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First published online 17 October 2007
doi: 10.1242/dev.005389

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Photoactivatable GFP resolves Drosophila mesoderm migration behaviour

The ARC Special Research Centre for the Molecular Genetics of Development and Molecular Genetics and Evolution Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT, 2601, Australia.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Models for mesoderm spreading in Drosophila. (A) Following invagination, the mesoderm forms an epithelial tube. (B) The cells then undergo an EMT and divide once. (C) The mesodermal cells then collapse down onto the ectoderm and begin to spread out. We notionally divide cells into outer cells adjacent to the ectoderm (grey) and inner cells (white). Three possible cellular mechanisms for spreading are depicted [adapted from Wilson and Leptin (Wilson and Leptin, 2000)]. In the Chemotaxis model, a chemoattractant emanating from the dorsal part of the ectoderm (red) attracts mesodermal cells dorsally. In the Differential Affinity model mesodermal cells have more affinity for the ectoderm (blue) than for each other, and seek to maximise their contact with the ectoderm. In this model, activation of the FGF receptor Htl would simply impart a degree of motility to cells allowing inner cells to move over, and in between, existing outer cells until they were able to find contact with the ectoderm. In the Convergent Extension model, inner and outer cells move towards each other (arrows) and intercalate, resulting in a net, lateral spreading of the tissue. (D) Eventually the mesoderm forms a single layer of cells.

View larger version (66K): [in this window] [in a new window]   Fig. 2. Photoactivation of PAGFP-Tub84D-expressing embryos. Photoactivation in Drosophila embryos derived from cogGAL4VP16/+;NGT40/+;nanos-GAL4VP16/UASp-PAGFP-Tub84D females crossed to UASp-PAGFP-Tub84D homozygous males. (A) Photoactivation using Hg 405/20 nm light with a 60x objective. Post-activation fluorescence is approximately proportional to exposure time up to 30 seconds. (B) Photoactivation using UV confocal laser light (both 351 and 364 nm) and a 63x objective. Fluorescence is approximately proportional to zoom level up to 16x. (C) Photoactivation is possible with 351 nm UV laser light, stronger with 364 nm light, and strongest with both. (D) UV laser activation of a patch of epidermal cells. PAGFP-Tub is cytoplasmic, excluded from nuclei during interphase, and correctly localises to spindle poles (arrowheads) and mitotic spindle (arrow). (E) A gastrulating embryo after 60 seconds photoactivation using Hg 405/20 nm light. (F,G) z-series reconstruction (F) and z-projection (G) of same embryo imaged using confocal microscopy 15 minutes later, showing that the entire mesoderm is strongly labelled and easily distinguishable from the ectoderm. Scale bars: 10 µm in D; 20 µm in F-G. e, ectoderm; m, mesoderm.

View larger version (131K): [in this window] [in a new window]   Fig. 3. Overview of mesoderm development in Drosophila. Typical time-lapse sequence of an embryo with three to four segments of photoactivated mesoderm showing cells undergoing two divisions as they spread out into a monolayer (see Movie 1 in the supplementary material). (A) At 15 minutes post-gastrulation, the mesoderm shows a medial seam (arrowhead), indicating that the invaginated mesoderm is still arranged as an epithelial tube. (B) By 33 minutes, the EMT has occurred and the cells undergo a synchronous division, as evidenced by the appearance of mitotic spindle poles (arrowheads) and spindles (arrows). (C) At 1:03, the cells have re-entered interphase and are migrating out over the ectoderm. (D) At 1:19, the second division is in progress (arrows indicate spindles). During this division the mesoderm rapidly extends laterally. (E,F) By 2:15 the monolayer has been achieved and the embryo (E) shows a similar segmentally repeated striped pattern to a control embryo (F) (twist::CD2) (arrowheads). The segmentally repeated variation in brightness is due to the changing thickness of the epidermal/neural tissue, which attenuates the signal from the photoactivated mesoderm (data not shown). (G,H) z-series reconstruction at the end of time-lapse acquisition (G), showing that the mesoderm has adopted the typical monolayer arrangement seen in fixed twist::CD2 control embryos (H). Scale bars: 20 µm.

