The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis

The Drosophila imaginal cells that produce epidermal hairs, the shafts of sensory bristles and the lateral extensions of the arista are attractive model systems for studying the morphogenesis of polarized cell extensions. We now report the identification and characterization of furry, an essential Drosophila gene that is involved in maintaining the integrity of these cellular extensions during morphogenesis. Mutations in furry result in the formation of branched arista laterals, branched bristles and a strong multiple hair cell phenotype that consists of clusters of epidermal hairs and branched hairs. By following the morphogenesis of arista laterals in pupae, we have determined that the branched laterals are due to the splitting of individual laterals during elongation. In genetic mosaics furry was found to act cell autonomously in the wing. The phenotypes of double mutant cells argue that furry functions independently of the frizzled planar polarity pathway and that it probably functions in the same pathway as the tricornered gene. We used a P-element insertion allele as a tag to clone the furry gene and found it to be a large and complicated gene that encodes a pair of large conserved proteins of unknown biochemical function.     

The genetic control of arista lateral morphogenesis in Drosophila

The sensory bristles and epidermal hairs of Drosophila have proven to be valuable model cell types for studying the role of the cytoskeleton in cellular morphogenesis. We have recently begun to use the arista laterals as a third model cell type. The laterals display a combination of bristle and hair characteristics and provide a system where we can compare the relative importance of specific genes and subcellular structures for the morphogenesis of different polarized cellular extensions. We have characterized the lateral phenotype of a collection of mutations selected because of their phenotypes in hairs and bristles. In many but not all ways the lateral phenotypes are similar to the hair and bristle phenotypes. We provide compelling genetic evidence for the importance of the actin cytoskeleton in lateral elongation, shaping and integrity. Our observations provide evidence that defects in actin bundling can destabilize laterals so that they split during growth. Temperature shift experiments suggest that a defect in lateral initiation can lead to subsequent splitting. These observations provide a link between multiple hair and lateral cells forming by both multiple initiation events and by the splitting of individual cellular extensions. We also found that mutations that lead to lateral splitting typically alter the stereotypic arrangement of actin filament bundles and microtubules in laterals.     

The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase

During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.     

The flare gene, which encodes the AIP1 protein of Drosophila, functions to regulate F-actin disassembly in pupal epidermal cells

Adult Drosophila are decorated with several types of polarized cuticular structures, such as hairs and bristles. The morphogenesis of these takes place in pupal cells and is mediated by the actin and microtubule cytoskeletons. Mutations in flare (flr) result in grossly abnormal epidermal hairs. We report here that flr encodes the Drosophila actin interacting protein 1 (AIP1). In other systems this protein has been found to promote cofilin-mediated F-actin disassembly. In Drosophila cofilin is encoded by twinstar (tsr). We show that flr mutations result in increased levels of F-actin accumulation and increased F-actin stability in vivo. Further, flr is essential for cell proliferation and viability and for the function of the frizzled planar cell polarity system. All of these phenotypes are similar to those seen for tsr mutations. This differs from the situation in yeast where cofilin is essential while aip1 mutations result in only subtle defects in the actin cytoskeleton. Surprisingly, we found that mutations in flr and tsr also result in greatly increased tubulin staining, suggesting a tight linkage between the actin and microtubule cytoskeleton in these cells.     

IKK epsilon regulates F actin assembly and interacts with Drosophila IAP1 in cellular morphogenesis

Differentiated cells assume complex shapes through polarized cell migration and growth. These processes require the restricted organization of the actin cytoskeleton at limited subcellular regions. IKK epsilon is a member of the IkappaB kinase family, and its developmental role has not been clear. Drosophila IKK epsilon was localized to the ruffling membrane of cultured cells and was required for F actin turnover at the cell margin. In IKK epsilon mutants, tracheal terminal cells, bristles, and arista laterals, which require accurate F actin assembly for their polarized elongation, all exhibited aberrantly branched morphology. These phenotypes were sensitive to a change in the dosage of Drosophila inhibitor of apoptosis protein 1 (DIAP1) and the caspase DRONC without apparent change in cell viability. In contrast to this, hyperactivation of IKK epsilon destabilized F actin-based structures. Expression of a dominant-negative form of IKK epsilon increased the amount of DIAP1. The results suggest that at the physiological level, IKK epsilon acts as a negative regulator of F actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis. This IKK epsilon function involves the negative regulation of the nonapoptotic activity of DIAP1.     

Drosophila Mob family proteins interact with the related tricornered (Trc) and warts (Wts) kinases

The function of Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase, is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. In this report, we provide genetic and biochemical data that Drosophila Trc also interacts with and is activated by Drosophila Dmob proteins. In addition, Drosophila Mob proteins appear to interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) was only seen in genetic mosaics and not when the entire animal was mutant. We conclude that unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and that individual NDR family kinases interact with multiple Mob proteins. We further provide evidence that Mo25, the Drosophila homolog of the RAM pathway hym1 gene does not function along with Trc.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development

The Trc/Ndr/Sax1/Cbk1 family of ser/thr kinases plays a key role in the morphogenesis of polarized cell structures in flies, worms, and yeast. Tricornered (Trc), the Drosophila nuclear Dbf2-related (Ndr) serine/threonine protein kinase, is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites. We obtained in vivo evidence that Trc function was regulated by phosphorylation and that mutations in key regulatory sites resulted in dominant negative alleles. We found that wild-type, but not mutant Trc, is found in growing hairs, and we failed to detect Trc in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have previously been implicated as being essential for the function of the Ndr kinase family. We found that Drosophila furry (Fry) also is found in growing hairs, that its subcellular localization is dependent on Trc function, and that it can be coimmunoprecipitated with Trc. Our data suggest a feedback mechanism involving Trc activity regulates the accumulation of Fry in developing hairs.     

