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First published online 1 October 2003
doi: 10.1242/dev.00867

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Segment boundary formation in Drosophila embryos

National Institute for Medical Research, The Ridgeway Mill Hill, London NW7 1AA, UK

View larger version (98K): [in a new window]   Fig. 1. Morphological changes during segmental boundary formation. (A) Schematic drawing of gene expression patterns in a horizontal section through one segmental unit. The position of the segmental boundary is marked with a vertical bar. (B) Schematic drawing of the fusion protein used to label cell outlines under the EM (under UAS control). It comprises the signal peptide from Wingless, human CD2 (without signal peptide) and HRP. (C-K) TEM images showing the changes in cell morphology as segmental grooves form and regress. Embryos were stained with DAB and sectioned horizontally through the ventral aspect of the embryo. Although some staining appears at the surface of non-expressing cells (maybe as a result of membrane shedding from expressing cells), we were able to confidently identify expressing cells after a bit of practice. An annotated version of this figure highlighting expressing cells is provided at http://dev.biologists.org/supplemental/. (C) Shortly after germ-band retraction is initiated, a small dip (arrow) appears between engrailed-expressing and non-expressing cells. (D) Apical contact appears to loosen (arrow). (E) The most posterior engrailed-positive cell constricts apically and moves inwards in relation to surrounding cells. (F) This cell finds itself at the bottom of the forming groove and neighbouring cells follow this inward motion. (G) More cells have moved in and the groove is now two to three cell diameters deep. (H) The groove at its deepest reaches at least three cell diameters in depth. (I) At this stage the bottom engrailed-expressing cell is bottle shaped and severely constricted apically (arrow). (J) The disappearance of grooves is a very rapid event, which allows the cells to return to their original position. (K) The embryo eventually becomes almost flat ventrally. (L-N) Grooves are much deeper laterally (L) than ventrally (M), and posterior grooves (between abdominal segments 2 and 3; N) are not as deep as anterior ones (between abdominal segments 7 and 8; M). Scale bars: 500 nm.

View larger version (38K): [in a new window]   Fig. 2. Loss of engrailed expression in the `bottle cells'. (A-C) Lateral view (focused on the ventral midline) of wild-type embryos stained with anti-Engrailed (red) and anti-HRP (green). (A,B) At stages 12 and 13, Engrailed and HRP immunoreactivity co-localises (although this is not clear at all focal planes). (C) By contrast, at stage 14 the so-called bottle cell downregulates Engrailed expression although it remains labelled with HRP (white arrow). No attempt was made to identify the staining detected inside the embryo, which could be in the mesoderm or the nervous system.

View larger version (95K): [in a new window]   Fig. 3. Persistence of segmental grooves is affected by dorsal closure. (A-D) Wild type and zipper mutants, which are defective in dorsal closure, at stage 15 and oriented such that the ventral midline is at the bottom. (A) Wild-type embryo stained with anti-Engrailed (black). (B) Wild-type embryo as seen by scanning electron microscopy (SEM). The ventral epidermis is almost flat (black arrow), whereas shallow grooves are still present laterally (white arrow). (C) Brightfield image of a zipper mutant embryo stained with anti-Engrailed (black). Note that grooves persist ventrally (black arrow). (D) Persistent ventral grooves can also be seen by SEM (black arrow). Moreover, lateral grooves appear deeper (white arrow) than in the wild type.

View larger version (79K): [in a new window]   Fig. 4. Boundary formation requires Hedgehog and Ci. (A-H) Stage 13+ embryos stained with anti-Engrailed (black or brown). (A,B) Wild-type embryos. Deep grooves are easily seen (black arrows) in the lateral view in A, while the ventral view shows the normal stripes of Engrailed expression (two to three cell diameters wide). (C,D) Embryos lacking hedgehog but continuing to express engrailed (full genotype is shown). No groove form as seen from the lateral view focused on the ventral midline (C) and stripes of engrailed-expressing cells are broken up into clumps as seen in the ventral view (D). (E,F) In ci94 embryos (with artificially maintained Engrailed), grooves form (black arrows in E) and Engrailed stripes appear normal (F). (G,H) In ciCell embryos (with artificially maintained Engrailed), grooves do not form (G) and there is moderate disruption of the Engrailed stripes (H). (I-L) Cartoons summarising the results shown in panels above. (M,N) SEM of stage 13 embryos. Compare the wild type in M with a hedgehog mutant (with artificially maintained Engrailed) in N.

