Socket Cells Research Articles

Socket Array Irregularities and Wing Membrane Distortions at the Eyespot Foci of Butterfly Wings Suggest Mechanical Signals for Color Pattern Determination.

Eyespot foci on butterfly wings function as organizers of eyespot color patterns during development. Despite their importance, focal structures have not been examined in detail. Here, we microscopically examined scales, sockets, and the wing membrane in the butterfly eyespot foci of both expanded and unexpanded wings using the Blue Pansy butterfly Junonia orithya. Images from a high-resolution light microscope revealed that, although not always, eyespot foci had scales with disordered planar polarity. Scanning electron microscopy (SEM) images after scale removal revealed that the sockets were irregularly positioned and that the wing membrane was physically distorted as if the focal site were mechanically squeezed from the surroundings. Focal areas without eyespots also had socket array irregularities, but less frequently and less severely. Physical damage in the background area induced ectopic patterns with socket array irregularities and wing membrane distortions, similar to natural eyespot foci. These results suggest that either the process of determining an eyespot focus or the function of an eyespot organizer may be associated with wing-wide mechanics that physically disrupt socket cells, scale cells, and the wing membrane, supporting the physical distortion hypothesis of the induction model for color pattern determination in butterfly wings.

Glial regulators of ions and solutes required for specific chemosensory functions in Caenorhabditis elegans.

Glia and accessory cells regulate the microenvironment around neurons and primary sensory cells. However, the impact of specific glial regulators of ions and solutes on functionally diverse primary cells is poorly understood. Here, we systemically investigate the requirement of ion channels and transporters enriched in Caenorhabditis elegans Amsh glia for the function of chemosensory neurons. Although Amsh glia ablated worms show reduced function of ASH, AWC, AWA, and ASE neurons, we show that the loss of glial enriched ion channels and transporters impacts these neurons differently, with nociceptor ASH being the most affected. Furthermore, our analysis underscores the importance of K+, Cl-, and nucleoside homeostasis in the Amphid sensory organ and uncovers the contribution of glial genes implicated in neurological disorders. Our findings build a unique fingerprint of each glial enriched ion channel and transporter and may provide insights into the function of supporting cells of mammalian sensory organs.

Structural Analysis of the Caenorhabditis elegans Dauer Larval Anterior Sensilla by Focused Ion Beam-Scanning Electron Microscopy.

At the end of the first larval stage, the nematode Caenorhabditis elegans developing in harsh environmental conditions is able to choose an alternative developmental path called the dauer diapause. Dauer larvae exhibit different physiology and behaviors from non-dauer larvae. Using focused ion beam-scanning electron microscopy (FIB-SEM), we volumetrically reconstructed the anterior sensory apparatus of C. elegans dauer larvae with unprecedented precision. We provide a detailed description of some neurons, focusing on structural details that were unknown or unresolved by previously published studies. They include the following: (1) dauer-specific branches of the IL2 sensory neurons project into the periphery of anterior sensilla and motor or putative sensory neurons at the sub-lateral cords; (2) ciliated endings of URX sensory neurons are supported by both ILso and AMso socket cells near the amphid openings; (3) variability in amphid sensory dendrites among dauers; and (4) somatic RIP interneurons maintain their projection into the pharyngeal nervous system. Our results support the notion that dauer larvae structurally expand their sensory system to facilitate searching for more favorable environments.

The color pattern inducing gene wingless is expressed in specific cell types of campaniform sensilla of a polka-dotted fruit fly, Drosophila guttifera.

A polka-dotted fruit fly, Drosophila guttifera, has a unique pigmentation pattern on its wings and is used as a model for evo-devo studies exploring the mechanism of evolutionary gain of novel traits. In this species, a morphogen-encoding gene, wingless, is expressed in species-specific positions and induces a unique pigmentation pattern. To produce some of the pigmentation spots on wing veins, wingless is thought to be expressed in developing campaniform sensillum cells, but it was unknown which of the four cell types there express(es) wingless. Here we show that two of the cell types, dome cells and socket cells, express wingless, as indicated by in situ hybridization together with immunohistochemistry. This is a unique case in which non-neuronal SOP (sensory organ precursor) progeny cells produce Wingless as an inducer of pigmentation pattern formation. Our finding opens a path to clarifying the mechanism of evolutionary gain of a unique wingless expression pattern by analyzing gene regulation in dome cells and socket cells.

