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Developmental regulation of the cell cycle transition from genomic to site-specific DNA replication in Drosophila
ABSTRACTS / Developmental Biology 306 (2007) 333–338
surface expression, we examined DeltaD distribution after Notch signaling was blocked by a gamma-secretase inhibitor, DAPT or by morpholinos directed against Notch. Both manipulations resulted in exaggerated expression of deltaD. In DAPT-treated embryos some DeltaD accumulated on the plasma membrane, however, most was in cytoplasmic puncta. In contrast, there was a significant increase in DeltaD surface expression in Notch morphants. This suggests that exaggerated expression of deltaD transcripts resulting from failure of Notch signaling per se contributes minimally to increased surface expression. Furthermore, the significant increase in Delta surface expression in Notch morphants suggests that endocytosis of DeltaD is not only dependent on Mib function but also on interactions with Notch. doi:10.1016/j.ydbio.2007.03.175
Program/Abstract # 118 Targeting of Sanpodo to asymmetric pericentrosomal early endosomes regulates Notch signaling in Drosophila sensory organ precursor cells Fabrice Roegiers, Xin Tong, Diana Zitserman Fox Chase Cancer Center, Philadelphia, PA, USA Asymmetric cell division is an evolutionarily conserved strategy to generate cell fate diversity during development. In Drosophila, dividing sensory organ precursor (SOP) cells segregate the membrane-associated cell fate determinant Numb, an antagonist of the Notch pathway, asymmetrically into the pIIb daughter cell. Notch signaling in the pIIb sister cell, pIIa, requires Sanpodo, a transmembrane protein. We showed previously that Numb is required for targeting Sanpodo to vesicles in the pIIb cell cytoplasm. Using in vivo imaging of a Sanpodo-GFP fusion protein, we now show that Numb is required for Sanpodo to be efficiently sequestered from the plasma membrane to a subpopulation of pericentrosomal Rab5-positive early endosomes in the pIIb cell within 10 min of the completion of SOP asymmetric cell division. Blocking formation of the early endosome compartment by inhibiting endocytic vesicle fusion causes failure of Sanpodo accumulation in large endosomes and results in cell fate switching of the pIIb cell due to increased Notch activity. We are currently exploring the mechanism of asymmetric recruitment of Rab5-positive early endosomes to the centrosome in the pIIb cell and are determining the temporal requirement for Notch signaling following SOP division by using a temperature-sensitive allele of Notch. We hypothesize that Notch signaling is regulated by sorting of Sanpodo, and perhaps other Notch pathway components, from the plasma membrane into an asymmetric endocytic node that forms around the centrosome in the pIIb daughter cell shortly after SOP cell mitosis. doi:10.1016/j.ydbio.2007.03.176
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Program/Abstract # 119 Characterization of the protein localization of pyramus and thisbe, Drosophila FGF ligands Sarah L. Payne, Angela M. Stathopoulos Dept. of Biology, California Institute of Technology, Pasadena, CA, USA Fibroblast Growth Factors (FGFs) are involved in important developmental processes including mesoderm induction and patterning, organ formation, and neuronal differentiation. A pressing question in the FGF field is how specificity is achieved and distinct cellular responses accomplished with so few receptors compared to ligands. The FGF family of ligands has two relatively new members in Drosophila, pyramus (pyr) and thisbe (ths), both of which activate the heartless (htl) FGFR. Two FGF ligands, pyr and ths, activate the htl receptor, a simplified system compared to the situation in vertebrates where often 4 or more ligands can activate a single receptor. We use this model to investigate how signaling by pyr versus ths could give rise to different cellular responses. We made HA-tagged versions of Pyr and Ths proteins and have shown these proteins to be functional in the embryo. These epitope tags have allowed us to visualize the proteins using GAL4 lines to overexpress them in the mesoderm (twist) and neuroectoderm (69B). These patterns will be compared with those in a htl deficiency background to see if the ligand–receptor complex is required to stabilize the protein. Further investigation of the proteins in a pyr/ths deficiency background will allow us to see whether the protein localization of one ligand is affected when the other is not present. doi:10.1016/j.ydbio.2007.03.177
Program/Abstract # 120 Developmental regulation of the cell cycle transition from genomic to site-specific DNA replication in Drosophila Alexander J. Armento, Jianjun Sun, Wu-Min Deng Department of Biological Sciences, Florida State University, Tallahassee, FL, USA Cell-cycle regulation plays a crucial role in the development of virtually all multicellular organisms. Drosophila oogenesis is an excellent model system to study developmental regulation of cell cycle programs, because the epithelial follicle cells of the egg chamber undergo two temporally regulated cell-cycle transitions. The first switch, from the archetypical mitotic cycle to the endocycle, a cell-cycle variant in which DNA is replicated but cell division is bypassed, is induced by Notch activation. The regulation mechanisms of the second transition, from the endocycle to a site-specific gene amplification cycle, are unclear. Here we show that Notch activity is down-regulated at stage 10B, when the follicle cells transit from the endocycle to site-specific gene amplification. Using a constitutively active form of Notch, Notch Intracellular Domain (NICD), we show that extended Notch signaling in follicle cells causes defects in the transition to the site-specific gene amplification cycle.
