Periodic Reporting for period 1 - MitoScaling (Mitotic scaling to cell size diversity during vertebrate embryonic development)
Reporting period: 2019-09-01 to 2021-08-31
Multicellular eukaryotes reproduce through the combination of a male and a female gamete, that once combined generate a one cell entity called the zygote. From this stage onwards, the zygote goes through multiple rounds of mitotic divisions to increase cell number that later will differentiate in different tissues and organs that will constitute the adult organism. Several studies have shown that human and mice early embryonic divisions are highly error prone and this could be associated with embryo malformation and potential miscarriage. Therefore, the first rounds of division of an embryo are decisive to insure its viability and it is of great interest to understand how they are regulated.
Early embryonic development is still a mysterious process, mainly because of the difficulty (technical and ethical) in obtaining early embryos from mammalian organisms, such as mice or humans. In this project we use zebrafish embryos as a eukaryotic model system that develops outside the mother and can be easily observed under the microscope. These have two particularities that are of interest in this project: first, embryos are very big (approximately half a millimeter, 10 times bigger than a cell in the human body) and second, during the first 2 hours after fertilization, zebrafish embryos go through 10 rounds of synchronous mitotic divisions without growth. This means that cells become exponentially smaller in every division cycle.
Therefore, in this project we asked the questions: how does cell division occur in an extremely big cell such as the 2 cell-stage zebrafish embryo? and which adaptation mechanisms the mitotic machinery uses to adjust to the fast decrease in cell size?
Particularly in this project, we are focused on how chromosome segregation, the movement of replicated DNA into the two new daughter cells, is controlled in order to ensure equal distribution of the genetic material. During my PhD, I showed that cultured cells have a molecular mechanism based on the phosphorylation activity of the kinase Aurora B. The kinase activity creates a phosphorylation gradient between the two sets of chromosomes during anaphase and the phosphorylation/dephosphorylation balance of specific substrates regulates the distance of chromosome separation before nuclear envelop reformation (NER). Again, culture cells have approximately 20μm in diameter whereas the 2 cell-stage embryo is 10 bigger. Our objectives are:
- Are the molecular mechanisms that control chromosome separation conserved in other systems?
- How does this mechanism adapt to a 10 times bigger cell?
- How does this mechanism changes with a fast decrease in cell size?
So far, we observed that indeed the same phosphorylation activity exist in zebrafish embryos and controls the distance of chromosome separation before DNA decondensation. We have also observed that the mechanism adjusts to the decreasing cell size, essentially the phosphorylation activity of Aurora B scales with cell size. And finally, we observed that the scaling capacity is a function of changes in chromosome velocity for the different cell sizes. We are now addressing the question of how does the velocity of chromosome segregation scales with cell size.
Early embryonic development is still a mysterious process, mainly because of the difficulty (technical and ethical) in obtaining early embryos from mammalian organisms, such as mice or humans. In this project we use zebrafish embryos as a eukaryotic model system that develops outside the mother and can be easily observed under the microscope. These have two particularities that are of interest in this project: first, embryos are very big (approximately half a millimeter, 10 times bigger than a cell in the human body) and second, during the first 2 hours after fertilization, zebrafish embryos go through 10 rounds of synchronous mitotic divisions without growth. This means that cells become exponentially smaller in every division cycle.
Therefore, in this project we asked the questions: how does cell division occur in an extremely big cell such as the 2 cell-stage zebrafish embryo? and which adaptation mechanisms the mitotic machinery uses to adjust to the fast decrease in cell size?
Particularly in this project, we are focused on how chromosome segregation, the movement of replicated DNA into the two new daughter cells, is controlled in order to ensure equal distribution of the genetic material. During my PhD, I showed that cultured cells have a molecular mechanism based on the phosphorylation activity of the kinase Aurora B. The kinase activity creates a phosphorylation gradient between the two sets of chromosomes during anaphase and the phosphorylation/dephosphorylation balance of specific substrates regulates the distance of chromosome separation before nuclear envelop reformation (NER). Again, culture cells have approximately 20μm in diameter whereas the 2 cell-stage embryo is 10 bigger. Our objectives are:
- Are the molecular mechanisms that control chromosome separation conserved in other systems?