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.

Prickle isoforms determine handedness of helical morphogenesis.

Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. Here, we describe a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. We show that the Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, we show that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. We provide evidence that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pkpk or Pksple predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes.

Complex genetic interactions of novel Suppressor of Hairless alleles deficient in co-repressor binding.

Throughout the animal kingdom, the Notch signalling pathway allows cells to acquire diversified cell fates. Notch signals are translated into activation of Notch target genes by CSL transcription factors. In the absence of Notch signals, CSL together with co-repressors functions as a transcriptional repressor. In Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless (Su(H)) and the Notch (N) antagonist Hairless (H) that together form a repressor complex. Guided by crystal structure, three mutations Su(H)LL, Su(H)LLF and Su(H)LLL were generated that specifically affect interactions with the repressor H, and were introduced into the endogenous Su(H) locus by gene engineering. In contrast to the wild type isoform, these Su(H) mutants are incapable of repressor complex formation. Accordingly, Notch signalling activity is dramatically elevated in the homozygotes, resembling complete absence of H activity. It was noted, however, that heterozygotes do not display a dominant H loss of function phenotype. In this work we addressed genetic interactions the three H-binding deficient Su(H) mutants display in combination with H and N null alleles. We included a null mutant of Delta (Dl), encoding the ligand of the Notch receptor, as well as of Su(H) itself in our genetic analyses. H, N or Dl mutations cause dominant wing phenotypes that are sensitive to gene dose of the others. Moreover, H heterozygotes lack bristle organs and develop bristle sockets instead of shafts. The latter phenotype is suppressed by Su(H) null alleles but not by H-binding deficient Su(H) alleles which we attribute to the socket cell specific activity of Su(H). Modification of the dominant wing phenotypes of either H, N or Dl, however, suggested some lack of repressor activity in the Su(H) null allele and likewise in the H-binding deficient Su(H) alleles. Overall, Su(H) mutants are recessive perhaps reflecting self-adjusting availability of Su(H) protein.

A model study of the morphogenesis of D. melanogaster mechanoreceptors: the central regulatory circuit.

Macrochaetes (large bristles) are sensor organs of the Drosophila peripheral nervous system with a function of mechanoreceptors. An adult mechanoreceptor comprises four specialized cells: shaft (trichogen), socket (tormogen), neuron, and glial cell (thecogen). All these cells originate from a single cell, the so-called sensor organ precursor (SOP) cell. Separation of the SOP cell from the encompassing cells of the imaginal disc initiates a multistage process of sensory organ development. A characteristic feature of the SOP cell is the highest amount of the proneural proteins AS-C as compared with the encompassing ectodermal cells. The accumulation of proneural proteins and maintenance of their amount in the SOP cell at a necessary level is provided by the gene network with the achaete-scute gene complex (AS-C) as its key component. The activity of this complex is controlled by the central regulatory circuit (CRC). The CRC comprises the genes hairy, senseless (sens), charlatan (chn), scratch (scrt), daughterless (da), extramacrochaete (emc), and groucho (gro), coding for the transcription factors involved in the system of direct links and feedbacks and implementation of activation-repression relationships between the CRC components. The gene phyllopod (phyl), involved in degradation of the AS-C proteins, is also associated with the CRC functioning. In this paper, we propose a mathematical model for the CRC functioning as a regulator of the amount of proneural AS-C proteins in the SOP cell taking into account their degradation. The modeling has demonstrated that a change in the amount of proneural proteins in the SOP cell is stepwise rather than strictly monotonic. This prediction can be tested experimentally.

Caenorhabditis elegans male sensory-motor neurons and dopaminergic support cells couple ejaculation and post-ejaculatory behaviors

The circuit structure and function underlying post-coital male behaviors remain poorly understood. Using mutant analysis, laser ablation, optogenetics, and Ca2+ imaging, we observed that following C. elegans male copulation, the duration of post-coital lethargy is coupled to cellular events involved in ejaculation. We show that the SPV and SPD spicule-associated sensory neurons and the spicule socket neuronal support cells function with intromission circuit components, including the cholinergic SPC and PCB and the glutamatergic PCA sensory-motor neurons, to coordinate sex muscle contractions with initiation and continuation of sperm movement. Our observations suggest that the SPV and SPD and their associated dopamine-containing socket cells sense the intrauterine environment through cellular endings exposed at the spicule tips and regulate both sperm release into the hermaphrodite and the recovery from post-coital lethargy.

