A dual function of phyllopod in Drosophila external sensory organ development: cell fate specification of sensory organ precursor and its progeny

During Drosophila external sensory organ development, one sensory organ precursor (SOP) arises from a proneural cluster, and undergoes asymmetrical cell divisions to produce an external sensory (es) organ made up of different types of daughter cells. We show that phyllopod (phyl), previously identified to be essential for R7 photoreceptor differentiation, is required in two stages of es organ development: the formation of SOP cells and cell fate specification of SOP progeny. Loss-of-function mutations in phyl result in failure of SOP formation, which leads to missing bristles in adult flies. At a later stage of es organ development, phyl mutations cause the first cell division of the SOP lineage to generate two identical daughters, leading to the fate transformation of neurons and sheath cells to hair cells and socket cells. Conversely, misexpression of phyl promotes ectopic SOP formation, and causes opposite fate transformation in SOP daughter cells. Thus, phyl functions as a genetic switch in specifying the fate of the SOP cells and their progeny. We further show that seven in absentia (sina), another gene required for R7 cell fate differentiation, is also involved in es organ development. Genetic interactions among phyl, sina and tramtrack (ttk) suggest that phyl and sina function in bristle development by antagonizing ttk activity, and ttk acts downstream of phyl. It has been shown previously that Notch (N) mutations induce formation of supernumerary SOP cells, and transformation from hair and socket cells to neurons. We further demonstrate that phyl acts epistatically to N. phyl is expressed specifically in SOP cells and other neural precursors, and its mRNA level is negatively regulated by N signaling. Thus, these analyses demonstrate that phyl acts downstream of N signaling in controlling cell fates in es organ development.     

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. Here we show 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, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling.     

Lateral inhibition and the development of the sensory bristles of the adult peripheral nervous system of Drosophila

Cells in the neurectoderm of Drosophila face a choice between neural and epidermal fates. On the notum of the adult fly, neural cells differentiate sensory bristles in a precise pattern. Evidence has accumulated that the bristle pattern arises from the spatial distribution of small groups of cells, proneural clusters, from each of which a single bristle will result. One class of genes, which includes the genes of the achaete-scute complex, is responsible for the correct positioning of the proneural clusters. The cells of a proneural cluster constitute an equivalence group, each of them having the potential to become a neural cell. Only one cell, however, will adopt the primary, dominant, neural fate. This cell is selected by means of cellular interactions between the members of the group, since if the dominant cell is removed, one of the remaining, epidermal, cells will switch fates and become neural. The dominant cell therefore prevents the other cells of the group from becoming neural by a phenomenon known as lateral inhibiton. They, then, adopt the secondary, epidermal, fate. A second class of genes, including the gene shaggy and the neurogenic genes mediate this process. There is some evidence that a proneural cluster is composed of a small number of cells, suggesting a contact-based mechanism of communication. The molecular nature of the protein products of the neurogenic genes is consistent with this idea.     

Lethal giant larvae acts together with numb in notch inhibition and cell fate specification in the Drosophila adult sensory organ precursor lineage

The tumor suppressor genes lethal giant larvae (lgl) and discs large (dlg) act together to maintain the apical basal polarity of epithelial cells in the Drosophila embryo. Neuroblasts that delaminate from the embryonic epithelium require lgl to promote formation of a basal Numb and Prospero crescent, which will be asymmetrically segregated to the basal daughter cell upon division to specify cell fate. Sensory organ precursors (SOPs) also segregate Numb asymmetrically at cell division. Numb functions to inhibit Notch signaling and to specify the fates of progenies of the SOP that constitute the cellular components of the adult sensory organ. We report here that, in contrast to the embryonic neuroblast, lgl is not required for asymmetric localization of Numb in the dividing SOP. Nevertheless, mosaic analysis reveals that lgl is required for cell fate specification within the SOP lineage; SOPs lacking Lgl fail to specify internal neurons and glia. Epistasis studies suggest that Lgl acts to inhibit Notch signaling by functioning downstream or in parallel with Numb. These findings uncover a previously unknown function of Lgl in the inhibition of Notch and reveal different modes of action by which Lgl can influence cell fate in the neuroblast and SOP lineages.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

The conserved c2 domain protein lethal (2) giant discs regulates protein trafficking in Drosophila

Drosophila sensory organ precursor (SOP) cells undergo several rounds of asymmetric cell division to generate the four different cell types that make up external sensory organs. Establishment of different fates among daughter cells of the SOP relies on differential regulation of the Notch pathway. Here, we identify the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. We show that lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes. When Lgd function is compromised, Notch and other transmembrane proteins accumulate in enlarged early endosomal compartments. These enlarged endosomes are positive for Rab5 and Hrs, a protein involved in trafficking into the degradative pathway. Our experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs.     

Notch signaling represses the glial fate in fly PNS

By using gain-of-function mutations it has been proposed that vertebrate Notch promotes the glial fate. We show in vivo that glial cells are produced at the expense of neurons in the peripheral nervous system of flies lacking Notch and that constitutively activated Notch produces the opposite phenotype. Notch acts as a genetic switch between neuronal and glial fates by negatively regulating glial cell deficient/glial cells missing, the gene required in the glial precursor to induce gliogenesis. Moreover, Notch represses neurogenesis or gliogenesis, depending on the sensory organ type. Numb, which is asymmetrically localized in the multipotent cell that produces the glial precursor, induces glial cells at the expense of neurons. Thus, a cell-autonomous mechanism inhibits Notch signaling.     

The Drosophila IgC2 domain protein Friend-of-Echinoid,a paralogue of Echinoid,limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway

The Notch signaling pathway is critical in cell fate specification throughout development. In the developing wing disc, single sensory organ precursors (SOPs) are selected from proneural clusters via a process of lateral inhibition mediated by the Notch signaling pathway. The epidermal growth factor receptor (EGFR) pathway has also been implicated in SOP formation. Here, we describe the Drosophila melanogaster gene friend of echinoid (fred), a paralogue of echinoid (ed), a gene recently identified as a negative regulator of the EGFR pathway. fred function was examined in transgenic flies by using inducible RNA interference (RNAi). Suppression of fred in developing wing discs results in specification of ectopic SOPs, additional microchaeta, and cell death. In eye-antennal discs, fred suppression causes a rough eye phenotype. These phenotypes are suppressed by overexpression of Notch, Suppressor of Hairless [Su(H)], and Enhancer of split m7. In contrast, overexpression of Hairless, a negative regulator of the Notch pathway, and decreased Su(H) activity enhance these phenotypes. Thus, fred acts in close concert with the Notch signaling pathway. Dosage-sensitive genetic interaction also suggests a close relationship between fred and ed.     

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate.

BACKGROUND: The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS: Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS: Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.     

The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis

We have studied the role of Notch-1 and its antagonist Numb in the activation of satellite cells during postnatal myogenesis. Activation of Notch-1 promoted the proliferation of myogenic precursor cells expressing the premyoblast marker Pax3. Attenuation of Notch signaling by increases in Numb expression led to the commitment of progenitor cells to the myoblast cell fate and the expression of myogenic regulatory factors, desmin, and Pax7. In many intermediate progenitor cells, Numb was localized asymmetrically in actively dividing cells, suggesting an asymmetric cell division and divergent cell fates of daughter cells. The results indicate that satellite cell activation results in a heterogeneous population of precursor cells with respect to Notch-1 activity and that the balance between Notch-1 and Numb controls cellular homeostasis and cell fate determination.     

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. Here, we describe the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. We propose a model in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP.     

Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.