The shavenoid gene of Drosophila encodes a novel actin cytoskeleton interacting protein that promotes wing hair morphogenesis

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular- and tissue-level morphogenesis. The developing hairs are filled with F-actin and microtubules and the activity of these cytoskeletons is important for hair morphogenesis. On the basis of mutant phenotypes several genes have been identified as playing a key role in stimulating hair formation. Mutations in shavenoid (sha) (also known as kojak) result in a delay in hair morphogenesis and in some cells forming no hair and others several small hairs. We report here the molecular identification and characterization of the sha gene and protein. sha encodes a large novel protein that has homologs in other insects, but not in more distantly related organisms. The Sha protein accumulated in growing hairs and bristles in a pattern that suggested that it could directly interact with the actin cytoskeleton. Consistent with this mechanism of action we found that Sha and actin co-immunopreciptated from wing disc cells. The morphogenesis of the hair involves temporal control by sha and spatial control by the genes of the frizzled planar polarity pathway. We found a strong genetic interaction between mutations in these genes consistent with their having a close but parallel functional relationship.     

The frizzled pathway regulates the development of arista laterals.

Background  

The frizzled pathway in Drosophila has been studied intensively for its role in the development of planar polarity in wing hairs, thoracic bristles and ommatidia. Selected cells in the arista (the terminal segment of the antenna) elaborate a lateral projection that shares characteristics with both hairs and bristles.     

The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells

Background  

The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal) cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear.     

Establishing cell polarity in development

Polarity is a common feature of many different cell types, including the Caenorhabditis elegans zygote, the Drosophila oocyte and mammalian epithelial cells. The initial establishment of cell polarity depends on asymmetric cues that lead to reorganization of the cytoskeleton and polarized localization of several cortical proteins that act downstream of the polarization cues. The past year revealed that homologs of the C. elegans par (partitioning defective) genes are also essential for establishing polarity in Drosophila and vertebrate cells. There is growing evidence that the proteins encoded by these genes interact with key regulators of both the actin and the microtubule cytoskeletons.     

Planar signaling and morphogenesis in Drosophila

The regulatory mechanisms governing the parallel alignment of hairs, bristles, and ommatidia in Drosophila have all served as model systems for studying planar signaling and tissue level morphogenesis. Polarity in all three systems is mediated by the serpentine receptor Frizzled and a number of additional gene products. The localized accumulation of these proteins within cells plays a key role in the development of planar polarity. A comparison of the function of these gene products in the different cell types suggests cell-specific modifications of the pathway.     

Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. We have identified a novel role for the actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. We show that Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator gamma-tubulin. Moreover, this change in gamma-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that gamma-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons.     

Phenotypic and genotypic structure of a natural Drosophila population for meristic morphologic characters and its seasonal changes

A natural population of Drosophila was genetically heterogeneous in the number of sternopleural bristles and the number of arista branches. In the summer, these characters had a low mean value and high coefficient of variation: in autumn, this relationship was reversed; in late spring, the values of the mean and coefficient of variation were intermediate. The dynamics of the character mean was determined by changes in phenotypic and genotypic composition of the population, which was examined during different seasons of the year. On average, the number of sternopleural bristles was higher, and the number of arista branches was lower in females than in males. The number of sternopleural bristles exhibited a higher phenotypic and genotypic variation than the number of arista branches.     

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.     

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.     

Spatial control of the actin cytoskeleton in Drosophila epithelial cells

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.     

Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division.

BACKGROUND: Both symmetric and asymmetric cell divisions are required for the generation of appropriate cell lineages during development. Wild-type Schizosaccharomyces pombe cells divide in a symmetric fashion to produce two similar rod-shaped daughter cells. Formins are proteins with conserved roles in cell polarity, cytokinesis, and the regulation of actin and microtubule cytoskeletons. RESULTS: Here, we identify and characterize a new S. pombe formin, for3p. for3 Delta mutant cells divide in an asymmetric manner; a mother cell divides medially to produce one daughter cell that develops into a monopolar cell and one daughter that develops into a bipolar cell. Both daughter cells recapitulate similar asymmetric lineages themselves. Inheritance of the bipolar pattern correlates with inheritance of the recent birth scar, not with asymmetry in the spindle pole bodies. for3 Delta mutants lack interphase actin cables and have delocalized actin patch and myo52p (type V myosin) distributions. for3 Delta cells have normal microtubule dynamics and cortical interactions but have defects in microtubule organization and increased numbers of microtubule bundles. for3p-GFP is localized at both cell tips in an actin-dependent manner and at the cell division site. CONCLUSIONS: for3p is a cell polarity factor required for interphase actin cable formation and microtubule organization. The for3 Delta phenotype suggests that cells are able to grow in a polarized manner even in the absence of functional actin cables and polarized distribution of actin patches. for3p and possibly actin cables are part of a regulatory network that ensures that cell divisions are symmetric.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.