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The expression of pannier and achaete-scute homologues in a mosquito suggests an ancient role of pannier as a selector gene in the regulation of the dorsal body pattern

Corinna Wülbeck^* and Pat Simpson^,

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France and Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
^* Present address: Institut für Zoologie, Lehrstuhl für Entwicklungsbiologie, Universität Regensburg, 93040 Regensburg, Germany
Present address: Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK

View larger version (84K): [in a new window]   Fig. 1. (A) The complete amino acid sequence of Ag-ASH is compared with that of the achaete-scute homologue of the butterfly Precis coenia (Galant et al., 1998) and with lethal of scute from Drosophila (D.m. L’scute) (Villares and Cabrera, 1987). Differences are shown in black. The start site is indicated by a green circle. Conservation is highest in the bHLH domain, which is boxed. The shaded blue box indicates a conserved stretch in the C-terminal domain. (B) The complete amino acid sequence of Ag-Pnr is compared with that of the Pannier protein of Drosophila melanogaster and Ceratitis capitata. Differences are shown in black. The start site is indicated by a green circle. The two zinc fingers are boxed. The helical structures are shown as shaded blue boxes. The nuclear localisation signal (NLS) is indicated by the shaded grey box.

View larger version (135K): [in a new window]   Fig. 2. Expression of Ag-ASH and Ag-pnr in Drosophila. (A) A wild-type notum. (B) The GAL4 line pnrMD237 was used to drive Ag-ASH expression in the medial notum of Drosophila; this leads to the segregation of many ectopic bristles in the medial domain of the notum. Note that the slight midline cleft is a mutant phenotype associated with the pnrMD237 insert. (C) The results of ectopic expression of Ag-pnr in the lateral notum with the driver C765. Note the presence of additional, large, (dorso-central) bristles on the lateral posterior scutum. (D) The notum of a fly in which early ubiquitous expression of Ag-pnr over the entire dorsal notum was driven by Gal4ap. Elements of the lateral pattern are absent, but the medial pattern is formed normally apart from some extra bristles along the lateral margin.

View larger version (112K): [in a new window]   Fig. 3. Sections of larvae illustrating the development of the imaginal notum. (A) A section through a second instar larva, part of which is enlarged in B, showing the leg and wing buds and a small patch of thickened epithelial cells (imaginal cells) at the junction between the larval epithelium and the wing bud. The notum is derived from this structure. (C) Section through a fourth instar larva. The wing and leg buds have evaginated and lie flat against the body wall. (D) An enlargement of the dorsal area of this section at the level indicated. The imaginal epidermis (star) has replaced the larval one and dorsal closure is complete. Remnants of the degenerating cells of the larval epidermis can be seen as small black spots (arrowhead).

View larger version (121K): [in a new window]   Fig. 4. The bristle pattern of the imaginal notum. (A) A camera lucida drawing. The large sensory bristles are indicated by black circles and the sensory scales by red circles. The acrostichal, dorsocentral, prescutellar and scutellar bristles are indicated as AC, DC, PST, SC respectively. Their number varies considerably from one animal to another. (B) A camera lucida drawing of a bristle and a scale illustrating the difference in size and shape. (C) Part of a pupal epithelium stained with anti-HRP antibody that labels neurons. Neurons and axons of the AC and DC bristles can be seen.

View larger version (90K): [in a new window]   Fig. 5. Domains of expression on the developing Drosophila notum. (Top left) A schematic diagram of the Drosophila notum, indicating the extent of the expression domain of pannier (green), achaete-scute (blue) and the positions of the macrochaetes (red). (A-D) Ag-ASH and (E-H) Ag-pnr expression. The spatial expression of the two genes in the medial half of the notum is identical. Expression of both genes starts in two broad bands, one on either side of the midline. This is best seen in the late fourth instar larva in E, where staining is seen beneath the larval cuticle which has a ridged appearance. The cuticle was removed for all other preparations shown. The two early bands are just becoming visible in A, where the positions of the future acrostichal (AC) and dorsocentral (DC) bristle rows are indicated by dashed lines. Stained areas round the edges of this and other preparations are artefacts due to the folded cut edge of the epithelium. Although the larval epidermis is lost, a number of larval sense organs (LSO, arrows) and larval sensory hairs (LSH, circles) persist and remain just above the imaginal epithelium. They are out of focus in some of the photographs. The larval sensory hair provides a useful positional marker with respect to which the anterior limit of the succeeding triangular and kidney-shaped domains can be measured. The full extent of the triangular domain of staining on the posterior scutum just above the scutellar suture can be seen in F. In this image, and others, some of the acrostichal and dorsocentral bristle precursors can be distinguished. The triangle has already started to shrink from its anterior limit in B and is progressively transformed to the kidney-shaped domain in C, G and H. The crescent-shaped domain on the scutellum can be seen below the LSH in D. (Note some damage to this preparation that distorts the picture of the kidney-shaped domain). A patch of staining on the midline at the anterior edge of the notum is seen in B,C,F,G and H (unlabelled). It is a consistent feature but we have been unable to find a morphological correlate to this domain of expression. The correspondence between the expression domains and the positions of the future bristles is indicated in the schematic drawings below. The acrostichal (AC) and dorsocentral (DC) rows are drawn in green and red, the prescutellar (PST) and scutellar (SC) bristles are shown in blue.

View larger version (108K): [in a new window]   Fig. 6. Expression of (A,B) Ag-pnr and (C) Ag-ASH in segregating sensory organ precursors. Precursors arise as the domains of expression fade and can be seen as spaced single cells. Precursors of bristles of the acrostichal (AC) and dorsocentral (DC) rows arise first, indicated by arrowheads; they are large and arranged in poorly defined rows. Scale precursors in the vicinity of the AC and DC rows arise at the same time, indicated by the arrow in C. The scale precursors are smaller. The triangular domain of expression is visible at the bottom in B. The epithelium is somewhat creased at these stages distorting the spatial arrangements. Precursors of bristles and scales at other positions arise slightly later. Within the rows and at different spatial locations, there is no apparent order to precursor segregation: the distribution of stained cells is variable between individuals. LSH, persistent larval sensory hair. (D) A fourth instar larva stained with anti-HRP to visualise the neurons. (Precursor segregation is accomplished before pupation in some individuals). Neurons corresponding to some of the AC and DC bristles have appeared in this individual, as well as some of the lateral antealar (AA) neurons. LSN, larval sensory neurons.