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Live imaging-based model selection reveals periodic regulation of the stochastic G1/S phase transition in vertebrate axial development.

Sugiyama M, Saitou T, Kurokawa H, Sakaue-Sawano A, Imamura T, Miyawaki A, Iimura T - PLoS Comput. Biol. (2014)

Bottom Line: This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells.To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data.This approach may have implications for the characterization of the pathophysiological tissue growth mode.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

ABSTRACT
In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.

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Progressive mode of the stochastic G1/S transition in the notochordal cells during embryonic body elongation.(A) Fucci (fluorescent ubiquitination-based cell cycle indicator) labels nuclei of G1 phase cells red (orange) and those of cells in the S/G2/M phase green. During the cell cycle of cells expressing Fucci, two chimeric proteins, mKO2-zCdt1 and mAG-zGeminin, reciprocally accumulate in the G1 and S/G2/M phases [48], [49] (see [48] for detailed molecular construction). (B) Cecyil (cell cycle illuminated: a zebrafish line producing zFucci) embryos at the 17 somite stage. A Z-stacking lateral view of optical sections. The anterior view is to the left, the posterior is to the right. (C) A mid-sagittal optical section of a fixed Cecyil embryo at 22 hpf counterstained with phalloidin Alexa Fluor 647. (D, E) Mid-sagittal optical section of a fixed Cecyil embryo at 18 hpf. A DIC (differential integrative phase contrast) image (D) and fluorescence image (E) are shown. The posterior-most green cell (PGC) and anterior-most red cell (ARC) in the upper and lower notochordal cells are indicated by green and red arrowheads, respectively. The posterior tip of the precursor pool of the notochord and the last formed somitic border are marked by sky blue and blue arrowheads, respectively. (F–K) Mid-sagittal optical sections of a developing Cecyil embryo. DIC images obtained at three different time points (F–H) and their corresponding fluorescence images are shown (I–K). The colored arrowheads indicate the same structures of cells as shown in D and E. The white arrow indicates a single cell whose cell cycle phase was transitioning from G1 (I) to S (J and K). Embryonic stages are indicated by somite stages (s.s.). (L) The temporal changes in the red and green fluorescence intensity of the cell indicated by the white arrow in G–H are plotted. The time course of the live imaging observations and embryonic stages is indicated in minutes and somite stages (s.s.), respectively. The vertical broken lines indicate the observation times corresponding to the images in g–i. The crossing time point of the red and green lines is indicated by the yellow point (115 min, in 18 somite stages). This time was selected as the time at which the G1/S transition occurred (see Methods). White scale bars: 100 µm. Anterior to the left, posterior to the right. Dorsal to the top, ventral to the bottom. s.s, somite stage.
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pcbi-1003957-g001: Progressive mode of the stochastic G1/S transition in the notochordal cells during embryonic body elongation.(A) Fucci (fluorescent ubiquitination-based cell cycle indicator) labels nuclei of G1 phase cells red (orange) and those of cells in the S/G2/M phase green. During the cell cycle of cells expressing Fucci, two chimeric proteins, mKO2-zCdt1 and mAG-zGeminin, reciprocally accumulate in the G1 and S/G2/M phases [48], [49] (see [48] for detailed molecular construction). (B) Cecyil (cell cycle illuminated: a zebrafish line producing zFucci) embryos at the 17 somite stage. A Z-stacking lateral view of optical sections. The anterior view is to the left, the posterior is to the right. (C) A mid-sagittal optical section of a fixed Cecyil embryo at 22 hpf counterstained with phalloidin Alexa Fluor 647. (D, E) Mid-sagittal optical section of a fixed Cecyil embryo at 18 hpf. A DIC (differential integrative phase contrast) image (D) and fluorescence image (E) are shown. The posterior-most green cell (PGC) and anterior-most red cell (ARC) in the upper and lower notochordal cells are indicated by green and red arrowheads, respectively. The posterior tip of the precursor pool of the notochord and the last formed somitic border are marked by sky blue and blue arrowheads, respectively. (F–K) Mid-sagittal optical sections of a developing Cecyil embryo. DIC images obtained at three different time points (F–H) and their corresponding fluorescence images are shown (I–K). The colored arrowheads indicate the same structures of cells as shown in D and E. The white arrow indicates a single cell whose cell cycle phase was transitioning from G1 (I) to S (J and K). Embryonic stages are indicated by somite stages (s.s.). (L) The temporal changes in the red and green fluorescence intensity of the cell indicated by the white arrow in G–H are plotted. The time course of the live imaging observations and embryonic stages is indicated in minutes and somite stages (s.s.), respectively. The vertical broken lines indicate the observation times corresponding to the images in g–i. The crossing time point of the red and green lines is indicated by the yellow point (115 min, in 18 somite stages). This time was selected as the time at which the G1/S transition occurred (see Methods). White scale bars: 100 µm. Anterior to the left, posterior to the right. Dorsal to the top, ventral to the bottom. s.s, somite stage.

