Does a morphogen gradient slow the segmentation clock? A live fluorescent reporter based biophysical investigation
Final Report Abstract
The regularly-spaced units of the vertebrate backbone are formed during embryogenesis. The precursors of each vertebral body, termed somites, are formed rhythmically and sequentially as the embryo’s body extends. Each newly forming somite buds off from the anterior end of the presomitic mesoderm, a population of progenitor cells. When this process does not occur properly, the embryo develops with congenital scoliosis, a permanent malformation of the backbone. The cells of the presomitic mesoderm contain genetic oscillators that synchronize locally with their neighbors. Waves of oscillating gene expression sweep from the tail end of the presomitic mesoderm to the anterior, where they arrest. The position and timing of the arrest mark the formation of the newly forming somite. This system is known as the segmentation clock. The waves are thought to arise at a cellular level from the gradual slowing of their oscillations, and then their arrest. However, it is not understood how this slowing and arrest is controlled. Some clues have come from signaling molecules that are released from the tail end and can diffuse through the presomitic mesoderm, creating a gradient of signal. Alterations in these molecules can change the position where the oscillators arrest and where the new somite boundary forms, creating segments of different lengths. This project aimed to test whether the diffusible gradients directly controlled the slowing and arrest of oscillating cells. We developed transgenes and microscopy set-up that allowed us to observe the gradient of the Wnt8 signaling molecule in a living embryo. We wrote software that allowed us to follow the genetic oscillations at a tissue level and to extract the phase of the oscillations. We observed that the period of segmentation is influenced by the wave-patterns; both the movement of the anterior end of the tissue into the waves and the gradual change in the wave patterns over longer developmental times contributes to the period measured at the anterior end. This surprising result indicates that the wave pattern is itself an active part of the timing mechanism, and provides new relevance to the slowing and arrest of individual cells. We used a method to transiently reduce the level of Wnt signaling, by temporarily blocking the Wnt receptor, and observed changes to the position of oscillator arrest as well as a change in the segment length. We were not able to directly compare the local level of Wnt8 and the oscillation frequency. However, we observed that the time taken for the reduced Wnt effect to move across the tissue correlated simply with the time taken for a cell to move across the tissue. This surprising result suggests that the information about when the cell should arrest oscillations is set in the very tailend of the tissue, and may be carried intrinsically within the cell thereafter. In summary, the work emerging from this project has helped to change our understanding of the wave patterns that characterize the vertebrae segmentation clock. Future work inspired by this project will include attempts to observe the slowing and arrest program in individual cells isolated from the segmentation clock, a novel assay that should allow us to cleanly dissect intrinsic and extrinsic influences on the frequency of oscillations.
Publications
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A Doppler Effect in Embryonic Pattern Formation. Science. 2014 July 11;345, 222-225
Soroldoni D, Jörg DJ, Morelli LG, Richmond DL, Schindelin J, Jülicher F, Oates, AC
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Chevron formation of the zebrafish muscle segments. Journal of Experimental Biology. 2014 Sept; 217, 3870-3882
Rost F, Eugster C, Schröter C, Oates AC, Brusch L
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Wnt-regulated dynamics of positional information in vertebrate segmentation. Development. 2014 Mar;141(6):1381-91
Bajard L, Morelli LG, Ares S, Pecreaux J, Jülicher F, Oates AC
