Single Sensory Organ Precursor Research Articles

Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection

The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. We show here that a critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands, Delta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, we probe the role of CI in vivo. We develop a mathematical model for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. We show theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. Our results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.

Corrigendum to: Single-cell visualization of mir-9a and Senseless co-expression during Drosophila melanogaster embryonic and larval peripheral nervous system development.

The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disc, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a.

Single-cell visualization of mir-9a and Senseless co-expression during Drosophila melanogaster embryonic and larval peripheral nervous system development

The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disc, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a.

Transcriptional profiling of olfactory system development identifies distal antenna as a regulator of subset of neuronal fates

Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1–4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. Here we use transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. We surveyed four critical developmental stages of the olfactory system: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. We focused on the expression profiles of olfactory receptor genes and transcription factors—the two main classes of genes that regulate the sensory identity of ORNs. We identify distinct clusters of genes that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. We show that the expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Insensible is a novel nuclear inhibitor of Notch activity in Drosophila.

Notch signalling regulates a wide range of developmental processes. In the Drosophila peripheral nervous system, Notch regulates a series of binary fate decisions that lead to the formation of regularly spaced sensory organs. Each sensory organ is generated by single sensory organ precursor cell (SOP) via a series of asymmetric cell divisions. Starting from a SOP-specific Cis-Regulatory Module (CRM), we identified insensible (insb), a.k.a CG6520, as a SOP/neuron-specific gene encoding a nuclear factor that inhibits Notch signalling activity. First, over-expression of Insb led to the transcriptional repression of a Notch reporter and to phenotypes associated with the inhibition of Notch. Second, while the complete loss of insb activity had no significant phenotype, it enhanced the bristle phenotype associated with reduced levels of Hairless, a nuclear protein acting as a co-repressor for Suppressor of Hairless. In conclusion, our work identified Insb as a novel SOP/neuron-specific nuclear inhibitor of Notch activity in Drosophila.

Drosophila mechanoreceptors as a model for studying asymmetric cell division

Asymmetric cell division (ACD) is one of the processes creating the overall diversity of cell types in multicellular organisms. The essence of this process is that the daughter cells exit from it being different from both the parental cell and one another in their ability to further differentiation and specialization. The large bristles (macrochaetae) that are regularly arranged on the surface of the Drosophila adult function as mechanoreceptors, and since their development requires ACD, they have been extensively used as a model system for studying the genetic control of this process. Each macrochaete is composed of four specialized cells, the progeny resulting from several ACDs from a single sensory organ precursor (SOP) cell, which differentiates from the ectodermal cells of the wing imaginal disc in the third-instar larva and pupa. In this paper we review the experimental data on the genes and their products controlling the ACDs of the SOP cell and its daughter cells, and their further specialization. We discuss the main mechanisms determining the time when the cell enters ACD, as well as the mechanisms providing for the structural characteristics of asymmetric division, namely, polar distribution of protein determinants (Numb and Neuralized), orientation of the division spindle relative to these determinants, and unequal segregation of the determinants specifying the direction of daughter cell 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.

Both inhibition and activation of Notch signaling rely on a conserved Neuralized-binding motif in Bearded proteins and the Notch ligand Delta

Lateral inhibition is one of the key functions of Notch signaling during animal development. In the proneural clusters that give rise to Drosophila mechanosensory bristles, Delta (Dl) ligand in the sensory organ precursor (SOP) cell is targeted for ubiquitination by the E3 ligase Neuralized (Neur), resulting in activation of Dl's capacity to signal to the Notch receptor on neighboring cells. The cells that receive this signal activate a genetic program that suppresses their SOP fate potential, insuring that only a single SOP develops within each cluster. Using multiple lines of investigation, we provide evidence that members of the Bearded family of proteins (BFMs) inhibit Dl activation in non-SOP cells by binding to Neur and preventing it from interacting with Dl. We show that this activity of BFMs is dependent on the conserved NXXN motif, and report the unexpected finding that several BFMs include multiple functional copies of this motif. We find that a conserved NXXN motif in the intracellular domain of Dl is responsible for its interaction with Neur, indicating direct competition between Dl and BFMs for binding to Neur, and we show that Neur-dependent endocytosis of Dl requires the integrity of its NXXN motif. Our results illuminate the mechanism of an important regulatory event in Notch signaling that appears to be conserved between insects and crustaceans.