An Enhancer Composed of Interlocking Submodules Controls Transcriptional Autoregulation of Suppressor of Hairless

Positive autoregulation is an effective mechanism for the long-term maintenance of a transcription factor's expression. This strategy is widely deployed in cell lineages, where the autoregulatory factor controls the activity of a battery of genes that constitute the differentiation program of a postmitotic cell type. In Drosophila, the Notch pathway transcription factor Suppressor of Hairless activates its own expression, specifically in the socket cell of external sensory organs, via an autoregulatory enhancer called the ASE. Here, we show that the ASE is composed of several enhancer submodules, each of which can independently initiate weak Su(H) autoregulation. Cross-activation by these submodules is critical to ensure that Su(H) rises above a threshold level necessary to activate a maintenance submodule, which then sustains long-term Su(H) autoregulation. Our study reveals the use of interlinked positive-feedback loops to control autoregulation dynamically and provides mechanistic insight into initiation, establishment, and maintenance of the autoregulatory state.

Planar Polarized Protrusions Break the Symmetry of EGFR Signaling during Drosophila Bract Cell Fate Induction

Secreted signaling molecules typically float in the outer leaflet of the plasma membrane or freely diffuse away from the signaling cell, suggesting that a signal should be sensed equally by all neighboring cells. However, we demonstrate that Spitz (Spi)-mediated epidermal growth factor receptor (EGFR) signaling is spatially biased to selectively determine the induction of a single bract cell on the proximal side of eachmechanosensory organ on the Drosophila leg. Dynamic and oriented cellular protrusions emanating from the socket cell, the source of Spi, robustly favor the Spi/EGFR signaling response in a particular cell among equally competent neighbors. We propose that these protrusive structures enhance signaling by increasing contact between the signaling and responding cells. The planar polarized direction of the protrusions determines the direction of the signaling outcome. This asymmetric cell signaling serves as a developmental mechanism to generate spatially patterned cell fates.

Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules

In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities.

Dynamics of Endoreplication during Drosophila Posterior Scutellar Macrochaete Development

Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level.

DVL genes play a role in the coordination of socket cell recruitment and differentiation

Specialized plant cells arise from undifferentiated cells through a series of developmental steps. The decision to enter into a certain differentiation pathway depends in many cases on signals from neighbouring cells. The ability of cells to engage in short-range intercellular communication permits the coordination of cell actions necessary in many developmental processes. Overexpression of genes from the DEVIL/ROTUNDIFOLIA (DVL/ROT) family results in severe developmental alterations, but very little is known about their mechanism of action. This work presents evidence that suggests a role for these genes in local signalling, specifically in the coordination of socket cell recruitment and differentiation. Overexpression of different DVL genes results in protuberances at the base of the trichomes surrounded by several rows of elongated epidermal cells, morphologically similar to socket cells. Localized overexpression of DVL4 in trichomes and socket cells during early developmental stages activates expression of socket cell markers in additional cells, farther away from the trichome. The same phenomenon is observed in an activation tagged line of DVL1, which also shows an increase in the number of socket cells in contact with the trichome. The roles of individual DVL genes have been difficult to discover since their overexpression phenotypes are quite similar. In gl1 leaves that lack trichomes and socket cells DVL1 expression shows a 69% reduction, suggesting that this gene could be involved in the coordination of socket cell development in wild-type plants.

