title: Imaging the onset of the segmentation clock during mouse gastrulation
creator: Falk, Henning Johannes
subject: 500
subject: 500 Natural sciences and mathematics
subject: 570
subject: 570 Life sciences
description: A fundamental requirement for embryonic development is tight spatiotemporal coordination between a steadily increasing number of cells. A notable example of highly coordinated cellular activity occurs during body axis segmentation of the vertebrate embryo. Organized oscillatory signaling activity produces regular waves of gene expression that traverse the tissue. These oscillations are thought to define the timing of segmentation and the positioning of embryonic segments, the somites. The striking rhythmicity of the process is ascribed to a molecular oscillator in undifferentiated cells, the “segmentation clock”, involving the Notch, Wnt and FGF signaling pathways in mouse. While the segmentation clock and its intercellular coordination mechanism have been studied in some detail, the start of the process, the establishment of an initial synchrony between several hundreds of presomitic cells, remains largely unknown.   I chose a live imaging approach to examine the onset of synchronous signaling oscillations in the developing embryo. To this end, I established a mounting and culture method to enable long-term multi-sample post-implantation mouse embryo imaging on a light-sheet microscope. This new setup enables routine embryo culture and imaging from early gastrulation on embryonic day 6.5 for up to two days, closely recapitulating in utero development. Using genetically encoded signaling reporters to visualize the cellular oscillation status, I was able to capture the very first observable coherent oscillations of the cyclic gene Lfng as well as the preceding dynamics. I found concurrent upregulation of Lfng expression across nascent mesoderm and the primitive streak at mid gastrulation, marking the first synchronous activity pattern of this segmentation clock gene. This expression pulse was also seen for the FGF signaling target Dusp4 but not the Wnt target Axin2. Despite Lfng being a Notch signaling target in the context of somitogenesis, I found the Lfng pulse to occur in the presence of Notch signaling inhibitor DAPT as well as in the absence of Notch transcription factor RBP-J κ and the Notch signaling core oscillator HES7. These findings argue for an initial synchronization signal independent of the Notch pathway. Quantification of early wave dynamics identified a gradual buildup of phase waves and revealed that a period gradient, which is present from the very first observable oscillation onwards, functionally underlies the establishment of waves in the gastrulating embryo.  Taken together, this study provides detailed and quantitative insight into the dynamics of the early segmentation clock in the mouse embryo, pioneering future studies on the molecular mechanism of the synchronization process. The post-implantation embryo culture and imaging method described here opens up new possibilities for the study of the gastrulation-to-organogenesis stages, which in the past have been difficult to follow in real time. Further technical developments of multi-sample mammalian embryo microscopy, which I also discuss in this work, will continue to make an important contribution to the investigation of the establishment of the segmentation clock, and beyond can help to better understand many aspects of mammalian embryonic development.
date: 2019
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
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identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/24446/10/Movie_9_Control_DAPT_RBP.avi
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identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/24446/11/Movie_10_Dusp4Lfng.avi
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identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/24446/12/Movie_11_AxinLfng.avi
identifier: DOI:10.11588/heidok.00024446
identifier: urn:nbn:de:bsz:16-heidok-244465
identifier:   Falk, Henning Johannes  (2019) Imaging the onset of the segmentation clock during mouse gastrulation.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/24446/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng