- Email: [email protected]
P02. Migration of human fibrosarcoma cells toward the blood vessel when transplanted into chicken embryo
Differentiation 80 (2010) S17–S63
Contents lists available at ScienceDirect
Differentiation journal homepage: www.elsevier.com/locate/diff
Abstracts
Poster presentations
P01 Modelling of differentiation and malignant transformation in 1D and 3D culture of canine kidney epithelial MDCK cells with inducible Src activation
¨ b, A. Mannine b, Sinikka Eskeline a J. Capra a, S. Myllymak
¨ environment at + 35 1C (Toyli et al., 2010). Survivin is a highly expressed protein in almost all malignant tumours and completely missing in adult healthy tissues. It is an essential regulator of mitosis, inhibits apoptosis and promotes migration of transformed cells. Thus, survivin may serve as a possible Src oncogene target at various steps of carcinogenesis. Reference
a
Biocenter Oulu and the Institute of Diagnostics, University of Oulu, Finland b Biocenter Oulu and the Institute of Biomedicine, University of Oulu, Finland E-mail address: sinikka.eskelinen@oulu.fi (S. Eskeline)
¨ Toyli, M., Rosberg-Kulha, L., Capra, J., Vuoristo, J., Eskelinen, S., 2010. Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays. Lab. Invest. 90, 915–928. doi: 10.1016/j.diff.2010.09.007
MDCK cells expressing a temperature sensitive mutant of v-Src provide a well-suited model system to study malignant transformation. When cultivated at 40.5 1C, they behave as normal epithelial cells, whereas after a shift to 35 1C, Src tyrosine kinase is activated and the transformation process begins. Src-transformed MDCK cells grown at + 40.5 1C in 3-D Matrigel form polarized cysts with apical surface facing an internal lumen and basal surface facing the extracellular matrix (ECM). Upon Src-activation at + 35 1C these cells form lumenless spherical ¨ cysts without apico-basal polarity (Toyli et al., 2010). In order to monitor the mechanism of lumen filling in 3D culture we transfected Src-MDCK cells with GFP cadherin. These cells were cultivated in Matrigel at + 40.5 1C until they formed lumina, and subsequently monitored under a microscope adjusted at + 35 1C to follow the transformation process for up to 24 h. GFP cadherin and Hoechst stain served as markers for cell polarization and transformation. In 1–2 h the cells began to lose their apico-basal polarity and some cells were observed inside the lumen. In 6 h the luminal space was filled, but the cyst size was not increased. When incubated overnight at + 35 1C, the cells formed large irregular clusters. We conclude that the initial mechanism of filling the luminal space was cell migration. Next, we cultivated the MDCK cells on a non-adhesive surface in the presence of a function-blocking beta1-integrin antibody. In these 1D conditions both untransformed and v-Src-transformed MDCK cells formed aggregates floating in the suspension without any apico-basal polarity. Interestingly, the cells survived several days without entering apoptosis. The results show that ECM-contact and integrin signalling are crucial for formation of apico-basal axis and central lumen. However, additional signals are needed to trigger apoptosis in cells without ECM contact in suspension or trapped within the 3D cell cluster. We observed a strong expression of survivin in ts-Src MDCK cells grown in 3D
P02 Migration of human fibrosarcoma cells toward the blood vessel when transplanted into chicken embryo
Taiji Yasue, Ryosuke Tadokoro, Yoshiko Takahashi Nara Institute of Science and Technology, Nara, Japan E-mail address: [email protected] (T. Yasue)
Cell migration is one of the most important phenomena not only for development but also for cancer metastasis. When cells migrate in the body, their surrounding tissues (environment) might be influential on the migratory behavior. However, little is known about interactions between migrating cells and their environment in the actual body. We have recently established a novel experimental system in which cultured line-cells are transplanted into early chicken embryos, where their migratory behaviors are directly assessed in vivo. We have examined 12 different cell lines, including 4 non-cancer cell lines (chicken DF-1, mammalian COS-7, NIH3T3 and C2C12) and 8 human cancer cell lines (HT1080, MCF-7, MDA-MB-231, Y-1, Panc-1, A375, Mewo and A2058). These cells are manufactured in vitro to stably express EGFP prior to transplantation into the dorsal aspect of the somitic mesoderm of E2 chicken embryo. Among the 12 cell lines, HT1080 cells (highly invasive human sarcoma) are found to actively migrate to the dorsal aorta whereas the others do not.
