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

Abstract

Hox genes determine positional codes along the head-to-tail axis. Here, we replaced the entire Drosophila melanogaster proboscipedia (pb) Hox locus, which controls the development of the proboscis and maxillary palps, with that from Drosophila mimica, a related species with highly modified mouthparts. The D. mimica replacement rescues most aspects of adult proboscis morphology; however, the shape and orientation of maxillary palps were modified, resembling D. mimica and closely related species. Expressing the D. mimica Pb protein in the D. melanogaster pattern fully rescued D. melanogaster morphology. However, the expression pattern directed by D. mimica pb cis-regulatory sequences differed from that of D. melanogaster pb in cells that produce altered maxillary structures, indicating that pb regulatory sequences can evolve in related species to alter morphology.

Fig. 1.. Mouthparts of D.mel and D.mim and Hox gene expression in maxillary and labellar region.

(A) Scanning electron microscopy (SEM) image of D.mel and D.mim male adult head with mouthparts. (B) SEM image of D.mel and D.mim female adult head with mouthparts. False color was assigned to highlight maxillary palps (green) and labellar lobes (tan). (C) Bright-field image of the D.mel head. Dotted lines highlight the perpendicular orientation of maxillary palps (Mx) relative to the labellum (Lb). (D) Bright-field image of the D.mim head. Dashed lines indicate the parallel orientation of the maxillary palps and labellum. (E) Top: Schematic representation of Hox gene expression patterns for pb, Dfd, and Scr in D.mel. Bottom: Gal4-tagged pb, Dfd, and Scr driving expression of UAS-nlsGFP in adult mouthparts. Scale bars, 50 μm.

Fig. 2.. Replacement of D.mel pb genomic locus with that of D.mim.

(A) Diagram of the pb genomic locus from D.mel (38 kb) and D.mim (44 kb). pb-Fragments 1, 2, and 3 were sequentially replaced to generate a full locus replacement with pb-mim sequences. (B) Bright-field image of female and male heads. The dashed lines highlight the relative orientations of maxillary palps and labellum in D.mel, pb-mim replaced, and D.mim. False color highlights the maxillary palps (green). (C) SEM image of D.mel, pb-mim replaced, and D.mim female adult heads with mouthparts. False colors were also assigned to highlight the maxillary palp (green) and the labellar lobes (tan). Scale bars, 50 μm. (D) Female and male maxillary palps of D.mel, pb-mim replaced, and D.mim. Scale bars, 25 μm. Asterisks here and in panel G indicate notch on the inner surface. (E) Phylogenetic tree of three Drosophila species based on conserved amino acids based on the maximum likelihood method using the CLC Main Workbench program. Bars indicate evolutionary distance. (F) Sketch of a D. lobatopalpus male maxillary palp (provided by S. Roy). (G) For D.mel and pb-mim replaced, the neurons of the female and male maxillary palps were stained with anti-Elav antibodies. Dotted white lines denote the boundaries of the maxillary palps. Scale bars, 25 μm. The graph at the bottom shows the number of neurons on the medial surfaces (left side of dotted line) of maxillary palps in D.mel and pb-mim replaced flies. Error bars indicate SD; number of maxillary palps scored (n) = 7. ****P < 0.0001.

Fig. 3.. pb expression pattern changes in concert with changes in maxillary palp structure and orientation in pb-mim replaced flies.

(A) Schematic of the pb genomic region incorporating a Gal4-T2A insertion at the pb start codon (endogenous) followed by the cDNAs encoding D.mel:Pb, D.mim:Pb, or the human Hox pb ortholog (H.sap:HoxA2) with transcription terminated by two stop codons. (B) Homozygous endogenous Pb, D.mel:Pb cDNA, D.mim:Pb cDNA, and H.sap:HoxA2 cDNA fly heads. Scale bar, 50 μm. (C) Schematic of the pb D.mel genomic region with a Gal4-T2A insertion at the pb start codon (melPb-G4, top) and the pb-mim replaced genomic region with a Gal4-T2A insertion at the pb start codon (mimPb-G4, bottom). (D) Expression patterns provided by melPb-G4 and mimPb-G4 are shown as marked by their activation of UAS-nlsGFP in maxillary palps and proboscis in late pupae; the dashed box indicates the region of Pb expression changed between melPb-G4 and mimPb-G4. Scale bar, 50 μm. (E) Graph represents normalized mean GFP intensity between dashed box region with labellum in melPb-G4 and mimPb-G4 female (n = 30) and male (n = 22) late pupae. Error bars indicate SD. *P < 0.05, and ****P < 0.0001. (F) Representative bright-field image of heads of flies with either melPb-G4 controls (left) or melPb-G4>UAS-Dfd with tub-Gal80^ts female (center) and male (right) head. Scale bar, 50 μm.

Fig. 4.. Summary diagram.

Diagram illustrates full replacement of the endogenous pb locus of D.mel (fly head and gene locus on right) with orthologous sequences from D.mim (fly head and gene locus on left) and the effects of this replacement on adult mouthpart structures and pb expression (bottom).