Presentation on theme: "Tribolium Segmentation: What’s Going On In the Posterior Growth Zone? Ayaki Nakamoto 1, S.D. Hester 1, S.J. Constantinou 2, W.G. Blaine 2, A.B. Tewksbury."— Presentation transcript:
Tribolium Segmentation: What’s Going On In the Posterior Growth Zone? Ayaki Nakamoto 1, S.D. Hester 1, S.J. Constantinou 2, W.G. Blaine 2, A.B. Tewksbury 2, M.T. Matei 1, Anjin Singh 1, L.M. Nagy 1, T.A. Williams 2 * 1 Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 2 Department of Biology, Trinity College, Hartford, CT The Tribolium segmentation “clock” doesn’t tick at a regular pace Summary Abstract ´ This research is supported by a grant from NSF-BSF to TAW and LMN. Sequential segmentation depends on posterior elongation of the growing embryo. In malacostracan crustaceans, posterior growth originates from posterior stem cells (12). In centipedes, it is known that the posterior growth zone is replenished through migration of cells from outside the growth zone (13). For most arthropod species the behavior of cells in the posterior that gives rise to sequential segmentation is not known, but any number of models shown above (or combinations thereof) are conceivable. Even in the best studied sequentially segmenting arthropod, Tribolium castaneum, how the embryo elongates is not known. While cell division can be detected throughout the germband, no one has established a significant role for cell division in elongation. More recently, high-resolution live imaging has shown a role for cell movements and cell rearrangements in Tribolium elongation (11,14,15), but the relative contributions of cell rearrangement and cell division are not known. Stem cells Posterior Proliferation Specified Pre-pattern Migration Generalized Proliferation Cell rearrangements Elongation can be modeled in the absence of cell division. We injected DMNB caged fluorescein (Invitrogen) and Fluoro Ruby into syncytial stage embryos. The dye was uncaged in small clusters of cells with UV irradiation during the blastoderm stage. Embryos were allowed to develop varying amounts of time, fixed and stained with engrailed, DAPI and anti-fluorescein (to increase the signal from the injected dye) to visualize the resultant clone in later stage embryos. We traced clones of cells marked in the blastoderm – in both wild type and eve knock-down embryos. Posterior clones give rise to more segments and elongate disproportionately compared to anterior clones (Panel A and B). All clones divide 2.4 times on average- regardless of their initial position (Panel C). Labeled ectodermal clones rarely mix with non-labeled cells (Panel E) Instead, cells within a clone maintain cell contact along the length of the clone. Because the pair-rule gene eve is required for cell rearrangements that drive germband elongation in Drosophila (17), we labeled blastoderm clones in embryos injected with dseve RNA in those early blastoderm (Panel F). Clones at 60-80% EL did not differ from controls in amount of cell division (Panel D) but failed to elongate along the AP axis. Overall germband area does not increase significantly – a high degree of posterior proliferation is not needed Posterior blastoderm clones behave differently, and don’t divide more than anterior clones Adding segments all at once or one by one. Segmented animals have been very successful evolutionarily and are found in major clades as phylogenetically distant as vertebrates and arthropods. Developing body segments during embryogenesis has typically been viewed as a regular process, relying on a segmentation clock to pattern budding segments and high levels of mitosis in the posterior to drive axial elongation. Our analysis of the flour beetle, Tribolium, throughout the entire period of segment addition, demonstrates that segmentation is an irregular process. Segments are added at varying rates. Furthermore, much of the rapid segment addition is driven by unexpectedly high rates of cell rearrangement, demonstrated by the differential behavior of marked anterior and posterior blastoderm cells. Although our computer model of the posterior growth zone without any cell division successfully mimics the pulse of rapid segmentation late in germband elongation, pharmacological experiments with inhibitors of cell division suggest that cell division is nonetheless required for both segmentation and elongation. This work was funded by NSF. Segments are made nearly simultaneously in Drosophila melanogaster. But nearly all other arthropods, vertebrates and add their segments sequentially. Sequential segmentation is best studied in vertebrates, where it relies on regulatory interactions between a segmentation clock that oscillates in the posterior growth zone and a posteriorly moving wavefront that stabilizes the readout of the clock, leading to segmentation(1,2,3). The spatial pattern of segmentation derives from a temporal periodic pattern of oscillating gene expression – the “segmentation clock” in the growth zone. The time it takes for a segment formation is species specific (4) – e.g. 30 min in zebrafish, 120 min in mouse, but within an individual the time to from a segment is relatively constant. Support for a segmentation clock in arthropods is growing (5-11), but species specific variation in the time to form a segment are not well characterized. To understand how the Tribolium embryo elongates, we quantified dimensional changes in the developing embryo. The early germband is short and broad, and through a combination of elongation and mesoderm ingression, narrows as segments are added. During this time the length of the germband increase 137% while the area only increase 41% (Fig. 1A). As segments are added, the growth zone – the region behind the last EN stripe – gets smaller (Fig 1B). The length- but not the width- of the most recently added segment stays the same- perhaps indicative of a mechanism to measure the distance from an anterior position or the immediately anterior segment. References We analyzed the rate of segment addition throughout the period of segmentation. We find a striking variability in rates: segments are added in rapid bursts followed by slower rates of addition (Panel A). At 16h AEL segment addition slows until 18h AEL. Then there is a burst of 5-6 segments added over 2h, almost twice as many as during the 12-14h and 14- 16h intervals and five times as many as during the 16- 18h interval (Panel C). The abdominal segments formed during the rapid burst of segmentation are the same segments to which the dramatically elongated posterior clones contribute. Can posterior elongation occur without cell division? We built a computational model using the cell-based Compu- Cell3D Simulation softwarev(16). The initial conditions (Panel 1A,B) matched dimension of the 18hr Tribolium embryo. Cells in the model do not divide. They have both an intrinsic random motility and a position-dependent directional movements based on measurements from live-imaging. Simulations that produce fie additional segments (Fig. 4C) produce elongated germband and reduced growth zones similar to the 20hr Tribolium embryo (Fig. 4D). This simulation supports the notion that posteriorly localized division is not required to form these abdominal segments. The Tribolium segmentation clock ‘ticks’ with a varying rate. Segmentation has phases of variable periodicity during which segments are added at consistently different rates. The growth zone of Tribolium diminishes over time and changes in the overall surface area of the germband do not support a significant role for high rates of proliferation in the posterior growth zone. A cell based computational model of the elongating germband during the rapid segmentation successfully reproduces elongation through cell rearrangement in the absence of cell division. During the rapid phase of posterior segment addition, elongation is driven by higher rates of cell rearrangement. Temporal variation coincides with the switch from forming thoracic to forming abdominal segments. Unlike current steady-state models of sequential segmentation, our results indicate a more complex phenomenon in which cell behaviors are dynamic and variable, corresponding to differences in segmentation rate that give rise to morphologically distinct regions of the embryo. 1.Aulehla, A. & Herrmann, B. G. Segmentation in vertebrates: clock and gradient finally joined. Genes Dev. 18, 2060–2067 (2004); 2.Ozbudak, E. M. & Pourquié, O. The vertebrate segmentation clock: the tip of the iceberg. Curr. Opin. Genet. Dev. 18, 317–323 (2008); 3. Oates, A. C., Morelli, L. G. & Ares, S. 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