0301-4681/$ - see front matter & 2010 Published by Elsevier Ltd. on behalf of International Society of Differentiation Join the International Society for Differentiation (www.isdifferentiation.org) doi:
S18
Abstract / Differentiation 80 (2010) S17–S63
Time-lapse imaging with confocal laser microscopy allows us to observe numerous filopodia extending from migrating cells. Such migratory behavior is dependent on the activity of matrix metalloproteinase (MMP), known to degrade extracellular matrix proteins. When overexpressed with MMP inhibitor Timp-2 or Reck, the migration has been impaired. Following the migration, HT1080 cells accumulate around the aortic vessel. Interestingly, the dorsal and ventral aspects of this vessel provide differential environment to the HT1080: in the dorsal region cells undergo intravasation (breaking the blood vessel to invade into the blood stream) whereas the ventral region allows pronounced accumulation of cells. We will discuss how cells interact with their surrounding tissues for cell migration and intravasation events. doi: 10.1016/j.diff.2010.09.008
P03 Ex vivo live imaging at high resolution to directly visualize melanin transfer from melanocytes to keratinocytes
Ryosuke Tadokoro, Kenichiro Sakai, Hidetaka Murai, Yoshiko Takahashi Graduate School of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0192, Japan E-mail address: [email protected] (R. Tadokoro) Melanin pigmentation in the skin is physiologically important to protect our body from UV irradiation. Melanocytes are known as melanin-producing cells in which melanin is synthesized in melanosomes. In the tanning processes, melanocytes extend numerous dendrites that are attached to surrounding keratinocyes, and subsequently melanin granules are transferred through melanocyte dendrites to keratinocytes. Although several models for intercellular melanin-transfer have been proposed by in vitro experiments and electron microscopic observations, the mechanisms of melanin-transfer in vivo remain controversial. We have developed time-lapse imaging techniques that directly visualize the melanin-transfer in an ex vivo cultured skin of chicken embryo, where epidermal cells and melanocytes are expected to behave in a similar way to in vivo. In this system, embryonic melanocytes are genetically and stably labeled with a membranebound EGFP using transposon-mediated gene transfer technique (Sato et al., 2007. Dev. Biol.). This manipulation directly visualizes fine structures of dendrites of differentiated melanocytes. At early stages of melanin-synthesis, melanocytes actively elongate and continuously change their shape with dendrites both extending and retracting. As melanin-synthesis proceeds, these dendrites form blebs on the plasma membrane, and subsequently they release small membrane vesicles (0.5–1 mm in diameter) to the extracellular space. Importantly, these vesicles contain melanin granules, and become incorporated into adjacent keratinocytes. Thus, melanin granules are transferred from melanocytes into keratinocytes via membrane vesicles. This is the first demonstration that the process of melanin transfer is directly visualized in the developing skin. doi: 10.1016/j.diff.2010.09.009
P04 Dissecting the contribution of non-neural ectoderm to the vertebrate neural tube closure
H. Morita a,b, N. Ueno a,b a
The Graduate University for Advanced Studies (Sokendai), Hayama, Japan b National Institute for Basic Biology, Okazaki, Japan E-mail address: [email protected] (H. Morita) Neural tube closure is one of the most prominent morphogenetic events during vertebrate development with dynamic cell shape changes and reorganization of cells. These cell behaviors are observed not only in a forming neural tube (neural ectoderm) itself but also in epidermal (non-neural) ectoderm. Cells in the neural ectoderm undergo apical constriction and mediolateral intercalation, which allow the neural tissue to form a groove along the anteroposterior axis and converge toward the midline, respectively. Although the knowledge on cellular and molecular mechanisms within neural ectoderm during vertebrate neurulation is accumulating, contribution of non-neural ectoderm is not well understood. In tissue level, nonneural ectoderm intensively