Mentions: Generally, during in vivo tissue development, biochemical phenomena are intrinsically associated with stochasticity, in which fluctuations in cellular responses are observed in populations of cells exposed to the same environmental conditions [38], [44]–[47]. In multicellular organism development, heterogeneous cellular behavior, such as the G1/S transition, possibly blinds regulatory events. Therefore, it is relevant to develop novel approaches to numerically estimate noise strength or the probability of a stochastic cellular response in multicellular organisms, which would unveil a regulatory mode of tissue growth associated with morphogenesis and patterning. Our research group previously reported the findings of whole-view imaging of cell cycle progression during embryonic development obtained using fluorescence live imaging of Cecyil, a zebrafish line that genetically carries a fluorescence cell cycle indicator, Fucci [48]. The Fucci system applies ubiquitin-based cell cycle phase-specific oscillators that are fused to distinct colored fluorescence proteins [49], which demarcates cell nuclei in the G1 and S/G2/M phases in red and green, respectively (Figures 1A, B). By taking advantage of the Fucci system, this study aimed to establish a method to clarify the regulatory mode of cell cycle progression veiled in the stochastic behavior of developing tissue. Quantitative analyses of our live imaging data combined with mathematical modeling demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion with doubled periodicity of neighboring paraxial mesoderm segmentation. The results of our analyses are expected to benefit the characterization of the regulatory mode of tissue growth, in both physiological and pathological development, such as that involving tumor formation.


Live imaging-based model selection reveals periodic regulation of the stochastic G1/S phase transition in vertebrate axial development.

Sugiyama M, Saitou T, Kurokawa H, Sakaue-Sawano A, Imamura T, Miyawaki A, Iimura T - PLoS Comput. Biol. (2014)

