuronal cell fates: the EW1 interneuron and the GW motoneuron. In this division, genetic alteration in the expression of Notch leads to switching between these two cell fates. A loss of Notch activity in spdo mutants leads to two Ddc/Hb-expressing EW1 cells and the overexpression of Notch leads to two Zfh-1 expressing GW cells. Therefore, Notch signaling must be inactivated during development of the EW1 neuron. Numb appears to have a minor role in this inactivation. In a numb1 mutant, 7% of the hemisegments do not develop an EW1 neuron, and a similar number of numb1 hemisegments show two Zfh-1-expressing GW cells. This transformation from an EW1 cell fate to a GW cell fate is what one would expect if Numb were inhibiting Notch. However, most EW1 neurons develop normally in a numb1 mutant and do not convert to the GW cell fate. Therefore, inactivation of Notch signaling in EW1 is mostly independent of Numb function. One possible explanation is that EW1 has a factor that is redundant for Numb function, which can inhibit Notch signaling and is capable of masking the effect of a numb1 mutation in most hemisegments. The unique expression of Hb in GMC-1 progeny could be responsible for establishing this redundancy. However, if a redundant Numb-like factor does exist, it is insufficient to protect EW1 during expression of the UAS-NotchACT transgene (Lundell, 2003).

During the divisions of GMC-2 and GMC-3, genetic alterations in the expression of Notch lead to a switching between a neuronal cell fate and apoptosis. A reduction of Notch signaling with either spdoG104 or UAS-Numb embryos produces ectopic NB7-3 cells that express Zfh-2. Conversely, the overexpression of Notch in either UAS-NotchACT or numb1 embryos led to an increase in TUNEL labeling of GMC-2 and GMC-3 progeny. Additionally, inhibiting apoptosis with UAS-p35 or reducing Notch activity with spdoG104 can rescue the numb1 phenotype. It is hypothesized that during the divisions of GMC-2 and GMC-3, Numb partitions asymmetrically into EW2 and EW3 where it inactivates Notch signaling and leads to neuronal development. The mitotic sisters of EW2 and EW3 do not receive Numb, maintain Notch signaling and undergo apoptosis. The difficulty in detecting wild-type hemisegments that have more than four immunoreactive Eg cells, suggests that any other cells produced during divisions of the NB7-3 lineage quickly undergo apoptosis (Lundell, 2003).

Ectopic Eg cells in the NB7-3 lineage can be induced at stage 15 by H99, UAS-Numb, spdoG104 and UAS-p35. However, the ability of these alleles to produce ectopic Ddc and corazonin-containing neurons at later stages is variable. No significant ectopic Ddc or corazonin-containing cells were detected in either H99 or UAS-Numb CNS. For UAS-Numb it was shown that the ectopic Eg cells detected at stage 15 can undergo apoptosis. spdoG104 mutants produce only ectopic Ddc cells, but the reduction in the number of corazonin-containing cells in general suggests that either GMC-3 does not consistently form in these mutants or that GMC-3 progeny may convert from a corazonin-containing cell fate to a serotonergic cell fate. UAS-p35 mutants produce both ectopic Ddc and corazonin-containing cells at low frequency, but the allele is much more efficient at rescuing the EW neurons in numb1 and UAS-Notch mutants. Therefore, apoptosis is harder to reverse in cells that normally undergo apoptosis, than in the cells genetically induced to undergo apoptosis. The ability of these various alleles to produce ectopic Ddc- and corazonin-containing cells could be influenced by mutant effects they cause outside the NB7-3 lineage or may reflect different roles they have in the apoptotic pathway. The mechanism by which Notch induces apoptosis in the NB7-3 lineage remains to be determined, but the apoptotic genes reaper, grim and hid may be involved because all three of these genes are deleted in the H99 allele (Lundell, 2003).