moves toward the dorsal side of the embryo, probably contributing to the actual closure event, and it has been shown by studies with chick embryos that explants of neural ectoderm isolated from non-neural ectoderm fail to form neural tube, demonstrating the importance of non-neural ectoderm for neural tube closure. To clarify cellular and molecular basis of non-neural ectoderm that contributes to neural tube closure, we have analyzed possible mechanisms that may generate a force causing the movements, using Xenopus laevis embryos as a vertebrate model system. We first tested a contribution of cell divisions by treating the embryos with hydroxyurea and aphidicolin (HUA), inhibitors for cell cycle, throughout neurulation. Time-lapse images and sections of fixed embryos revealed that neural tube closure of the HUA-treated embryos were indistinguishable from the untreated controls with cell divisions suppressed significantly, suggesting that cell divisions may be dispensable for the tube closure. We are currently examining other cellular mechanisms, including surface expansion, mediolateral and/or anteroposterior rearrangement, and radial intercalation of non-neural ectoderm cells, using digital scanned laser light sheet microscopy (DSLM) as one of powerful tools enabling live imaging of cell morphogenesis in neural tube closure. doi: 10.1016/j.diff.2010.09.010
P05 Mechanical force generated by leading edge mesoderm modulates collective cell polarization in axial mesoderm during Xenopus gastrulation
Yusuke Hara a,b, Makoto Suzuki a,b, Kazuaki Nagayama c, Takeo Matsumoto c, Naoto Ueno a,b a
Division of Morphogenesis, National Institute for Basic Biology, Aichi, Japan b The Graduate University for Advanced Studies (Sokendai), Kanagawa, Japan c Biomechanics Laboratory, Nagoya Institute of Technology, Aichi, Japan E-mail address: [email protected] (Y. Hara)
Gastrulation is one of the most important processes during the morphogenesis of early embryo, involving dynamic cell shape change
Contents lists available at ScienceDirect
Differentiation journal homepage: www.elsevier.com/locate/diff
Abstracts
Poster presentations
P01 Modelling of differentiation and malignant transformation in 1D and 3D culture of canine kidney epithelial MDCK cells with inducible Src activation
¨ b, A. Mannine b, Sinikka Eskeline a J. Capra a, S. Myllymak
¨ environment at + 35 1C (Toyli et al., 2010). Survivin is a highly expressed protein in almost all malignant tumours and completely missing in adult healthy tissues. It is an essential regulator of mitosis, inhibits apoptosis and promotes migration of transformed cells. Thus, survivin may serve as a possible Src oncogene target at various steps of carcinogenesis. Reference
a
Biocenter Oulu and the Institute of Diagnostics, University of Oulu, Finland b Biocenter Oulu and the Institute of Biomedicine, University of Oulu, Finland E-mail address: sinikka.eskelinen@oulu.fi (S. Eskeline)
¨ Toyli, M., Rosberg-Kulha, L., Capra, J., Vuoristo, J., Eskelinen, S., 2010. Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays. Lab. Invest. 90, 915–928. doi: 10.1016/j.diff.2010.09.007
MDCK cells expressing a temperature sensitive mutant of v-Src provide a well-suited model system to study malignant transformation. When cultivated at 40.5 1C, they behave as normal epithelial cells, whereas after a shift to 35 1C, Src tyrosine kinase is activated and the transformation process begins. Src-transformed MDCK cells grown at + 40.5 1C in 3-D Matrigel form polarized cysts with apical surface facing an internal lumen and basal surface facing the extracellular matrix (ECM). Upon Src-activation at + 35 1C these cells form lumenless spherical ¨ cysts without apico-basal polarity (Toyli et al., 2010). In order to monitor the mechanism of lumen filling in 3D culture we transfected Src-MDCK cells with GFP cadherin. These cells were cultivated in Matrigel at + 40.5 1C until they formed lumina, and subsequently monitored under a microscope adjusted at + 35 1C to follow the transformation process for up to 24 h. GFP cadherin and Hoechst stain served as markers for cell polarization and transformation. In 1–2 h the cells began to lose their apico-basal polarity and some cells were observed inside the lumen. In 6 h the