- How does this mechanism adapt to a 10 times bigger cell?
- How does this mechanism changes with a fast decrease in cell size?
So far, we observed that indeed the same phosphorylation activity exist in zebrafish embryos and controls the distance of chromosome separation before DNA decondensation. We have also observed that the mechanism adjusts to the decreasing cell size, essentially the phosphorylation activity of Aurora B scales with cell size. And finally, we observed that the scaling capacity is a function of changes in chromosome velocity for the different cell sizes. We are now addressing the question of how does the velocity of chromosome segregation scales with cell size.
Our first goal in the project was to understand if the molecular mechanisms that control the distance of chromosome separation are conserved in other model systems. We started by imaging live zebrafish embryos under the microscope during the first 2 hours of development, which allows us to follow mitotic divisions from a 2-cell stage embryo to a 512-cell stage embryo, where cell size decreases 6x. We defined anaphase duration as the moment from anaphase onset until nuclear envelope reformation (NER) and measured both duration and distance of chromosome segregation for different cell sizes. We found that the duration of anaphase is constant for all cell sizes but the distance that chromosomes migrate to opposite poles before NER is different: bigger cells separate more their chromosomes then smaller cells.
Since the phosphorylation gradient of Aurora B has been shown to define the distance at which the nuclear envelope reforms we tested if the gradient could also scale. Indeed, the gradient scales with cell size but because the rates of phosphorylation/dephosphorylation do not scale, the gradient and NER must scale as a function of chromosome separation velocity. Finally, we measured chromosome velocity and this scales with cell size. Therefore, for the same anaphase duration chromosomes migrate longer distances because they separate faster.
Several mechanisms have been described to contribute for chromosome segregation during anaphase and interestingly different mechanisms can have different importance in different cell types. In the zebrafish embryos we have found the presence of flows in the cytoplasm that emerge specifically during anaphase and scale with cell size. We are currently addressing if theses flows can account for the scaling of chromosome velocity and constitute an adaptation mechanism for chromosome segregation in big cells.
The work performed under this grant has been presented at the following conferences:
03/2021 short talk. Conference: “Mitotic spindle: From living and synthetic systems to theory”. Online event
06/2021 poster presentation. Conference: “Physics of living systems: From molecules to tissues. Online event
Since the phosphorylation gradient of Aurora B has been shown to define the distance at which the nuclear envelope reforms we tested if the gradient could also scale. Indeed, the gradient scales with cell size but because the rates of phosphorylation/dephosphorylation do not scale, the gradient and NER must scale as a function of chromosome separation velocity. Finally, we measured chromosome velocity and this scales with cell size. Therefore, for the same anaphase duration chromosomes migrate longer distances because they separate faster.
Several mechanisms have been described to contribute for chromosome segregation during anaphase and interestingly different mechanisms can have different importance in different cell types. In the zebrafish embryos we have found the presence of flows in the cytoplasm that emerge specifically during anaphase and scale with cell size. We are currently addressing if theses flows can account for the scaling of chromosome velocity and constitute an adaptation mechanism for chromosome segregation in big cells.
The work performed under this grant has been presented at the following conferences:
03/2021 short talk. Conference: “Mitotic spindle: From living and synthetic systems to theory”. Online event
06/2021 poster presentation. Conference: “Physics of living systems: From molecules to tissues. Online event
So far, zebrafish embryos have been used to study early development, from 5 hours post fertilization (hpf) onwards. In this project, we have successfully established zebrafish embryos as model system for early embryonic divisions (0hpf-3hpf). Furthermore, we have established tools and markers to study anaphase in a living organism in its natural context, providing us with a unique set of information.
With this project we open not only the possibility to further understand early embryonic divisions but also to study other model systems where the same mitotic machinery must adapt to the different context of the tissues and organs cells are part of.
With this project we open not only the possibility to further understand early embryonic divisions but also to study other model systems where the same mitotic machinery must adapt to the different context of the tissues and organs cells are part of.