View larger version (169K): [in this window] [in a new window]   Fig. 4. Outer cells migrate dorsolaterally as a group. (A) Overview of a time-lapse sequence of a Drosophila embryo showing the initial epithelial tube (0:15:00), the EMT and first round of division (0:21:00), a migratory phase (0:31:00) and the onset of the second division (0:51:00). Lower panels give reconstructed cross-sectional views. (B) Following the first division, there are typically three to four rows of outer cells (i.e. cells that are in contact with the ectoderm) on either side of the midline, which move laterally over the ectoderm. Two cells on either side of the midline (white dots) are tracked for a period of 12 minutes, as they move apart (see Movie 2 in the supplementary material). This image (and C and E) show a single focal plane. Cells marked with a black dot have, by the last panel, moved dorsolaterally into a deeper focal plane (data not shown). (C) A more superficial focal plane showing the movement of outer cells in regions closer to the midline. The positions of the cells marked in B (white dots) (not visible in this focal plane) are here again depicted with white dots. The original location of these cells with respect to the ectoderm was tracked and is indicated with a black dot. As the marked cells move laterally, other cells (down arrowheads) move laterally to occupy the original positions of the marked cells on the ectoderm. Times are as in B. (D) A reconstructed cross-section showing the uneven nature of the ectodermal surface over which the cells migrate laterally. Rounded regions appear to be nascent neuroblasts (asterisks). The indicated cell (0:43:00, arrowhead) corresponds to the cell in C (arrowheads on right-hand side). Times are as in B. (E) Enlarged view of the sequence in B showing that the marked cell (white dot) maintains its relationship to neighbouring cells (arrowheads) during migration. The cell to the lower right of the marked cell moves into a deeper focal plane (data not shown). Scale bars: 20 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Inner cells can move over outer cells. A time-lapse series of a Drosophila embryo showing inner cells (those not adjacent to the ectoderm) moving over outer cells (see Movie 3 in the supplementary material). (A) A 2 µm z-series at 0:31:00 showing a labelled cell (white dot and arrow) that is, at this time, the most laterally placed cell. (B) A reconstructed cross-sectional time-series in which the labelled cell (white dot and arrows) is overtaken by inner cells (arrowheads). (C) A z-series at end of the time-lapse showing that at deeper z-slices new cells (arrowhead) are now more dorsolaterally placed than the labelled cell (arrow). Scale bar: 20 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 6. Inner medial cells become dispersed and intercalate towards the ectoderm. (A) We define inner medial (IM) cells as cells at the centre of the furrow, which will be situated at the innermost point (top) of the invaginated epithelial tube (dark grey) after gastrulation. (B) Photoactivated IM cell at gastrulation. The midline is indicated by the radial line. (C) The same cell 5 minutes later is now situated at the top of the invaginated epithelial tube. Photoactivated fluorescence (red), Neurotactin (green). (D) Before gastrulation, embryos were monitored using the low levels of pre-activation fluorescence until the onset of furrowing was detected. The furrow was first detected as a slight flattening at the anterior and posterior ends of the nascent furrow. (E) Two cells at the centre of the developing furrow were first scanned with 488 nm laser at zoom x14.5. A polygon was then drawn around them and they were scanned with the UV laser. (F) This resulted in the two cells being brightly labelled, with some neighbouring cells also being weakly labelled. The dotted line shows the midline. (G,G') One minute 45 seconds later the furrow has formed and the photolabelled cells are internalised. This morphological stage is used as the zero time-point for all timings in movies. A reconstructed cross-section through the dotted line is shown in G'. (H-J) A time-lapse sequence at a constant focal plane showing the appearance of inner cell progeny at approximately 1 hour post-gastrulation. (H'-J') Reconstructed cross-sections of z-series in upper panels. (H) At 0:46:00 the inner medial cell progeny are too internal to be detected. White blurred areas on the right are yolk. (I) At 0:56:00 minutes the inner medial cell progeny are first detected. (J) By 1:06:00 the cells are clearer, and are dispersed across the mesoderm. As seen in the reconstructed cross-section panel below, the cells are at a depth typical of cells adjacent to the ectoderm. (K) An isolated inner cell in a fixed control embryo shortly before the second division. Dotted line shows midline. Scale bars: 20 µm in B-D,F-K; 2 µm in E.

