Movies Notch Neural Zebrafish. A merge of a and b is shown in c. Colcemide Sigma was prepared as a 0. Injections of fluorescently labeled RNA were carried out at the 8—64 cell stage to ensure a mosaic expression pattern. BDM was added at the beginning of the movies and not removed. Mitosis in the neural tube.
B Images from Movie S The pink dot labels the nucleus that migrates apically. Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. D Images of Movies S7 and S8. It is possible that most stochastic movements derive from the nudges of neighboring cells. However, staining of stable MTs with anti-acetylated tubulin shows a striking difference between centrin2 morphants and the control embryos.
Therefore, it is a good candidate to investigate whether actomyosin networks play a role in IKNM. Acetylated tubulin stains basal bodies asterisksbut no stabilized MTs are observed spanning the epithelium a.
A closer look at actomyosin forces in individual cells might help to answer these questions. To explore this possibility, we depleted cells of all MTs by adding Colcemide.
These results raise questions about how the movements of IKNM correlate with the particular phases interkinftic the cell cycle, and whether the movements are really monotonic, as Sauer proposed. For the stochastic phases of movement, the velocity histogram of Colcemide-treated nhclear is slightly broader, perhaps because of a lack of mechanical constraints of a MT cytoskeleton.
Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed.
Injections of fluorescently labeled RNA were carried out at the 8—64 cell stage to ensure a mosaic expression pattern.
Heartbeat was still observed during the analysis time of the movies. D Stochastic motion velocity distribution for pooled nuclear movement in control embryos with a normal fit. The four-dimensional data thus obtained was processed and analyzed with the Volocity software Improvision, Coventry, UK. A Three-dimensional reconstruction of MTs that envelope a nucleus is shown c. To explore whether this effect was due to the slightly thinner epithelium in centrin morphants, we analyzed the consequences of centrin2 knockdown in mosaic epithelia.
Supplemental Data Document S1.
Though we have identified actomyosin as the prime mover of IKNM, many questions remain. An epithelium is a sheet of polarized cells that extend from an apical surface to a basal lamina. The embryos is at 28 hpf and the movie plays at ten frames per second, covering 6 min at 6 s intervals.
We incorporated a threshold based on simulated one-dimensional random walk analyses to control for false positives Saxton and Jacobson, Author information Article notes Copyright and License information Knterkinetic.
D Images from Movie S BDM was added at the beginning of the movie and not removed. Colcemide was added at the beginning of the movie and not removed. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
EB3 starts to disappear min after addition and cannot be seen moving into an apicobasal direction min and more after drug addition. E Velocity distribution of nuclear movements in DNpexpressing cells curves show nonparametric density estimates scaled in proportion to the number of nuclear displacements representing the motion type; persistent motion curves are enlarged by a factor of ten. Immunolabeling, electron microscopy, and live imaging studies reveal oriented MTs that extend from the apically located centrosome along the apicobasal axis of neuroepithelial cells, passing within molecular distances of the nucleus.
Pulling usually requires centrosome-microtubule collaboration in proximity to the nucleus. Between drinks the partygoers jostle around the room, but when they get thirsty again, they return to the bar in a fast and directed manner.
Shortly after drug addition, spindles are still formed and cells divide arrow. BDM was added at the beginning of the movies and not removed.
All secondary antibodies were used in a 1: Adams for encouraging him to pursue the collaboration. Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells. Centrosome composition and microtubule anchoring mechanisms.
Pink dot labels nucleus that migrates apically. The movie plays at nine frames per jigration, covering 3 hr 48 min of development at 4 min intervals.
Stages of embryonic development of the zebrafish. See the Supplemental Experimental Procedures for details. Arrows label activated Myosin Light Chain dots that move apically basal of the nucleus.
C Velocity distribution of pooled nuclear movements in control embryos curves show nonparametric density estimates scaled in proportion to the number of nuclear displacements representing the motion type, persistent motion curves are enlarged by a factor of ten.
Link2 and William A. The nucleus moves from interkinetjc cell apex toward the base and then returns to the apex when it then divides. Positions were measured at time intervals of 2, or 5 min, for periods of 1 to 13 hr. Nuclei still undergo rapid apical migration before forming a metaphase plate as seen in the control cells. Centrin2-MOs were injected at a concentration of 2—3 ng.
Starting at 28—32 hours postfertilization hpfwe tracked the location of nuclei, marked by H2B-RFP in the developing zebrafish retina Figures 1 A and 1Ba.
The embryo is 28 hpf at the start of the movie. In addition, not a single event of rapid apical migration could be found in all the BDM and Blebbistatin movies.
Statistical Analysis of Nuclear Motion Apicobasal nuclear position trajectories were generated where nuclear position at each time point p i is defined as the straight line distance between the point of cell contact with the basal lamina, and the basal extent of the nucleus. Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration IKNM.
No obvious intensity changes can be observed. This construct enables focal labeling of actin and characterization of cytosolic actin mobilization.
Nuclei labeled by H2B-RFP move in apical and basal direction in centrin-2 morphants, indicated by white lines between false color red and yellow nuclei and the basal membrane of the epithelium.