Dye-filling of the amphid sheath glia: Implications for the functional relationship between sensory neurons and glia in Caenorhabditis elegans

The nervous system is composed of cells including neurons and glia. It has been believed that the former cells play central roles in various neural functions while the latter ones have only supportive functions for neurons. However, recent findings suggest that glial cells actively participate in neural activities, and the cooperation between neurons and glia is important for nervous system functions. In Caenorhabditis elegans, amphid sensory organs in the head also consist of sensory neurons and glia-like support cells (amphid socket and amphid sheath cells). Ciliary endings of some sensory neurons exposed to the environment detect various chemicals, molecules and signals, and the cilia of some neurons can also take up fluorescent dyes such as DiI. Here, we show that the amphid sheath glia are also stained with DiI and that its uptake by the amphid sheath cells correlates with DiI-filling of sensory neurons, suggesting that the amphid sheath glia might interact with sensory neurons. Furthermore, the localization of the amphid sheath cell reporter F52E1.2SP::YFP is abnormal in che-2 mutants, which have defective cilia. These findings imply that sensory neurons might affect amphid sheath glia functions in the amphid sensory organ of C. elegans.

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.

DREF is Critical for Drosophila Bristle Development by Regulating Endoreplication in Shaft Cells

DREF (DNA replication-related element-binding factor) plays important roles in replication and proliferation in vivo by regulating transcription of various genes. However, due to a lack of appropriate cell biological studies in vivo, roles of DREF during a single cell development are poorly understood. To address this question, we focused our attention on macrochaetes bristle development system. Utilizing cell lineage analysis focusing on a single posterior scutellar (PSC) macrochaete sensory organ precursor (SOP) lineages in combination with GAL4/UAS targeted expression system for DREF double strand RNA, we revealed that DREF plays no apparent role in differentiation process during SOP formation. Rather, DREF regulates the timing of asymmetric cell division but perhaps plays no direct role in differentiation during asymmetric cell division. Most importantly, DREF affected replication and growth in shaft cells and/or socket cells. Further analysis revealed that DREF is necessary but not sufficient for nuclear growth and protein synthesis in shaft cells. Finally, it could be demonstrated that DREF plays a critical role in regulating pcna transcription in endocycling shaft cells. All these results provide evidence that DREF plays critical roles, especially in endoreplication process of bristle development, at least in part by regulating the pcna gene expression.

The evolutionarily conserved TSC/Rheb pathway activates Notch in tuberous sclerosis complex and Drosophila external sensory organ development

Mutations in either of the genes encoding the tuberous sclerosis complex (TSC), TSC1 and TSC2, result in a multisystem tumor disorder characterized by lesions with unusual lineage expression patterns. How these unusual cell-fate determination patterns are generated is unclear. We therefore investigated the role of the TSC in the Drosophila external sensory organ (ESO), a classic model of asymmetric cell division. In normal development, the sensory organ precursor cell divides asymmetrically through differential regulation of Notch signaling to produce a pIIa and a pIIb cell. We report here that inactivation of Tsc1 and overexpression of the Ras homolog Rheb each resulted in duplication of the bristle and socket cells, progeny of the pIIa cell, and loss of the neuronal cell, a product of pIIb cell division. Live imaging of ESO development revealed this cell-fate switch occurred at the pIIa-pIIb 2-cell stage. In human angiomyolipomas, benign renal neoplasms often found in tuberous sclerosis patients, we found evidence of Notch receptor cleavage and Notch target gene activation. Further, an angiomyolipoma-derived cell line carrying biallelic TSC2 mutations exhibited TSC2- and Rheb-dependent Notch activation. Finally, inhibition of Notch signaling using a gamma-secretase inhibitor suppressed proliferation of Tsc2-null rat cells in a xenograft model. Together, these data indicate that the TSC and Rheb regulate Notch-dependent cell-fate decision in Drosophila and Notch activity in mammalian cells and that Notch dysregulation may underlie some of the distinctive clinical and pathologic features of TSC.

Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases' role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death.

Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila

The adult Drosophila external sensory organ (ESO), comprising the hair, socket, neuron, sheath and glia cells, arises through the asymmetric division of sensory organ precursor cells (SOPs). In a mosaic screen designed to identify new components in ESO development, we isolated mutations in sequoia, which encodes a putative zinc-finger transcription factor that has previously been shown to have a role in dendritogenesis. Here, we show that adult clones mutant for seq exhibit a loss of hair cells and a gain of socket cells. We propose that the seq mutant phenotype arises, in part, owing to the loss of several crucial transcription factors known to be important in peripheral nervous system development such as D-Pax2, Prospero and Hamlet. Thus, Sequoia is a new upstream regulator of genes that orchestrates cell fate specification during development of the adult ESO lineage.