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Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo

¹ Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
² Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (22K): [in a new window]   Fig. 1. Two alternative hypotheses could explain the specification of leading edge cells. According to hypothesis 1, patterning of dorsal cell fates in the blastoderm stage embryo is achieved by interpretation of specific threshold levels of BMP activity. High activity is required for amnioserosa tissue (AS), intermediate activity specifies leading edge cells (LE) and low activity patterns the dorsal ectoderm (DE). Dorsalizing mutations that alter the shape or extent of the BMP activity gradient would be expected to expand dorsal cell fates including the LE. Alternatively, hypothesis 2 predicts that LE cells are not part of the blastoderm fate map but arise secondarily, possibly through inductive interactions between differentiating amnioserosa and dorsal ectoderm. According to this hypothesis, dorsalizing mutations would not be expected to expand LE cell fate beyond a single row.

View larger version (169K): [in a new window]   Fig. 2. An enhancer trap in the puckered locus, pucE69, is expressed in leading edge (le) cells of the dorsal ectoderm (de) during dorsal closure. When closure commences at stage 13, the LE appears as a single row of cells forming a ring around the amnioserosa (as), which becomes internalized by stage 16. Closure is complete when the LE cells meet and adhere at the dorsal midline. Panels are dorsolateral views with anterior towards the left.

View larger version (51K): [in a new window]   Fig. 3. Examination of amnioserosa and leading edge in dorsalized embryos. Embryos have been double immunolabeled for Kruppel (blue, alkaline phosphatase) to reveal the large amnioserosa cells and ß-gal (brown, horseradish peroxidase) to reveal puc enhancer expression in the LE (arrowheads). In wild-type embryos (A), a single row of LE cells is detected at the interface between amnioserosa and dorsal ectoderm. At this stage, Kruppel is also detected in segmentally repeated muscle precursors. Weakly dorsalized embryos are derived from mothers bearing a temperature-sensitive Tl mutation raised at 18°C (B). LE cells are detected as a single row located more ventrally than in wild type. Further dorsalization of embryos raised at 29°C results in amnioserosa tissue that encompasses the DV circumference of the embryo (C). LE cells are also reoriented circumferentially but remain as a single row of cells at the amnioserosa/ectoderm interface. Lateral views with anterior towards the left.

View larger version (91K): [in a new window]   Fig. 4. The leading edge is formed in dorsalized embryos despite disruption of amnioserosa and dorsal ectoderm. Here, dorsal ectoderm (DE), leading edge (LE) and amnioserosa (AS) of dorsalized embryos are examined in close detail with various combinations of antibodies as indicated in the panels. Embryos are from wild-type (A) or dorsal mutant mothers (B-G'). Leading edge cells (arrowhead) comprise the first row of ectodermal cells that abut the amnioserosa in wild-type embryos (A). LE cells express ß-gal (green) and Fasciclin III (red), which is asymmetrically distributed in these cells (A, inset). Independent of tissue size or position, wherever amnioserosa tissue and dorsal ectoderm are juxtaposed, LE cells are formed. (B) A dorsalized embryo with circumferential single cell wide rings of LE (arrowhead) surrounding amnioserosa tissue. (C) A single row of LE cells at the edge of Fasciclin III-positive ectoderm (arrowhead). In dorsalized embryos, islands of tissue occasionally form (C-F'). (C,E) Unlabeled islands of amnioserosa surrounded by Fasciclin III-positive dorsal ectoderm. Fasciclin III localization is asymmetric in cells adjacent to these islands (E), and these correspond to LE cells that express ß-gal (green in C, arrows). (D,E') Similarly, islands of ectoderm are surrounded by amnioserosa (both tissues labeled in D, only ectoderm labeled in E' to show asymmetric Fasciclin III). Within a sea of amnioserosa, islands of ectoderm are consistently bordered by ß-gal-expressing LE cells (F,F'). Finally, dpp RNA is detected in dorsalized embryos by whole-mount in situ hybridization (G,G'). dpp, a marker of LE cells, is also observed in rings (arrow) and stripes (arrowheads) consisting of a single row of cells like those seen using the puc enhancer. Thus, three LE markers demonstrate the presence of LE cells in dorsalized embryos at the interface between amnioserosa and dorsal ectoderm. Dorsal views with anterior towards the left.

View larger version (163K): [in a new window]   Fig. 5. The leading edge is proportionately lost with increasing ventralization. By enzymatic detection of ß-gal from the puc enhancer trap, we observe that LE cells are present as a single row (arrowhead) in weakly ventralized embryos derived from mothers heterozygous for a cactus deficiency (A). LE is absent, as is amnioserosa, in severely ventralized embryos derived from mothers homozygous for strong hypomorphic cactus alleles (B). A few ß-gal-positive cells are evident in these embryos, but they are not likely to be LE cells (see text). The embryo in A is oriented dorsal upwards and anterior towards the left.

View larger version (65K): [in a new window]   Fig. 6. Leading edge is not expanded in mutants that modulate the shape of the BMP activity gradient. Wild-type (A), brk mutant (B) and sog mutant (C) embryos were independently labeled with anti-ß-gal antibodies to reveal the cells of the LE (arrowheads). Despite significant cell fate changes in the mutant embryos, LE cells are observed in a single row at the interface between amnioserosa and dorsal ectoderm, indicating that LE differentiation is fundamentally normal. In the anterior of each embryo, LE cells interdigitate, causing the appearance of multiple rows. This phenomenon correlates with the dramatic cell movements of dorsal closure and is not specific to the mutant genotypes. All panels are dorsolateral views with anterior towards the left.

View larger version (110K): [in a new window]   Fig. 7. Leading edge markers are altered in U-shaped mutants in which amnioserosa is prematurely lost. ush and hnt embryos have been double immunolabeled for Fasciclin III (red) and ß-gal (green) to identify dorsal ectoderm and LE, respectively (A-C) or used for whole-mount in situ hybridization to reveal dpp transcripts (D-E'). In ush mutant embryos (A), ß-gal is never expressed at the LE (arrowheads). In contrast, hnt mutants exhibit ectopic ß-gal expression in the region of the dying amnioserosa (arrows) from stage 11 (B), before Fasciclin III expression levels peak, through stage 13 (C). Additionally by stage 13 (C), ß-gal expression is clearly evident at the edge of the ectoderm indicating that LE fates are present in hnt mutant embryos, although in a less uniform arrangement compared with wild-type (compare with Fig. 3A). Similar results are shown with dpp transcripts in the LE (D-E'). dpp expression in the LE is substantially reduced in ush mutant embryos (D), although some residual staining is apparent (arrowhead), suggesting that LE specification is compromised. LE expression of dpp in hnt mutant embryos is relatively normal (E,E', arrowhead). Higher magnification of the same embryo (E') reveals some ectopic dpp expression in the amnioserosa (arrow), however, these ectopic transcripts are detected in less than 10% of mutant embryos. Lateral views with anterior towards the left.

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