Dissecting the evolutionary role of the Hox gene proboscipedia in Drosophila mouthpart diversification by full locus replacement

Description


Phenotypes of intermediate constructs
Homozygous replacement of only the 5' portion of the D.mel pb locus with sequences from D.mim (fragment 1) resulted in flies with a completely normal labellum, but with notable alterations in maxillary palp morphology and bristle patterning ( fig. S4). Maxillary palps of fragment 1 replaced flies were bulbous with increased numbers of tufted bristles in the distal region of the maxillary palps ( fig. S4A). Correspondingly, we observed greater numbers of underlying ELAV positive staining neurons with a broader distribution in developing pupal maxillary palps ( fig. S4D), a phenotype reminiscent of that observed for the full pb-mim replacement (Fig. 2G). Next, we replaced the 3'-most portion of the pb locus (fragment 2) alone (fig. S5, A and B) or in combination with replacement of fragment 1 (fig. S5, C and D). Flies homozygous for fragment 2 only displayed a partial loss of the pb homeotic phenotype which consists of a transformation of labellum into arista (fig. S5, A and B). The hypomorphic fragment 2 phenotype dominated when combined with the fragment 1 replacement. In light of the fact that a replacement of the entire D.mel pb locus with that of D.mim. provides nearly full rescue of pb function, we speculate that long-range interactions between multiple species-specific cis-acting regulatory sequences are required to act in concert to provide full activity of the locus.
It is noteworthy that the phenotype of the full D.mim pb replacement differs from the sum of its component parts in that replacement of only 5' portion of the locus leads to alteration in bristle numbers and underlying neurons (also observed in the full replacement) but does not result in altered orientation of the maxillary palps nor heart-shape palps in males, while replacement of only the 3' portion of the locus results in a partial loss-of-function phenotype. These observations underscore the importance of replacing the entire locus to accommodate potential species-specific evolution of interactions among distant cis-regulatory elements.  green) driven by T2A-GAL4 insertions into these loci respectively (Fig. 3A) in labial discs, salivary glands, leg disc T1, leg disc T2, eye-antennal discs and the brain/central nervous system.      males (B). Asterisks indicate notch on inner surface. Scale bar, 25 μm.

Fig. S7: Comparison of Pb protein coding sequences and function in D.mel versus D.mim (A)
The Pb protein sequence alignment of D.mim compared to D.mel The bar graph represents the conservation percentage of amino acid sequences. (B) The Pb protein sequence alignment of D.mim compared to D.grim. The bar graph represents the conservation percentage between single amino acid. (C) Pb (green) and zen2 (gray) paralog protein sequence comparison plotting percentage of amino acid conserved between D. mim and D.mel (mim-mel), D.grim. and D.mel (grim-mel), and D.mim and D.grim. (mim-grim). (D) Phylogenetic tree of Pb protein sequences of six Drosophila species. The tree was made using the maximum likelihood method with the CLC Main Workbench program. Bar indicates distance. (E) Schematic of the pb genomic regions engineered to incorporate Gal4-T2A fused to HoxA2 or HoxB2 cDNAs followed by 2 stop codons. (F) Images of control (melPb-G4) and Gal4-T2A:HoxA2 cDNAs driving expression of UAS-nlsGFP in primordia of the maxillary palps (Mx) and proboscis (Lb) in late pupae. The HoxA2 proboscis shape is altered because it has been partially transformed to legs.     Movie S1: The expression of melPb-GAL4 during metamorphosis.

Movie S2:
The expression of Dfd-GAL4 during metamorphosis.

Movie S3:
The expression of Scr-GAL4 during metamorphosis.
Data S1: D.mim zen2 and Pb protein and DNA sequences