Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development

The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.Subject terms: Development, Genetic interaction     

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.     

The Transcription Factor Optomotor-Blind Antagonizes Drosophila Haltere Growth by Repressing Decapentaplegic and Hedgehog Targets

In Drosophila, decapentaplegic, which codes for a secreted signaling molecule, is activated by the Hedgehog signaling pathway at the anteroposterior compartment border of the two dorsal primordia; the wing and the haltere imaginal discs. In the wing disc, Decapentaplegic and Hedgehog signaling targets are implicated in cell proliferation and cell survival. However, most of their known targets in the wing disc are not expressed in the haltere disc due to their repression by the Hox gene Ultrabithorax. The T-box gene optomotor-blind escapes this repression in the haltere disc, and therefore is expressed in both the haltere and wing discs. Optomotor-blind is a major player during wing development and its function has been intensely investigated in this tissue, however, its role in haltere development has not been reported so far. Here we show that Optomotor-blind function in the haltere disc differs from that in the wing disc. Unlike its role in the wing, Optomotor-blind does not prevent apoptosis in the haltere but rather limits growth by repressing several Decapentaplegic and Hedgehog targets involved both in wing proliferation and in modulating the spread of morphogens similar to Ultrabithorax function but without disturbing Ultrabithorax expression.     

Serrano (Sano) Functions with the Planar Cell Polarity Genes to Control Tracheal Tube Length

Epithelial tubes are the functional units of many organs, and proper tube geometry is crucial for organ function. Here, we characterize serrano (sano), a novel cytoplasmic protein that is apically enriched in several tube-forming epithelia in Drosophila, including the tracheal system. Loss of sano results in elongated tracheae, whereas Sano overexpression causes shortened tracheae with reduced apical boundaries. Sano overexpression during larval and pupal stages causes planar cell polarity (PCP) defects in several adult tissues. In Sano-overexpressing pupal wing cells, core PCP proteins are mislocalized and prehairs are misoriented; sano loss or overexpression in the eye disrupts ommatidial polarity and rotation. Importantly, Sano binds the PCP regulator Dishevelled (Dsh), and loss or ectopic expression of many known PCP proteins in the trachea gives rise to similar defects observed with loss or gain of sano, revealing a previously unrecognized role for PCP pathway components in tube size control.     

To Cease or To Proliferate: New Insights into TCTP Function from a Drosophila Study