Progressive mode of the stochastic G1/S transition in the notochordal cells during embryonic body elongation.(A) Fucci (fluorescent ubiquitination-based cell cycle indicator) labels nuclei of G1 phase cells red (orange) and those of cells in the S/G2/M phase green. During the cell cycle of cells expressing Fucci, two chimeric proteins, mKO2-zCdt1 and mAG-zGeminin, reciprocally accumulate in the G1 and S/G2/M phases [48], [49] (see [48] for detailed molecular construction). (B) Cecyil (cell cycle illuminated: a zebrafish line producing zFucci) embryos at the 17 somite stage. A Z-stacking lateral view of optical sections. The anterior view is to the left, the posterior is to the right. (C) A mid-sagittal optical section of a fixed Cecyil embryo at 22 hpf counterstained with phalloidin Alexa Fluor 647. (D, E) Mid-sagittal optical section of a fixed Cecyil embryo at 18 hpf. A DIC (differential integrative phase contrast) image (D) and fluorescence image (E) are shown. The posterior-most green cell (PGC) and anterior-most red cell (ARC) in the upper and lower notochordal cells are indicated by green and red arrowheads, respectively. The posterior tip of the precursor pool of the notochord and the last formed somitic border are marked by sky blue and blue arrowheads, respectively. (F–K) Mid-sagittal optical sections of a developing Cecyil embryo. DIC images obtained at three different time points (F–H) and their corresponding fluorescence images are shown (I–K). The colored arrowheads indicate the same structures of cells as shown in D and E. The white arrow indicates a single cell whose cell cycle phase was transitioning from G1 (I) to S (J and K). Embryonic stages are indicated by somite stages (s.s.). (L) The temporal changes in the red and green fluorescence intensity of the cell indicated by the white arrow in G–H are plotted. The time course of the live imaging observations and embryonic stages is indicated in minutes and somite stages (s.s.), respectively. The vertical broken lines indicate the observation times corresponding to the images in g–i. The crossing time point of the red and green lines is indicated by the yellow point (115 min, in 18 somite stages). This time was selected as the time at which the G1/S transition occurred (see Methods). White scale bars: 100 µm. Anterior to the left, posterior to the right. Dorsal to the top, ventral to the bottom. s.s, somite stage.
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pcbi-1003957-g001: Progressive mode of the stochastic G1/S transition in the notochordal cells during embryonic body elongation.(A) Fucci (fluorescent ubiquitination-based cell cycle indicator) labels nuclei of G1 phase cells red (orange) and those of cells in the S/G2/M phase green. During the cell cycle of cells expressing Fucci, two chimeric proteins, mKO2-zCdt1 and mAG-zGeminin, reciprocally accumulate in the G1 and S/G2/M phases [48], [49] (see [48] for detailed molecular construction). (B) Cecyil (cell cycle illuminated: a zebrafish line producing zFucci) embryos at the 17 somite stage. A Z-stacking lateral view of optical sections. The anterior view is to the left, the posterior is to the right. (C) A mid-sagittal optical section of a fixed Cecyil embryo at 22 hpf counterstained with phalloidin Alexa Fluor 647. (D, E) Mid-sagittal optical section of a fixed Cecyil embryo at 18 hpf. A DIC (differential integrative phase contrast) image (D) and fluorescence image (E) are shown. The posterior-most green cell (PGC) and anterior-most red cell (ARC) in the upper and lower notochordal cells are indicated by green and red arrowheads, respectively. The posterior tip of the precursor pool of the notochord and the last formed somitic border are marked by sky blue and blue arrowheads, respectively. (F–K) Mid-sagittal optical sections of a developing Cecyil embryo. DIC images obtained at three different time points (F–H) and their corresponding fluorescence images are shown (I–K). The colored arrowheads indicate the same structures of cells as shown in D and E. The white arrow indicates a single cell whose cell cycle phase was transitioning from G1 (I) to S (J and K). Embryonic stages are indicated by somite stages (s.s.). (L) The temporal changes in the red and green fluorescence intensity of the cell indicated by the white arrow in G–H are plotted. The time course of the live imaging observations and embryonic stages is indicated in minutes and somite stages (s.s.), respectively. The vertical broken lines indicate the observation times corresponding to the images in g–i. The crossing time point of the red and green lines is indicated by the yellow point (115 min, in 18 somite stages). This time was selected as the time at which the G1/S transition occurred (see Methods). White scale bars: 100 µm. Anterior to the left, posterior to the right. Dorsal to the top, ventral to the bottom. s.s, somite stage.
Mentions: Generally, during in vivo tissue development, biochemical phenomena are intrinsically associated with stochasticity, in which fluctuations in cellular responses are observed in populations of cells exposed to the same environmental conditions [38], [44]–[47]. In multicellular organism development, heterogeneous cellular behavior, such as the G1/S transition, possibly blinds regulatory events. Therefore, it is relevant to develop novel approaches to numerically estimate noise strength or the probability of a stochastic cellular response in multicellular organisms, which would unveil a regulatory mode of tissue growth associated with morphogenesis and patterning. Our research group previously reported the findings of whole-view imaging of cell cycle progression during embryonic development obtained using fluorescence live imaging of Cecyil, a zebrafish line that genetically carries a fluorescence cell cycle indicator, Fucci [48]. The Fucci system applies ubiquitin-based cell cycle phase-specific oscillators that are fused to distinct colored fluorescence proteins [49], which demarcates cell nuclei in the G1 and S/G2/M phases in red and green, respectively (Figures 1A, B). By taking advantage of the Fucci system, this study aimed to establish a method to clarify the regulatory mode of cell cycle progression veiled in the stochastic behavior of developing tissue. Quantitative analyses of our live imaging data combined with mathematical modeling demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion with doubled periodicity of neighboring paraxial mesoderm segmentation. The results of our analyses are expected to benefit the characterization of the regulatory mode of tissue growth, in both physiological and pathological development, such as that involving tumor formation.

Bottom Line: This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells.To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data.This approach may have implications for the characterization of the pathophysiological tissue growth mode.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

ABSTRACT
In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.

Show MeSH
Related in: MedlinePlus