The Arp2/3 complex and WASp are required for apical trafficking of Delta into microvilli during cell fate specification of sensory organ precursors

Cell fate decisions mediated by the Notch signalling pathway require direct cell-cell contact between adjacent cells. In Drosophila melanogaster, an external sensory organ (ESO) develops from a single sensory organ precursor (SOP) and its fate specification is governed by differential Notch activation. Here we show that mutations in actin-related protein-3 (Arp3) compromise Notch signalling, leading to a fate transformation of the ESO. Our data reveal that during ESO fate specification, most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Using immunohistochemistry and transmission electron microscopy (TEM) analyses, we show that the ARS contains numerous microvilli on the apical surface of SOP progeny. In Arp2/3 and WASp mutants, the surface area of the ARS is substantially reduced and there are significantly fewer microvilli. More importantly, trafficking of Delta-positive vesicles from the basal area to the apical portion of the ARS is severely compromised. Our data indicate that WASp-dependent Arp2/3 actin polymerization is crucial for apical presentation of Delta, providing a mechanistic link between actin polymerization and Notch signalling.

How Drosophila melanogaster Forms its Mechanoreceptors.

A strictly determined number of external sensory organs, macrochaetes, acting as mechanoreceptors, are orderly located on drosophila head and body. Totally, they form the bristle pattern, which is a species-specific characteristic of drosophila.Each mechanoreceptor comprises four specialized cells derived from the single sensory organ precursor (SOP) cell. The conserved bristle pattern combined with a comparatively simple structure of each mechanosensory organ makes macrochaetes a convenient model for studying the formation spatial structures with a fixed number of elements at certain positions and the mechanism underlying cell differentiation.The macrochaete morphogenesis consists of three stages. At the first stage, the proneural clusters segregate from the massive of ectodermal cells of the wing imaginal disc. At the second stage, the SOP cell is determined and its position in the cluster is specified. At the third stage, the SOP cell undergoes two asymmetric divisions, and the daughter cells differentiate into the components of mechanoreceptor: shaft, socket, bipolar neuron, and sheath.The critical factor determining the neural pathway of cell development is the content of proneural proteins, products of the achaete-scute (AS-C) gene complex, reaching its maximum in the SOP cell.The experimental data on the main genes and their products involved in the control of bristle pattern formation are systematized. The roles of achaete-scute complex, EGFR and Notch signaling pathways, and selector genes in these processes are considered. An integral scheme describing the functioning of the system controlling macrochaete development in D. melanogaster is proposed based on analysis of literature data.

A mis-expression study of factors affecting Drosophila PNS cell identity

Drosophila PNS sense organs arise from single sensory organ precursor (SOP) cells through a series of asymmetric divisions. In a mis-expression screen for factors affecting PNS development, we identified string and dappled as being important for the proper formation of adult external sensory (ES) organs. string is a G2 regulator. dappled has no described function but is implicated in tumorigenesis. The mis-expression effect from string was analysed using timed over expression during adult ES-organ and, for comparison, embryonic Chordotonal (Ch) organ formation. Surprisingly, string mis-expression prior to SOP division gave the greatest effect in both systems. In adult ES-organs, this lead to cell fate transformations producing structural cells, whilst in the embryo organs were lost, hence differences within the lineages exist. Mis-expression of dappled, lead to loss and duplications of entire organs in both systems, potentially affecting SOP specification, in addition to affecting neuronal guidance.

Asymmetric Rab11 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.

Tramtrack co-operates to prevent inappropriate neural development in Drosophila

Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete–Scute complex (AS–C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. We demonstrate that the BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, we dissected functional domains of Ttk, confirming the importance of the bric-à-brac–tramtrack–broad complex (BTB) motif. We show that the Ttk BTB domain is a protein–protein interaction motif mediating tetramer formation.

Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling.

In Drosophila imaginal discs, the spatially restricted activities of the achaete (ac) and scute (sc) proteins, which are transcriptional activators of the basic-helix-loop-helix class, define proneural clusters (PNCs) of potential sensory organ precursor (SOP) cells. Here, we report the identification of several genes that are direct downstream targets of ac-sc activation, as judged by the following criteria. The genes are expressed in the PNCs of the wing imaginal disc in an ac-sc-dependent manner; the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY; where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including Bearded, Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate.

The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development.

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. We have found that the H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. Our results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H.