- Email: [email protected]
P04. Dissecting the contribution of non-neural ectoderm to the vertebrate neural tube closure
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
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