The tumor-like expansion of Ddc-expressing cells observed in heterozygous spdoG104 larvae suggests that Notch-induced apoptosis may be essential for regulating cell proliferation. This spdo phenotype is reminiscent of three tumor-suppressor genes; discs large (dlg), lethal giant larvae (lgl) and scribble (scrib), which produce tumors in the CNS and imaginal disks. Interestingly, these three genes work in a common pathway that regulates cell polarity, and lgl and dlg have been shown to be essential for the distribution of Numb and other asymmetric determinants. Further investigation will be necessary to determine if spdo is part of this same mechanism and exactly how spdo mutants inhibit Notch signaling. Spdo expression is ubiquitous throughout embryogenesis and persists through the larval stages and into adults. If a spdo mutation can alter the response of the Notch receptor to environmental cues that induce apoptosis, one would expect to see overproliferation in additional tissues (Lundell, 2003).

Numb/Notch signaling is also known to affect development of the midline dopaminergic cells. The expression of Ddc is essential to the biosynthesis of both serotonin and dopamine. Anti-Ddc antibody detects not only the serotonergic neurons, but also midline dopamine neurons. As a consequence of using Ddc as a marker for the serotonin lineage, a number of observations were made about the development of midline dopamine cells. In a numb1 mutant very few midline dopamine cells are detectable with Ddc. spdoG104 mutants produce ectopic dopamine cells and can rescue dopamine cells in the numb1 mutant phenotype. Thus, Numb/Notch signaling also has a role in the development of midline dopamine cells, but further investigation into the significance and whether apoptosis is involved in this lineage will require lineage analysis to determine the origin of the midline dopamine cells (Lundell, 2003).

Sanpodo: a context-dependent activator and inhibitor of Notch signaling during asymmetric divisions

Asymmetric cell divisions generate sibling cells of distinct fates ('A', 'B') and constitute a fundamental mechanism that creates cell-type diversity in multicellular organisms. Antagonistic interactions between the Notch pathway and the intrinsic cell-fate determinant Numb appear to regulate asymmetric divisions in flies and vertebrates. During these divisions, productive Notch signaling requires sanpodo, which encodes a novel transmembrane protein. This study demonstrates that Drosophila sanpodo plays a dual role to regulate Notch signaling during asymmetric divisions - amplifying Notch signaling in the absence of Numb in the 'A' daughter cell and inhibiting Notch signaling in the presence of Numb in the 'B' daughter cell. In so doing, sanpodo ensures the asymmetry in Notch signaling levels necessary for the acquisition of distinct fates by the two daughter cells. These findings answer long-standing questions about the restricted ability of Numb and Sanpodo to inhibit and to promote, respectively, Notch signaling during asymmetric divisions (Babaoglan, 2009).

Work from many labs indicates that the state of Notch signaling determines daughter cell fate during asymmetric divisions - high-level Notch signaling induces the 'A' fate; low-level Notch signaling permits the 'B' fate. In this context, the current work demonstrates that spdo acts in both daughter cells to accentuate the difference between Notch signaling levels in the two cells - amplifying Notch signaling in the absence of Numb in the 'A' cell, and enabling Numb to inhibit Notch signaling in the 'B' cell. By exerting opposite effects on Notch signaling in a Numb-dependent manner, Spdo simultaneously ensures that Notch signaling exceeds threshold levels in the 'A' cell, yet remains well below such levels in the 'B' cell, thus enabling the faithful execution of asymmetric divisions (Babaoglan, 2009).

Why Numb can inhibit Notch signaling during asymmetric divisions but no other Notch-dependent event has long remained unclear. Genetic data demonstrate that numb acts through spdo to inhibit Notch signaling. As spdo is expressed exclusively in asymmetrically dividing cells, and Numb segregates exclusively into the 'B' daughter cell during asymmetric divisions, these results account for the specific ability of Numb to inhibit Notch signaling in 'B' daughter cells — the only cell type in Drosophila that co-expresses spdo and numb. spdo does not appear to enable Numb to inhibit Notch signaling by regulating the localization of Numb, as Numb localization is grossly normal in spdo mutant embryos (Babaoglan, 2009).

