Seven Wnt homologues in Drosophila: a case study of the developing tracheae

Abstract

Sequencing of the Drosophila genome has revealed that there are "silent" homologues of many important genes-family members that were not detected by classic genetic approaches. Why have so many homologues been conserved during evolution? Perhaps each one has a different but important function in every system. Perhaps each one works independently in a different part of the body. Or, perhaps some are redundant. Here, we take one well known gene family and analyze how the individual members contribute to the making of one system, the tracheae. There are seven DWnt genes in the Drosophila genome, including wingless (wg). The wg gene helps to pattern the developing trachea but is not responsible for all Wnt functions there. We test each one of the seven DWnts in several ways and find evidence that wg and DWnt2 can function in the developing trachea: when both genes are removed together, the phenotype is identical or very similar to that observed when the Wnt pathway is shut down. DWnt2 is expressed near the tracheal cells in the embryo in a different pattern to wg but is also transduced through the canonical Wnt pathway. We find that the seven DWnt genes vary in their effectiveness in specific tissues, such as the tracheae, and, moreover, the epidermis and the tracheae respond to DWnt2 and Wg differently. We suggest that the main advantage of retaining a number of similar genes is that it allows more subtle forms of control and more flexibility during evolution.

Figure 1

The Wnt pathway is required for tracheal development. Lateral views of embryos at late stages of embryogenesis stained with mAb2A12 to highlight the lumen of the tracheae. In all of the figures, anterior is to the left and dorsal is above. (A) Wild-type embryo. DT, dorsal trunk (arrow); VB, visceral branch (arrowhead). (B) fz⁻fz2⁻ germ-line clones. The DT is completely missing apart from minute vestiges of DT material found in the posterior part. (C) wg⁻, a substantial amount of DT is formed (arrow). (D) Df(2L)RF, a similar phenotype to wg⁻ is observed (arrow points to DT). (E) Ectopic expression of wg in all tracheal cells—note hypertrophy of DT (arrow). (F) Ectopic expression of DWnt2 in all tracheal cells—note hypertrophy of DT (arrow).

Figure 2

Pattern of wg and DWnt2 expression with respect to Sal protein and the tracheal cells. (A and B) Three tracheal metameres of a 1-eve-1 embryo at stage 10 (A) or 12 (B) stained with a riboprobe for DWnt2 (green) and for Sal (in red) and β-Gal (blue) for the tracheal cells. Note that DWnt2 is expressed near the tracheal cells during stage 10 (A) in cells that also express sal. (C and D) Three tracheal metameres of a 1-eve-1 embryo at stage 10 (C) or 12 (D) stained with a riboprobe for wg (green) and for Sal (in red) and β-Gal (blue) for the tracheal cells. (E) Two tracheal metameres of a 1-eve-1 embryo at stage 11 stained with a riboprobe for DWnt2 (green) and for Wg (red) and β-Gal (blue) for the tracheal cells. Note that wg and DWnt2 differ in their pattern of expression. (F) Diagram summarizing the stainings in A–E. DWnt2 is expressed in the dorsal ectoderm near the tracheal cells that is in the most dorsal Sal-positive cells. wg expression alternates in stripes with Sal protein. Both genes are produced near the tracheal cells.

Figure 3

Tracheal requirements of wg and/or DWnt2. (A–C) Lateral views of embryos at late stages of embryogenesis stained with mAb2A12 to highlight the lumen of the tracheae. (A and B) wg⁻DWnt2⁻ double mutants, the DT is missing (A) or very much reduced (arrow in B). The arrowhead in A points to an incomplete VB. (C) DWnt2 combination, no tracheal defects are observed. Arrow points to DT. (D–I) Sal distribution at stages 12 (D–F) and 14/15 (G–I) in lateral views of embryos of the indicated genotypes. Note the absence of Sal in wg⁻DWnt2⁻ mutants (E, H, and Insets) as compared with WT (D, G, and Insets). Low levels of Sal still are observed in wg⁻ mutants (F, I, and Insets). (J and K) dpp expression at stage 11 in lateral views of embryos of the indicated genotypes. (L and M) Kni distribution at stage 14/15 in lateral views of embryos of the indicated genotypes.

Figure 4

(A and B) Lateral views of embryos of the indicated genotypes at late stages of embryogenesis stained with mAb2A12. Arrow in B points to the rescued DT. (C) Pattern of DWnt2 expression in wg⁻ embryo at stage 10. (D and F) Dark field images of larval cuticle preparations of the indicated genotypes. Overexpression of wg in the epidermis produces a naked cuticle phenotype, whereas overexpression of DWnt2 does not.

Figure 5

Rescue of DT in double mutants by DWnt proteins. Lateral views of embryos at late stages of embryogenesis stained with mAb2A12 to show the trachea. Each image shows a wg⁻DWnt2⁻ double mutant in which the DWnt indicated has been added in the pattern of the Gal4 line used. Note the rescue of DT (arrows) when wg, DWnt2, or DWnt8 are added to the embryo (A–F). Conversely, DWnt5 does not rescue the DT (G and H); the arrowhead in H points to a piece of the VB.