View larger version (36K): [in this window] [in a new window]   Fig. 7. Inner lateral cells move as a group laterally to dorsolateral positions. (A) We define inner lateral (IL) cells as cells adjacent to IM cells. (B) Photoactivated IL cell at gastrulation. The midline is indicated by the radial line. (C). The same cell 5 minutes later is situated just to the right of the midline in the invaginated epithelial tube. Photoactivated fluorescence (red), Neurotactin (green). (D) Two IL clusters photoactivated on either side of the midline. (E-G) Time-point 0' showing clusters internalising. Reconstructed cross-sections through the dotted lines are shown in F and G. (H) Following spreading, the two clusters have migrated to dorsolateral positions at opposite sides of the embryo (arrowheads). Dotted lines in B,D,F,G show the midline. Scale bars: 20 µm in B-E,H; 10 µm in F,G.

View larger version (73K): [in this window] [in a new window]   Fig. 8. Migration outcomes for inner medial and inner lateral cell clones. IM and IL cells were photoactivated in control and htlAB42 mutant Drosophila embryos (see Materials and methods for details) and development allowed to proceed until 90 minutes after gastrulation. The embryos were then fixed and immunostained for the mesoderm specific transcription factor Twist. By this stage, the monolayer was established and the photoactivated cells had divided twice to form clones of four cells. (A,C,E,G) Typical clones of photoactivated cells (red) within the mesoderm stained for Twist (green). (B,D,F,H) Schematics of IM/IL clone positions in control and htlAB42 embryos. Positions within the monolayer were calculated as a fraction of the full extent of the mesoderm, and are depicted as grey circles at proportional positions within the black boxes. The mean extent of the mesdoerm in control (143±15 µm, s.d.) versus htlAB42 (112±22 µm, s.d.) embryos is reflected by the width of the boxes. Boxes outlined in red correspond to the example embryos shown (A,C,E,G). (A) An IM clone arranged as two pairs of cells within the mesoderm monolayer. (B) IM clones spanned the full extent of the mesoderm and were typically arranged as two pairs of cells. Pairs could be widely separated and located on opposite sides of the midline. (C) An IL cell, to the right of the midline, has produced a clone of cells at the right hand, dorsalmost part of the mesoderm. (D) IL clones in control embryos are grouped into those arising from IL cells initially located to the right of the midline (n=8) and those arising from IL cells to the left of the midline (n=5). In all cases, clones did not cross the midline and were positioned in the dorsal part of the mesoderm. (E) A htlAB42 embryo in which the mesoderm has failed to spread into a monolayer. The clone of cells from an IM cell has failed to move away from the centre of the embryo. (F) IM clones in htlAB42 embryos tended to remain fairly central, and did not separate into two distinct pairs. (G) An IL cell to the left of the midline in a htlAB42 mutant, has produced a clone of cells that have not migrated out to the dorsal edge of the mesoderm. (H) IL clones in htlAB42 embryos tended to stay on the same side of the midline as the progenitor cell, but did not usually reach the dorsalmost regions of the mesoderm. Scale bars: 20 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 9. Model for mesodermal spreading in Drosophila embryos. (A) The invaginated epithelial tube before the EMT. (B) The mesodermal cells spread down onto the ectoderm as the EMT and first division occur. (C) Outer cells polarise and proceed to migrate dorsolaterally as a group (blue). As they move away, other cells take up their positions on the ectoderm (white arrows). Inner lateral cells (green) are attracted to the dorsal ectoderm and move over the outer cells. (D) During the second mitosis, inner medial cells (orange) that have failed to contact the mesoderm intercalate into the outer cell layer. (E) The monolayer is formed. See Discussion for details.