View larger version (83K): [in a new window]   Fig. 5. Wingless signalling inhibits segmental boundary formation. All embryos are at stage 13+ and stained by immunocytochemistry with anti-Engrailed (black). (A-D) Removal of Wingless (while maintaining engrailed expression) leads to duplication of segment boundaries. An `en face' view of the ventral area (A) shows that engrailed stripes are sharply delineated on both sides. In a side view of the ventral region (B), one can see grooves on both sides of engrailed stripe (e.g. black arrows). In the lateral region, an `en face' view (C) shows that Engrailed stripes are broken up into clumps. (D) Grooves are generated around the islands of engrailed-positive cells as seen in a side view. (E-G) In a double mutant (wingless– hedgehog–), no groove forms. (E) Engrailed stripes are disrupted throughout (en face view of the ventral region as in A). (F) Ventral grooves are no longer generated, as seen in a side view as in B. (G) Likewise no groove can be recognised laterally in a side view similar to that in D. (H,I) Schematic drawings summarising the results shown in A-G.

View larger version (60K): [in a new window]   Fig. 6. Hedgehog-independent requirement of Engrailed in groove formation. Engrailed is required in addition to Hedgehog for boundary formation. No grooves form in an engrailed mutant (or in an engrailed wingless double mutant). (A) Groove formation is rescued, at least in the lateral epidermis (see legend of Fig. 7), by expressing engrailed with paired-gal4, shown here in the wingless engrailed double mutant: grooves form on both sides (arrows) of the expression domain because of the absence of wingless. (B) By contrast, no such rescue is seen when Hedgehog is expressed in the same genetic background. (C,D) Diagrams summarising the results in A and B. (E) Stage 13 embryo expressing CiVP16 under the control of engrailed-Gal4 stained with anti-Engrailed (brown) and a ci RNA probe (purple). This embryo is expected to have active Hedgehog signalling on both sides of the presumptive boundaries. Boundary formation is not prevented. This is represented diagrammatically in F.

View larger version (85K): [in a new window]   Fig. 7. Continuous requirement of engrailed and hedgehog in groove formation. When driven by paired-gal4, expression of engrailed and hedgehog rescues segmental grooves only transiently (in an embryo lacking engrailed and also wingless). (A) Side view of such an embryo at stage 12, focused on the ventral midline. Shallow grooves can be seen (arrows). (B) At stage 13, these grooves are no longer visible. (C) Schematic representation of the domains of paired (Prd) and buttonhead (Btd) expression. Note that, in the ventral region, expression of buttonhead persists longer (up to stage 15) than that of paired (to stage 12+). (D) buttonhead-driven engrailed expression rescues groove formation (arrows) in an engrailed mutant even at stage 13 and beyond. This is not true of paired-gal4-driven engrailed (not shown).

View larger version (68K): [in a new window]   Fig. 8. stripe, rhomboid and spitz are not required for groove formation. Lateral views of stage 14 embryos. (A) Wild type. (B) stripeDG4 mutant embryos (here stained with anti-Engrailed) have grooves although they can be irregularly spaced. (C) Normal grooves form in a rhomboid7M43 mutant (also stained with anti-Engrailed). (D) spitz1 mutant. Again, grooves form although the epidermis can be disorganised

View larger version (14K): [in a new window]   Fig. 9. Morphological changes accompanying segmental groove formation. Schematic representation of the changes in cell morphology and genetic interactions before and during boundary formation. Here, drawings are oriented such that the apical side of the epithelium is upwards, according to convention. (A) Groove formation is initiated by Hedgehog signalling in cells adjoining the most posterior engrailed-expressing cells. Signalling by Hedgehog prevents repression by Ci[75], leading to the expression of gene(s) x. (B) The groove founder cells loose contact on their apical side and an unknown signal (Y) feeds back on the engrailed-expressing cell. (C) The most posterior engrailed-expressing cell constricts its apical surface and moves inwards. (D) It comes to lie at the bottom of the forming groove while continuing to constrict its apical surface. (E) As the groove reaches its deepest point, the most posterior engrailed-expressing cell acquires a bottle shape. At the same time, it turns off engrailed expression.