luminal space was filled, but the cyst size was not increased. When incubated overnight at + 35 1C, the cells formed large irregular clusters. We conclude that the initial mechanism of filling the luminal space was cell migration. Next, we cultivated the MDCK cells on a non-adhesive surface in the presence of a function-blocking beta1-integrin antibody. In these 1D conditions both untransformed and v-Src-transformed MDCK cells formed aggregates floating in the suspension without any apico-basal polarity. Interestingly, the cells survived several days without entering apoptosis. The results show that ECM-contact and integrin signalling are crucial for formation of apico-basal axis and central lumen. However, additional signals are needed to trigger apoptosis in cells without ECM contact in suspension or trapped within the 3D cell cluster. We observed a strong expression of survivin in ts-Src MDCK cells grown in 3D
P02 Migration of human fibrosarcoma cells toward the blood vessel when transplanted into chicken embryo
Taiji Yasue, Ryosuke Tadokoro, Yoshiko Takahashi Nara Institute of Science and Technology, Nara, Japan E-mail address: [email protected] (T. Yasue)
Cell migration is one of the most important phenomena not only for development but also for cancer metastasis. When cells migrate in the body, their surrounding tissues (environment) might be influential on the migratory behavior. However, little is known about interactions between migrating cells and their environment in the actual body. We have recently established a novel experimental system in which cultured line-cells are transplanted into early chicken embryos, where their migratory behaviors are directly assessed in vivo. We have examined 12 different cell lines, including 4 non-cancer cell lines (chicken DF-1, mammalian COS-7, NIH3T3 and C2C12) and 8 human cancer cell lines (HT1080, MCF-7, MDA-MB-231, Y-1, Panc-1, A375, Mewo and A2058). These cells are manufactured in vitro to stably express EGFP prior to transplantation into the dorsal aspect of the somitic mesoderm of E2 chicken embryo. Among the 12 cell lines, HT1080 cells (highly invasive human sarcoma) are found to actively migrate to the dorsal aorta whereas the others do not.
0301-4681/$ - see front matter & 2010 Published by Elsevier Ltd. on behalf of International Society of Differentiation Join the International Society for Differentiation (www.isdifferentiation.org) doi:
S18
Abstract / Differentiation 80 (2010) S17–S63
Time-lapse imaging with confocal laser microscopy allows us to observe numerous filopodia extending from migrating cells. Such migratory behavior is dependent on the activity of matrix metalloproteinase (MMP), known to degrade extracellular matrix proteins. When overexpressed with MMP inhibitor Timp-2 or Reck, the migration has been impaired. Following the migration, HT1080 cells accumulate around the aortic vessel. Interestingly, the dorsal and ventral aspects of this vessel provide differential environment to the HT1080: in the dorsal region cells undergo intravasation (breaking the blood vessel to invade into the blood stream) whereas the ventral region allows pronounced accumulation of cells. We will discuss how cells interact with their surrounding tissues for cell migration and intravasation events. doi: 10.1016/j.diff.2010.09.008
P03 Ex vivo live imaging at high resolution to directly visualize melanin transfer from melanocytes to keratinocytes
Ryosuke Tadokoro, Kenichiro Sakai, Hidetaka Murai, Yoshiko Takahashi Graduate School of Biological Science, Nara Institute of Science and Technology, Takayama, Ikoma, Nara 630-0192, Japan E-mail address: [email protected] (R. Tadokoro) Melanin pigmentation in the skin is physiologically important to protect our body from UV irradiation. Melanocytes are known as melanin-producing cells in which melanin is synthesized in melanosomes. In the tanning processes, melanocytes extend numerous dendrites that are attached to surrounding keratinocyes, and subsequently melanin granules are transferred through melanocyte dendrites to keratinocytes. Although several models for intercellular melanin-transfer have been proposed by in vitro experiments and electron microscopic observations, the mechanisms of melanin-transfer in vivo