338
ABSTRACTS / Developmental Biology 306 (2007) 333–338
During the second cell cycle transition, concurrent with Notch down-regulation, Ecdysone Receptor (EcR) expression is upregulated. EcR up-regulation appears to be independent of Notch signaling. Using an RNA interference construct against the EcR core domain to knock down EcR expression, the follicle cells fail to exit the endocycle properly. On the basis of these results, we hypothesize that Notch down-regulation and EcR activation signals the transition from the endocycle to gene amplification. doi:10.1016/j.ydbio.2007.03.178
Program/Abstract # 121 Interaction of multiple cell signaling pathways during follicle cell patterning in Drosophila oogenesis John S. Poulton, Wu-Min Deng Dept. of Biol. Sci., Florida State Univ., Tallahassee, FL, USA The patterning of the somatic follicle cells surrounding the Drosophila egg chamber is a vital aspect of oogenesis. The follicle cells differentiate into several cell types based on the activity patterns of Notch, JAK-STAT, and EGFR pathways. Previous research has shown that Notch is necessary for a
general differentiation to a mature cell fate. There is also a symmetrical gradient of JAK-STAT activity, with higher levels at the poles. JAK-STAT specifies the anterior follicle cells (AFC), whereas the posterior follicle cells (PFC) are further differentiated by EGFR activation exclusively at the posterior. We observed that a Notch-activity reporter displays an anterior– posterior gradient among the follicle cells, with highest levels in the AFC and declining towards to the posterior. We also found that PFC mutant for Ras, a component of the EGFR pathway, upregulates Notch activity, indicating that EGFR represses Notch in the PFC. Because JAK-STAT activity is effectively “unmasked” in PFC defective for EGFR, it is possible that JAKSTAT may drive Notch activity. We are currently testing this potential relationship between JAK-STAT and Notch activity. Previously we have shown that EGFR is necessary for downregulation of Dystroglycan, which partly mediates the effects of EGFR on oocyte polarity. We found that changes in Notch activity also caused changes in Dystroglycan levels. Further study will focus on whether changes in Notch activity levels mediate the effects of other signaling pathways on follicle cell and oocyte development. doi:10.1016/j.ydbio.2007.03.179
surface expression, we examined DeltaD distribution after Notch signaling was blocked by a gamma-secretase inhibitor, DAPT or by morpholinos directed against Notch. Both manipulations resulted in exaggerated expression of deltaD. In DAPT-treated embryos some DeltaD accumulated on the plasma membrane, however, most was in cytoplasmic puncta. In contrast, there was a significant increase in DeltaD surface expression in Notch morphants. This suggests that exaggerated expression of deltaD transcripts resulting from failure of Notch signaling per se contributes minimally to increased surface expression. Furthermore, the significant increase in Delta surface expression in Notch morphants suggests that endocytosis of DeltaD is not only dependent on Mib function but also on interactions with Notch. doi:10.1016/j.ydbio.2007.03.175
Program/Abstract # 118 Targeting of Sanpodo to asymmetric pericentrosomal early endosomes regulates Notch signaling in Drosophila sensory organ precursor cells Fabrice Roegiers, Xin Tong, Diana Zitserman Fox Chase Cancer Center, Philadelphia, PA, USA Asymmetric cell division is an evolutionarily conserved strategy to generate cell fate diversity during development. In Drosophila, dividing sensory organ precursor (SOP) cells segregate the membrane-associated cell fate determinant Numb, an antagonist of the Notch pathway, asymmetrically into the pIIb daughter cell. Notch signaling in the pIIb sister cell, pIIa, requires Sanpodo, a transmembrane protein. We showed previously that Numb is required for targeting Sanpodo to vesicles in the pIIb cell cytoplasm. Using in vivo imaging of a Sanpodo-GFP fusion protein, we now show that Numb is required for Sanpodo to be efficiently sequestered from the plasma membrane to a subpopulation of pericentrosomal Rab5-positive early endosomes in the pIIb cell within 10 min of the completion of SOP asymmetric cell division. Blocking formation of the early endosome compartment by inhibiting endocytic vesicle fusion causes failure of Sanpodo accumulation in large endosomes and results in cell fate switching of the pIIb cell due to increased Notch activity. We are currently exploring the mechanism of asymmetric recruitment of Rab5-positive early endosomes to the centrosome in the pIIb cell and are determining the temporal requirement for Notch signaling following SOP division by using a temperature-sensitive allele of Notch. We hypothesize that Notch signaling is regulated by sorting of Sanpodo, and perhaps other Notch pathway components, from the plasma membrane into an asymmetric endocytic node that forms around the centrosome in the pIIb daughter cell shortly after SOP cell mitosis. doi:10.1016/j.ydbio.2007.03.176
337