Tor (target of rapamycin) pathway underlies a major signaling mechanism for controlling cell growth and proliferation.¹ Rheb (Ras homolog enriched in brain) is a small GTPase in the Tor pathway.^2–4 Similar to other small GTPases, Rheb cycles between a GTP-bound active state and a GDP-bound inactive state. TSC2 (tuberous sclerosis complex 2), a gene mutated in an autosomal dominant disease tuberous sclerosis, was shown to be the Rheb-GAP (GTPase activating protein).^5,6 However, a guanine nucleotide exchange factor (GEF) for Rheb had been missing. Human TCTP (translationally controlled tumor protein) has been implicated in cancer, but its function in vivo has not been clearly elucidated. Recently we reported a molecular genetic characterization of TCTP function in Drosophila.⁷ Drosophila TCTP (dTCTP) displays GEF activity to Rheb and is essential for Rheb activation in organ growth. Thus, our study provides a tight linkage of dTCTP to the Rheb-TOR pathway. In this addendum, we will briefly overview our findings and discuss our perspectives for future research on TCTP.Key Words: TCTP, Tor pathway, Rheb, TSC, GEFTCTP is a highly conserved protein identified about 20 years ago as a translationally controlled protein P21 (or P23) enriched in tumor cell lines.⁸ Recently, this protein has drawn special interests because of its potential roles in tumorigenesis. TCTP is not only upregulated in a number of tumor cell lines but also downregulated during tumor reversion.^9,10 TCTP has also been implicated in a variety of intracellular and extracellular functions, including microtubule stabilization, cell cycle, apoptosis, and cytokine release.^11–16 However, these functions of TCTP have been inferred mainly from biochemical interactions and cell culture studies.To address the function of TCTP in vivo, we took a loss-of-function approach using Drosophila as a genetic model. Reduction of dTCTP by tissue-specific RNA interference (RNAi) or loss-of-function mutations resulted in smaller organs with reduction in both cell size and cell number, a phenotype often seen in mutations in Tor or insulin pathway. Our epistatic analysis suggested that dTCTP acts either downstream or in parallel to insulin receptor, TSC1, and dRheb, but upstream of dS6K. Despite the conserved sequence of TCTP proteins in a wide-range of species, TCTP has little similarity to the sequences of other protein families. However, the three-dimensional structure of fission yeast TCTP ortholog reveals similarities with a family of proteins that bind to the nucleotide-free form of Rab GTPases,¹⁷ providing a clue for its potential biochemical function. Consistent with our genetic evidence, our biochemical assays showed that dTCTP could facilitate the GDP/GTP exchange on dRheb both in vitro and in vivo. Our data led us to propose a model (Fig. 1) in which dTCTP regulates the Tor signaling pathway by directly interacting with dRheb GTPase as a GEF.Open in a separate windowFigure 1A model for dTCTP function in growth control. dRheb GTPase stimulates Tor signaling, which in turn activates dS6K and CycE to regulate cell growth and proliferation, respectively. dRheb GTPase is inactivated by the GAP function of TSC1/2 complex. In contrast, dTCTP activates dRheb GTPases by promoting GDP-GTP exchange. dTCTP might have additional functions independent of the Tor pathway, such as inhibiting cell death. It remains to be determined if dTCTP regulates cell proliferation and cell survival in part through the Hippo signaling.This study not only provides new insights into the mechanism of dTCTP function in regulation of dRheb activity but also reveals a complexity of dTCTP function in growth regulation. Firstly, dTCTP null mutant displays more severe phenotypes than loss of function mutations in the insulin or Tor pathways. For example, while dTCTP null mutant clones are eliminated during development, significant portions of dRheb and Tor null mutant clones can survive to form adult tissues. This argues against the model that dTCTP functions only in regulating the Rheb-Tor pathways. Although different maternal contribution of each gene and variations in genetic background might partially account for these differences, it is equally possible that dTCTP functions as a GEF for more than one GTPase targets. Conversely, other GEFs might exist for Rheb, as one small GTPase can be regulated by more than one GEFs.^18,19 Different GEFs might be required for dRheb-TOR function in other developmental events or growth-independent processes like axon guidance during neural development.²⁰Secondly, in contrast to the implicated role of TCTP in cancer,⁹ ubiquitous or tissue-specific overexpression of dTCTP was insufficient to cause overgrowth phenotypes in Drosophila. A plausible explanation is that the amount of dTCTP is in excess for dRheb activation in contrast to limited concentrations of TSC2 GAP in normal cells.⁵ Since overexpression of dTCTP alone does not result in tumorous overgrowth, it is unlikely that TCTP functions as an oncogene directly under normal condition. However, increased amounts of TCTP in tumor tissues may provide better potency for cells to undergo uncontrolled proliferation and massive overgrowth. In this regard, it will be intriguing to learn if dTCTP can act corporately with other oncogenes. Since the amino acid sequence identity of dTCTP and human TCTP is only 48%, it is also possible that a non-conserved region(s) of human TCTP may be required to induce tumors in human cells. Soon after the publication of our work, knockout of the mouse TCTP was reported. Remarkably, loss of TCTP in mice results in early lethality with smaller sizes of embryos.²¹ Developmental defects seen in mutant mice may be analogous to the phenotypes of dTCTP mutants in Drosophila, supporting the conserved function of TCTP in growth regulation. However, it is yet to be determined whether there is a common molecular basis for the developmental defects in both systems and whether overexpression of mouse TCTP in transgenic mice can induce tumors.Lastly, in addition to Tor and insulin signaling pathways, a third pathway consisting of Hippo-Warts protein kinase cascade controls organ size by affecting mainly the cell number.^22,23 The Hippo pathway regulates both cell proliferation and cell death by promoting cyclin E expression and downregulating Drosophila Inhibitor of Apoptosis 1 (DIAP1). It is worthy of note that dTCTP also regulates the cell number by affecting cell proliferation and apoptosis. It remains to be determined whether dTCTP and Hippo signaling pathways crosstalk or are independent of each other.Studies on TCTP suggest that its function is much more complex than what is known and its interactions with a multitude of proteins might underlie this complexity. Biochemical studies have identified several proteins interacting with TCTP. Given the feasibility of Drosophila genetics, the physiological relevance of these protein interactions can now be addressed in the context of normal development. It would also be powerful to take the advantage of Drosophila genetic screens for the identification of novel genes interacting with dTCTP. A more comprehensive understanding of TCTP functions and mechanistic explanations of its intriguing expression profiles in cancers can be expected in years to come.     