remain controversial. We have developed time-lapse imaging techniques that directly visualize the melanin-transfer in an ex vivo cultured skin of chicken embryo, where epidermal cells and melanocytes are expected to behave in a similar way to in vivo. In this system, embryonic melanocytes are genetically and stably labeled with a membranebound EGFP using transposon-mediated gene transfer technique (Sato et al., 2007. Dev. Biol.). This manipulation directly visualizes fine structures of dendrites of differentiated melanocytes. At early stages of melanin-synthesis, melanocytes actively elongate and continuously change their shape with dendrites both extending and retracting. As melanin-synthesis proceeds, these dendrites form blebs on the plasma membrane, and subsequently they release small membrane vesicles (0.5–1 mm in diameter) to the extracellular space. Importantly, these vesicles contain melanin granules, and become incorporated into adjacent keratinocytes. Thus, melanin granules are transferred from melanocytes into keratinocytes via membrane vesicles. This is the first demonstration that the process of melanin transfer is directly visualized in the developing skin. doi: 10.1016/j.diff.2010.09.009
P04 Dissecting the contribution of non-neural ectoderm to the vertebrate neural tube closure
H. Morita a,b, N. Ueno a,b a
The Graduate University for Advanced Studies (Sokendai), Hayama, Japan b National Institute for Basic Biology, Okazaki, Japan E-mail address: [email protected] (H. Morita) Neural tube closure is one of the most prominent morphogenetic events during vertebrate development with dynamic cell shape changes and reorganization of cells. These cell behaviors are observed not only in a forming neural tube (neural ectoderm) itself but also in epidermal (non-neural) ectoderm. Cells in the neural ectoderm undergo apical constriction and mediolateral intercalation, which allow the neural tissue to form a groove along the anteroposterior axis and converge toward the midline, respectively. Although the knowledge on cellular and molecular mechanisms within neural ectoderm during vertebrate neurulation is accumulating, contribution of non-neural ectoderm is not well understood. In tissue level, nonneural ectoderm intensively moves toward the dorsal side of the embryo, probably contributing to the actual closure event, and it has been shown by studies with chick embryos that explants of neural ectoderm isolated from non-neural ectoderm fail to form neural tube, demonstrating the importance of non-neural ectoderm for neural tube closure. To clarify cellular and molecular basis of non-neural ectoderm that contributes to neural tube closure, we have analyzed possible mechanisms that may generate a force causing the movements, using Xenopus laevis embryos as a vertebrate model system. We first tested a contribution of cell divisions by treating the embryos with hydroxyurea and aphidicolin (HUA), inhibitors for cell cycle, throughout neurulation. Time-lapse images and sections of fixed embryos revealed that neural tube closure of the HUA-treated embryos were indistinguishable from the untreated controls with cell divisions suppressed significantly, suggesting that cell divisions may be dispensable for the tube closure. We are currently examining other cellular mechanisms, including surface expansion, mediolateral and/or anteroposterior rearrangement, and radial intercalation of non-neural ectoderm cells, using digital scanned laser light sheet microscopy (DSLM) as one of powerful tools enabling live imaging of cell morphogenesis in neural tube closure. doi: 10.1016/j.diff.2010.09.010
P05 Mechanical force generated by leading edge mesoderm modulates collective cell polarization in axial mesoderm during Xenopus gastrulation
Yusuke Hara a,b, Makoto Suzuki a,b, Kazuaki Nagayama c, Takeo Matsumoto c, Naoto Ueno a,b a
Division of Morphogenesis, National Institute for Basic Biology, Aichi, Japan b The Graduate University for Advanced Studies (Sokendai), Kanagawa, Japan c Biomechanics Laboratory, Nagoya Institute of Technology, Aichi, Japan E-mail address: [email protected] (Y. Hara)
Gastrulation is one of the most important processes during the morphogenesis of early embryo, involving dynamic cell shape change