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First published online 25 July 2007
doi: 10.1242/dev.004911

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Oriented cell divisions in the extending germband of Drosophila

Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Cell divisions during the fast phase of GBE are predominantly oriented along the anteroposterior axis. (A) Side-view diagram illustrating the process of germband elongation (GBE). Images (C-F) were taken from the dorsal side of the embryo (eye icon). Notice how the posterior tip of the germband (red) moves towards the anterior of the embryo (left side). (B) Cell-division angles were measured relative to the ventral midline. (C-F) Cell divisions during the fast and slow phases of GBE. Examples of snapshots taken after 5 minutes (C) and 10 minutes (D) (during the fast phase), and at 45 minutes (E) and 50 minutes (F) (during the slow phase) are shown. The time when the pole cells become visible on the dorsal side was arbitrarily taken as t=0. Note how cell divisions tend to be longitudinally oriented during the fast phase (white lines). (G,H) Quantification of cell division angles during the two phases of GBE. The fast phase occurs during the first 25 minutes, whereas the slow phase takes place during the subsequent 70 minutes. A total of 50 and 100 cell divisions were counted (per embryo) for the fast and slow phases, respectively. Each bar represents the average obtained from five embryos. The standard error is also shown. Longitudinal divisions are predominant during the fast phase of GBE (white lines in D). A, anterior; P, posterior.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Quantification of GBE in wild-type and mutant backgrounds. Progression of the posterior tip of the germband in wild type (wt, A-D), and in string (E-H) and eve (I-L) mutants. Representative frames are shown at 5 (A,E,I), 15 (B,F,J), 25 (C,G,K) and 180 (D,H,L) minutes. (M-P) Quantification of germband elongation (GBE) progression. This was plotted as a percentage of egg length progressed over time. The fast phase occurs during the first 25 minutes, whereas the slow phase takes place during the subsequent 70 minutes. The average from five embryos is shown (with standard error). Notice that, in string mutants (N) (cell divisions are absent), the germband elongates more slowly than in wild-type embryos (M). Total elongation is less in eve embryos than in string mutants or in wild-type embryos (P). (P) Composite of M, N and O, with the error bars removed for clarity. (Q) Average velocity of the tip of the germband in the three genetic backgrounds during the first 45 minutes of extension. Fitting curves were drawn for a better view of the data.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Local tissue deformation in wild-type, eve and string embryos during the fast phase of germband extension.(A,B) Individual nuclei (and their progeny) at the posterior of a wild-type embryo were tracked during elongation. No intercalation was detected during this period despite clear tissue elongation. (C-H) In order to assess tissue deformation, a group of 25 nuclei in wild type (C,D), eve mutants (E,F) and string mutants (G,H) was outlined before (7 minutes after the posterior tip came into view; C,E,G) and after (13 minutes after the posterior tip came into view; D,F,H) a cluster of cell divisions. Deformation of the outline gives a visual indication of tissue deformation. To obtain a more quantitative assessment, a measure of the aspect ratio (AR) of the outlines was devised (as described in I). Overall, the data suggest that tissue elongation in this region of the germband is reduced in eve mutants and is nearly absent in string-deficient embryos.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Loss of longitudinally oriented cell divisions in segmentation mutants. (A-D) Pattern of cell divisions in an eve mutant embryo at 7 (A), 10 (B), 70 (C) and 75 (D) minutes after the onset of elongation. (E,F) Quantification of cell-division angles during 0-25 minutes. Out of the 260 cell divisions assessed (from five embryos), no significant longitudinal bias can be seen (E). For this data set, cell divisions were separately analysed in the medial (blue) and lateral (red) half of each hemisegment (F). A total of 130 mitoses were assessed for each domain. Some longitudinal bias can be seen for divisions occurring in the medial region (blue in F). Mild compensatory transversal bias in the lateral domain (red) might explain the lack of overall bias seen in E. (G) Quantification of cell division orientation during the 26-96 minute time period. Here, 150 divisions from five embryos were assessed. As in wild type, no longitudinal bias can be seen. (H-K) Orientation of cell divisions in embryos laid by bicoid nanos torso-like (BNT) females. Because these embryos do not undergo germband extension, the posterior region was observed from the ventral side of the egg. The portion of the embryo within view is shown as a white outline in the bottom right corner, with the double line indicating the ventral furrow. The first post-blastoderm divisions appear random in orientation (K; 150 divisions were assessed in three embryos) and cause an isotropic increase in tissue size (I,J). This increase reverts as other regions of the embryo undergo divisions, giving an impression of pulsatile behaviour in time-lapse recordings (see Movie 5 in the supplementary material).

View larger version (40K): [in this window] [in a new window]   Fig. 5. A PCP-specific mutation in dsh does not affect the orientation of cell divisions. In the same genetic background, wing hairs appear dishevelled, indicating a PCP defect (A). Nevertheless, denticle orientation appears normal in first instar larvae (B). (C,D) Orientation of cell divisions in dsh-deficient embryos during germband extension (GBE). Data for five embryos are shown with average and standard error. For each embryo, 50 and 100 (randomly chosen) divisions were counted for the fast (C) and slow (D) phases, respectively. Longitudinal divisions are predominant during the fast phase of GBE. As in wild-type embryos, a majority of fast-phase mitoses are oriented longitudinally in dsh embryos. For each embryo, we calculated an index of longitudinal bias as the absolute slope of the line relating the angle of division to the proportion of cells dividing along that angle (obtained from linear regression). We then compared the value of this index for five dsh and five wild-type embryos and found no statistically significant difference between the two groups (t-test, P>0.05). We conclude, therefore, that the orientation of cell divisions is unaffected in dsh mutant embryos.