Program/Abstract # 119 Characterization of the protein localization of pyramus and thisbe, Drosophila FGF ligands Sarah L. Payne, Angela M. Stathopoulos Dept. of Biology, California Institute of Technology, Pasadena, CA, USA Fibroblast Growth Factors (FGFs) are involved in important developmental processes including mesoderm induction and patterning, organ formation, and neuronal differentiation. A pressing question in the FGF field is how specificity is achieved and distinct cellular responses accomplished with so few receptors compared to ligands. The FGF family of ligands has two relatively new members in Drosophila, pyramus (pyr) and thisbe (ths), both of which activate the heartless (htl) FGFR. Two FGF ligands, pyr and ths, activate the htl receptor, a simplified system compared to the situation in vertebrates where often 4 or more ligands can activate a single receptor. We use this model to investigate how signaling by pyr versus ths could give rise to different cellular responses. We made HA-tagged versions of Pyr and Ths proteins and have shown these proteins to be functional in the embryo. These epitope tags have allowed us to visualize the proteins using GAL4 lines to overexpress them in the mesoderm (twist) and neuroectoderm (69B). These patterns will be compared with those in a htl deficiency background to see if the ligand–receptor complex is required to stabilize the protein. Further investigation of the proteins in a pyr/ths deficiency background will allow us to see whether the protein localization of one ligand is affected when the other is not present. doi:10.1016/j.ydbio.2007.03.177
Program/Abstract # 120 Developmental regulation of the cell cycle transition from genomic to site-specific DNA replication in Drosophila Alexander J. Armento, Jianjun Sun, Wu-Min Deng Department of Biological Sciences, Florida State University, Tallahassee, FL, USA Cell-cycle regulation plays a crucial role in the development of virtually all multicellular organisms. Drosophila oogenesis is an excellent model system to study developmental regulation of cell cycle programs, because the epithelial follicle cells of the egg chamber undergo two temporally regulated cell-cycle transitions. The first switch, from the archetypical mitotic cycle to the endocycle, a cell-cycle variant in which DNA is replicated but cell division is bypassed, is induced by Notch activation. The regulation mechanisms of the second transition, from the endocycle to a site-specific gene amplification cycle, are unclear. Here we show that Notch activity is down-regulated at stage 10B, when the follicle cells transit from the endocycle to site-specific gene amplification. Using a constitutively active form of Notch, Notch Intracellular Domain (NICD), we show that extended Notch signaling in follicle cells causes defects in the transition to the site-specific gene amplification cycle.
338
ABSTRACTS / Developmental Biology 306 (2007) 333–338
During the second cell cycle transition, concurrent with Notch down-regulation, Ecdysone Receptor (EcR) expression is upregulated. EcR up-regulation appears to be independent of Notch signaling. Using an RNA interference construct against the EcR core domain to knock down EcR expression, the follicle cells fail to exit the endocycle properly. On the basis of these results, we hypothesize that Notch down-regulation and EcR activation signals the transition from the endocycle to gene amplification. doi:10.1016/j.ydbio.2007.03.178
Program/Abstract # 121 Interaction of multiple cell signaling pathways during follicle cell patterning in Drosophila oogenesis John S. Poulton, Wu-Min Deng Dept. of Biol. Sci., Florida State Univ., Tallahassee, FL, USA The patterning of the somatic follicle cells surrounding the Drosophila egg chamber is a vital aspect of oogenesis. The follicle cells differentiate into several cell types based on the activity patterns of Notch, JAK-STAT, and EGFR pathways. Previous research has shown that Notch is necessary for a
general differentiation to a mature cell fate. There is also a symmetrical gradient of JAK-STAT activity, with higher levels at the poles. JAK-STAT specifies the anterior follicle cells (AFC), whereas the posterior follicle cells (PFC) are further differentiated by EGFR activation exclusively at the posterior. We observed that a Notch-activity reporter displays an anterior– posterior gradient among the follicle cells, with highest levels in the AFC and declining towards to the posterior. We also found that PFC mutant for Ras, a component of the EGFR pathway, upregulates Notch activity, indicating that EGFR represses Notch in the PFC. Because JAK-STAT activity is effectively “unmasked” in PFC defective for EGFR, it is possible that JAKSTAT may drive Notch activity. We are currently testing this potential relationship between JAK-STAT and Notch activity. Previously we have shown that EGFR is necessary for downregulation of Dystroglycan, which partly mediates the effects of EGFR on oocyte polarity. We found that changes in Notch activity also caused changes in Dystroglycan levels. Further study will focus on whether changes in Notch activity levels mediate the effects of other signaling pathways on follicle cell and oocyte development. doi:10.1016/j.ydbio.2007.03.179