Parvin Overexpression Uncovers Tissue-Specific Genetic Pathways and Disrupts F-Actin to Induce Apoptosis in the Developing Epithelia in Drosophila

Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1), high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.     

The influence of morphogene Wg on the formation of an ectopic eye in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Due to the ectopic expression of the ey gene in the wing imaginal disc under the action of the 1096-Gal4 driver, a part of the wing disc cells change their fate and become eye cells. Ectopic eyes are induced in definite regions of the wing disc and form a stable pattern on the wing of an adult fly. Here, we have shown that the ectopic expression of Wg inhibits the formation of ectopic eyes, and conversely the expression of Wg is reduced in the sites of ectopic Ey expression. Experiments with overexpression of the vesicular traffic protein Hrs capable of inhibiting the Wg signaling agree with the notion on antagonism of Wg and Ey in ectopic eyes. Our results confirm that the processes of formation of normal and ectopic eyes are principally similar with regard to genetic control.     

The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling

Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known N^spl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.     

Proteasome,but Not Autophagy,Disruption Results in Severe Eye and Wing Dysmorphia: A Subunit- and Regulator-Dependent Process in Drosophila

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx^MS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly’s eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing’s, but not eye’s, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development.     

Cullin-3 regulates pattern formation, external sensory organ development and cell survival during Drosophila development

Ubiquitin-mediated proteolysis regulates the steady-state abundance of proteins and controls cellular homoeostasis by abrupt elimination of key effector proteins. A multienzyme system targets proteins for destruction through the covalent attachment of a multiubiquitin chain. The specificity and timing of protein ubiquitination is controlled by ubiquitin ligases, such as the Skp1-Cullin-F box protein complex. Cullins are major components of SCF complexes, and have been implicated in degradation of key regulatory molecules including Cyclin E, beta-catenin and Cubitus interruptus. Here, we describe the genetic identification and molecular characterisation of the Drosophila Cullin-3 homologue. Perturbation of Cullin-3 function has pleiotropic effects during development, including defects in external sensory organ development, pattern formation and cell growth and survival. Loss or overexpression of Cullin-3 causes an increase or decrease, respectively, in external sensory organ formation, implicating Cullin-3 function in regulating the commitment of cells to the neural fate. We also find that Cullin-3 function modulates Hedgehog signalling by regulating the stability of full-length Cubitus interruptus (Ci155). Loss of Cullin-3 function in eye discs but not other imaginal discs promotes cell-autonomous accumulation of Ci155. Conversely, overexpression of Cullin-3 results in a cell-autonomous stabilisation of Ci155 in wing, haltere and leg, but not eye, imaginal discs suggesting tissue-specific regulation of Cullin-3 function. The diverse nature of Cullin-3 phenotypes highlights the importance of targeted proteolysis during Drosophila development.     

rap gene encodes Fizzy-related protein (Fzr) and regulates cell proliferation and pattern formation in the developing Drosophila eye-antennal disc

The rap (retina aberrant in pattern) gene encodes the Fizzy-related protein (Fzr), which as an activator of the ubiquitin ligase complex; APC/C (anaphase promoting complex/cyclosome) facilitates the cell cycle stage-specific degradation of cyclins. Loss-of-function mutations in rap cause unscheduled accumulation of cyclin B in the developing eye imaginal disc, resulting in additional mitotic cycles and defective patterning of the developing Drosophila eye. Targeted mis-expression of rap/fzr in the eye primordial cells causes precocious cell cycle exit, and smaller primordial eye fields, which either eliminate or drastically reduce the size of the adult eye. Although mitosis is inhibited in the mis-expression animals, cells with abnormally large nuclei form tumor-like structures from continued endoreplication, cell growth and retinal differentiation. Interestingly, overexpression of Rap/Fzr in the eye primordia also increases the size of the antennal primordium resulting in the induction of ectopic antennae. These results suggest that Rap/Fzr plays an essential role in the timely exit of precursor cells from mitotic cycles and indicate that mechanisms that regulate cell cycle exit are critical during pattern formation and morphogenesis.     

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.