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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00646

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The expression and function of the achaete-scute genes in Tribolium castaneum reveals conservation and variation in neural pattern formation and cell fate specification

¹ Program in Neuroscience, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA
² Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA
³ Division of Biology, Ackert Hall, Kansas State University, Manhattan, KS 66506, USA

View larger version (39K): [in a new window]   Fig. 1. Tribolium castaneum has two ac/sc homologs. (A) Comparison of the amino acid sequences from the bHLH domains of the Tribolium ac/sc genes (Tc-SH, Tc-Ase) with those from Drosophila (Dm-Ac, Dm-Sc, Dm-L'sc). Amino acids conserved in four or more proteins are highlighted in yellow and asterisks indicate residues conserved in all ac/sc bHLH domains. (B) Alignment of the C-terminal motif from Drosophila proneural ac/sc genes with the C-terminal motif of Tc-ASH. Conserved amino acids are highlighted in yellow. (C) Phylogenetic tree showing select invertebrate ac/sc genes; support values given at each node for distance (top) and parsimony (bottom) methods; nodes with support values less than 50 for either method have been collapsed, branch lengths are not to scale. Both distance and parsimony methods show that Tc-ASH groups within the proneural gene clade and Tc-Ase within the Asense clade. Ag-ASH, Anopheles gambiae Achaete-Scute Homolog; Ag-Ase, Anopheles gambiae Asense; Hv-ASH, Hydra vulgaris Achaete-Scute Homolog; Cs-ASH1, Cupiennius salei Achaete-Scute Homolog 1; Cs-ASH2, Cupiennius salei Achaete-Scute Homolog 2; Cc-Sc, Ceratitis capitata Scute; Cc-L'sc, Ceratitis capitata Lethal of scute; Cc-Ase, Ceratitis capitata Asense; Dm-Ac, Drosophila melanogaster Achaete; Dm-Sc, Drosophila melanogaster Scute; Dm-L'sc, Drosophila melanogaster Lethal of scute; Dm-Ase, Drosophila melanogaster Asense; Tc-ASH, Tribolium castaneum Achaete-Scute Homolog; Tc-Ase, Tribolium castaneum Asense.

View larger version (12K): [in a new window]   Fig. 2. Comparison of the Tribolium, Drosophila, and Anopheles achaete-scute complexes. (Top) The Tribolium ac/sc complex contains one proneural ac/sc gene, Tc-AS and one asense homolog, Tc-ase, that reside 55 kb apart. Cytochrome P450 (cyt P450) lies downstream of Tc-ase. (Middle) The Drosophila ac/sc complex contains three proneural genes ac, sc and l'sc and one asense gene located 45 kb downstream of l'sc. As in Tribolium, cyt P450 resides downstream of ase. (Bottom) The Anopheles ac/sc complex contains one proneural gene, Ag-ASH, and one asense gene, Ag-ase, that reside 22 kb apart. Unlike Tribolium and Drosophila, cyt P450 lies between the Anopheles ac/sc genes. Proneural genes are in shown in blue, asense genes in red, and cyt P450 in black. Arrows denote the direction of transcription. Drosophila map is adapted from Campuzano et al. (Campuzano et al., 1985) and the Anopheles map was constructed from available genome sequence data (Holt et al., 2002).

View larger version (80K): [in a new window]   Fig. 3. Tc-ASH exhibits a proneural expression pattern while Tc-ase expression is restricted to neural precursors. (A-D) Ventral views of 20-hour Tribolium embryos. (A,B) Tc-ASH in situ hybridization (blue) and Engrailed protein (brown). (C,D) Tc-ase in situ hybridization (blue) and Engrailed protein (brown). (A) Tc-ASH is expressed in cell clusters in developmentally younger segments at the posterior (arrow); in developmentally older anterior segments Tc-ASH expression has resolved to single cells, the CNS neural precursors (arrowhead). In each segment Tc-ASH is expressed in a transverse stripe posterior to the engrailed stripe (asterisk). (B) High magnification view of two segments that shows Tc-ASH expression in proneural clusters (arrow) and single neural precursors (arrowhead). (C) Tc-ase is expressed in neural precursors (arrowhead) but not in ectodermal cell clusters. Tc-ase staining is not detected outside of the neuroectoderm (asterisk). (D) High magnification view of two segments showing Tc-ase expression. Note the position of Tc-ase-expressing neural precursors in D is identical to that of the large Tc-ASH expressing cells in B. Scale bars: 100 µm (A,C); 25 µm (B,D). Images in A and C are photomontages. Anterior, left.

View larger version (111K): [in a new window]   Fig. 4. Tc-ASH is necessary and sufficient for neural precursor formation. (A-C) Tribolium embryos injected with (A) buffer or (B, C) Tc-ASH double stranded RNA stained for Tc-ase RNA (blue) and Engrailed protein (brown). (A) Buffer-injected embryo displays the wild-type pattern of neural precursors. (B,C) Tc-ASH RNAi-treated embryos display either (B) a complete or (C) a partial loss of neural precursors as judged by Tc-ase expression. (D-F) Dorsal view of adult Drosophila nota. (D) Wild-type pattern of sensory bristles. (E,F) Ectopic sensory bristles result from misexpression of either (E) UAS-Drosophila sc or (F) UAS-Tc-ASH under the control of the apterous-Gal4 driver line. A-C: anterior, left; D-F: anterior, up.

View larger version (18K): [in a new window]   Fig. 5. A Model for ac/sc gene evolution. The ac/sc genes, their developmental functions (proneural-like, blue or asense-like, pink), and existence in complexes have been mapped on a standard species tree (Maddison et al., 2001). Branch lengths are not drawn to scale. See discussion for detailed description.