Why does productive Notch signaling require spdo function in 'A' daughter cells during asymmetric divisions, but not during any other Notch-dependent event in Drosophila? It was found that in the absence of Numb, Spdo amplifies but is not obligately required for transduction of Notch signaling. Thus, while Notch signaling can occur in 'A' daughter cells in the absence of spdo, spdo function is normally required to enable signaling levels to exceed the threshold required to induce the 'A' fate (Babaoglan, 2009).

The results indicate that limiting levels or activity of the Notch receptor probably underlies the sub-threshold nature of Notch signaling in 'A' daughter cells in the absence of spdo. Notch levels or activity may be limiting in 'A' daughter cells owing to the downregulation of proteins that localize to adherens junctions in asymmetrically dividing cells. Notch has been shown to localize preferentially to adherens junctions in epithelial cells, and asymmetrically dividing cells display reduced levels of Notch as well as other proteins that normally localize to adherens junctions. Some of these other proteins, such as Echinoid, are known to facilitate Notch signaling during lateral inhibition and other Notch-dependent events. Thus, reduced levels of Notch and facilitators of Notch signaling in asymmetrically dividing cells may account for the specific requirement for Spdo to amplify Notch signaling levels during asymmetric divisions (Babaoglan, 2009).

Consistent with a role for spdo in simply amplifying Notch signaling levels in the absence of Numb, the Notch-dependent 'A' fate develops at low frequency in some lineages in the absence of spdo. Thus, in the absence of spdo, Notch signaling levels appear close to, but usually below, the threshold required to induce the 'A' fate. Surprisingly, rare instances where Numb-dependent 'B' daughter cells adopt the 'A' fate were also observed in spdo mutant embryos, specifically in the development of Svp+ heart cells at 18°C. Such events have not been observed in wild type, and indicate that Numb requires Spdo in the 'B' cell to maintain Notch signaling levels reliably below the threshold required for the 'A' fate. Thus, the dual and opposing roles of spdo in the regulation of Notch signaling levels during asymmetric divisions are crucial for the unerring ability of the two daughter cells to adopt distinct fates (Babaoglan, 2009).

What is the molecular mechanism through which spdo regulates Notch signaling during asymmetric divisions? The results indicate that any mechanistic model for spdo function must account for the ability of spdo to boost Notch signaling in the absence of Numb and to reduce Notch signaling in the presence of Numb. Present models of spdo function, such as a postulated role for Spdo in promoting recycling of Delta in the 'B', do not fully address the duality of spdo function in the two daughter cells. Rather the genetic data, together with prior work on Spdo physical interactions and Numb-dependent localization, lead to the idea that in the absence of Numb, Spdo localizes to the cell membrane of the 'A' cell, where it increases Notch association with effectors, and in so doing boosts Notch signaling levels (Babaoglan, 2009).

How could Numb convert Spdo from an activator to an inhibitor of Notch signaling? Numb binds directly to Spdo and regulates its subcellular localization, preventing Spdo from localizing to the cell membrane. If either Notch or an effector is internalized with Spdo by Numb, a quantitative decrease in Notch signaling would result. However, the levels of Notch at the cell membrane appear roughly equivalent between the two daughter cells, suggesting that if numb functions in this manner it may do so by targeting a Notch effector rather than Notch itself along with Spdo. Alternatively, small changes in Notch receptor levels may be sufficient to decrease signaling levels below the threshold required to induce the 'A' fate. The elucidation of the precise mechanism through which Spdo exerts opposite effects on Notch pathway activity in the two daughter cells probably awaits the systematic identification of the factors that physically interact with Spdo during asymmetric divisions (Babaoglan, 2009).

sanpodo: Biological Overview | Regulation | Developmental Biology | References

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