Hexagonal packing of Drosophila wing epithelial cells by the Planar Cell Polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but are poorly understood. Here we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented towards the distal wing tip. They are constructed by a hexagonal array of wing epithelial cells. We find that wing epithelial cells are irregularly arranged throughout most of development but become hexagonally packed shortly before hair formation. During the process, individual cell junctions grow and shrink, resulting in local neighbor exchanges. These dynamic changes mediate hexagonal packing and require the efficient delivery of E-cadherin to remodeling junctions; a process that depends on both the large GTPase Dynamin and the function of Rab11 recycling endosomes. We suggest that E-cadherin is actively internalized and recycled as wing epithelial cells pack into a regular hexagonal array. Hexagonal packing furthermore depends on the activity of the Planar Cell Polarity proteins. The Planar Cell Polarity group of proteins coordinates complex and polarized cell behavior in many contexts. No common cell biological mechanism has yet been identified to explain their functions in different tissues. A genetic interaction between Dynamin and the Planar Cell Polarity mutants suggests that the planar cell polarity proteins may modulate Dynamin-dependent trafficking of E-cadherin to enable the dynamic remodeling of junctions. We furthermore show that the Planar Cell Polarity protein Flamingo can recruit the exocyst component Sec5. Sec5 vesicles also co-localizes with E-cadherin and Flamingo. Based on these observations we propose that during the hexagonal repacking of the wing epithelium these proteins polarize the trafficking of E-cadherin-containing exocyst vesicles to remodeling junctions. The work presented in this thesis shows that one of the basic cellular functions of planar cell polarity signaling may be the regulation of dynamic cell adhesion. In doing so, the planar cell polarity pathway mediates the acquisition of a regular packing geometry of Drosophila wing epithelial cells. We identify polarized exocyst-dependent membrane traffic as the first basic cellular mechanism that can explain the role of PCP proteins in different developmental systems

Calcium spikes, waves and oscillations in a large, patterned epithelial tissue

While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-rang

Hexagonal packing of Drosophila wing epithelial cells by the Planar Cell Polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but are poorly understood. Here we investigate this problem in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented towards the distal wing tip. They are constructed by a hexagonal array of wing epithelial cells. We find that wing epithelial cells are irregularly arranged throughout most of development but become hexagonally packed shortly before hair formation. During the process, individual cell junctions grow and shrink, resulting in local neighbor exchanges. These dynamic changes mediate hexagonal packing and require the efficient delivery of E-cadherin to remodeling junctions; a process that depends on both the large GTPase Dynamin and the function of Rab11 recycling endosomes. We suggest that E-cadherin is actively internalized and recycled as wing epithelial cells pack into a regular hexagonal array. Hexagonal packing furthermore depends on the activity of the Planar Cell Polarity proteins. The Planar Cell Polarity group of proteins coordinates complex and polarized cell behavior in many contexts. No common cell biological mechanism has yet been identified to explain their functions in different tissues. A genetic interaction between Dynamin and the Planar Cell Polarity mutants suggests that the planar cell polarity proteins may modulate Dynamin-dependent trafficking of E-cadherin to enable the dynamic remodeling of junctions. We furthermore show that the Planar Cell Polarity protein Flamingo can recruit the exocyst component Sec5. Sec5 vesicles also co-localizes with E-cadherin and Flamingo. Based on these observations we propose that during the hexagonal repacking of the wing epithelium these proteins polarize the trafficking of E-cadherin-containing exocyst vesicles to remodeling junctions. The work presented in this thesis shows that one of the basic cellular functions of planar cell polarity signaling may be the regulation of dynamic cell adhesion. In doing so, the planar cell polarity pathway mediates the acquisition of a regular packing geometry of Drosophila wing epithelial cells. We identify polarized exocyst-dependent membrane traffic as the first basic cellular mechanism that can explain the role of PCP proteins in different developmental systems

Imaging Drosophila pupal wing morphogenesis.

International audienceDrosophila pupal (P) wing development entails a series of dynamic developmental events, such as epithelial and glial morphogenesis, that are of outstanding interest to cell biologists. Here, we first describe how to prepare P and prepupal (PP) wings for immunofluorescence microscopy. This protocol has been optimized to visualize wing epithelial architecture, such as polarized cortical domains of planar cell polarity proteins. We then provide a protocol to prepare pupae for whole mount live imaging of P wings. This procedure has allowed us to live-image glial cell migration and proliferation along wing sensory nerves

JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation

A tumor suppressor activity of Drosophila Polycomb genes mediated by JAK-STAT signaling.

A prevailing paradigm posits that Polycomb Group (PcG) proteins maintain stem cell identity by repressing differentiation genes, and abundant evidence points to an oncogenic role for PcG proteins in human cancer. Here we show using Drosophila melanogaster that a conventional PcG complex can also have a potent tumor suppressor activity. Mutations in any core PRC1 component cause pronounced hyperproliferation of eye imaginal tissue, accompanied by deregulation of epithelial architecture. The mitogenic JAK-STAT pathway is strongly and specifically activated in mutant tissue; activation is driven by transcriptional upregulation of Unpaired (Upd, also known as Outstretched, Os) family ligands. We show here that upd genes are direct targets of PcG-mediated repression in imaginal discs. Ectopic JAK-STAT activity is sufficient to induce overproliferation, whereas reduction of JAK-STAT activity suppresses the PRC1 mutant tumor phenotype. These findings show that PcG proteins can restrict growth directly by silencing mitogenic signaling pathways, shedding light on an epigenetic mechanism underlying tumor suppression

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models

Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies

DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis

Background: Tracking dynamic protein–chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches. Results: Here we adapt cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, we utilize ubiquitous DamID expression to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. We identify Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. We establish these findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae. Conclusions: Our study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.ISSN:1756-893