Gene duplication, lineage-specific expansion, and subfunctionalization in the MADF-BESS family patterns the Drosophila wing hinge

Abstract

Gene duplication, expansion, and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified, or altered to generate novel functions over evolutionary timescales. These features make gene duplication a powerful engine of evolutionary change. In this study, we explore these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains 16 similar members, each containing an N-terminal, DNA-binding MADF domain and a C-terminal, protein-interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, which we name hinge1, hinge2, and hinge3 are required for wing development, with a critical role in the wing hinge. hinge1 is a negative regulator of Winglesss expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax, and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing hinge, in spite of expansion and diversification for over 40 million years. The wing hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing hinge in a robust and fail-safe manner.

Keywords: BESS; MADF; Wnt/Wg; development; evolution.

Figure 1

The D. melanogaster MADF-BESS family codes for 16 members that are a consequence of gene duplication. (A) MADF-BESS family in D. melanogaster consists of 16 proteins coded by individual genes. The 16 proteins, represented in the figure, contain an N-terminal DNA-binding Myb/SANT like in Adf (MADF) domain and a C-terminal BEAF-32, Stonewall, Su (var) 3-7 homology (BESS) domain. MADF and BESS domains tend to be found together in a single polypeptide chain. Proteins labeled in orange have been characterized, while those in gray are the subject of this study and been named hinge genes based on their loss-of-function phenotype. (B) Maximum-likelihood phylogenetic tree of all MADF-BESS genes in dipterans. Branch lengths are proportional to mean substitutions per site. Orthologs of the 16 MADF-BESS genes cluster separately with the corresponding D. melanogaster gene (labeled on two-sided arrows), indicating that the duplication in the family occurred before the divergence of drosophilids. MADF-BESS genes from Culex, Anopheles, and Glossina morsitans have fewer orthologs and cluster separately (brown arrow) for the most part, indicating that, in their case, expansion in MADF-BESS was independent from that in drosophilids. The genes for different sequenced dipterans are color-coded to bring out this feature. The blue asterisk marks the genes that show short branch lengths and thus minimal sequence divergence.

Figure 2

MADF and BESS domains are expanded specifically in the Drosophila lineage. (A) MADF and BESS protein domains, when counted in representative members of the animal kingdom, indicate expansion in Drosophila lineage. (B) In arthropods, the number of individual MADF and BESS domains coded by the Drosophila genome is higher than in other sequenced species. The dipteran group is marked. (C) The expansion is dramatic in all members of the Drosophila lineage, confirming that that the expansion occurred in a common drosophilid ancestor. The Drosophilidae and the melanogaster group, which are referred to in the text, are marked.

Figure 3

Members of the MADF-BESS family (CG9437/hng1, CG8359/hng2, and CG13897/hng3) have roles in the development of the wing hinge. (A) Reduction of CG9437 transcripts in the wing-imaginal disc, by expressing UAS-CG9437 RNAi line, in the MS1096 expression domain leads to wing-hinge defects in the adult fly. Defects include a reduced/mispatterned alula, a bent hinge, and a disorganized costa region. The phenotype is 100% penetrant and is dose dependent with an increased knockdown leading to a stronger phenotype that also affects the more distal wing blade. (B) Reduction of hng1 transcripts in the more ubiquitous daughterless (Da) expression domain also lead to wing-hinge defects, indicating specific roles for hng1 in the hinge. (C) The defects can be rescued by co-expression of UAS-hng1 in the same expression domain. Expression of UAS-hng1 by itself does not affect normal wing development. (D) Two other genes (CG8359/hng2 and CG13897/hng3) in the family also show a similar phenotype on knockdown. The genes have been named as the hinge genes based on their loss-of-function phenotype. (E) Double knockdowns of hng1+hng2 or hng1+hng3 in a single dose each mimic the phenotypes seen in an increase dose of hng1 knockdowns. (F) Deficiency in the 85B1-85C2 genomic regions removes hng2 completely, and 68% of flies show the wing-hinge phenotype. This, when combined with hng1 (MS1096/+; UAS-hng1-RNAi/+) knockdown, gives enhancement in the Deficiency phenotype. Deficiency in the 57C3-57C7 genomic region removes hng1 completely and also shows enhancement when combined with hng1 (MS1096/+; UAS-hng1-RNAi/+) knockdown. (G) The VDRC KK lines used in this study are inserted into a single site in the regulatory region of the tio locus. A knockdown of tio does not enhance the hng1i phenotype, arguing against a contribution of the tio locus to the observed phenotype. (H) Parameters such as wing size (mm²) and alula size (mm²) are measured to quantify the phenotype in this and subsequent experiments. The phenotype (MS1096/+; UAS-hng1 RNAi/+) is enhanced in the presence of a Deficiency in the CG9437/hng1 locus and is rescued by expressing UAS-hng1. Arrowhead indicates the MS1096/+; UAS-hng1/+ line used as a control for statistical analyses. *P < 0.05, **P < 0.01, and ***P < 0.001. (I) In situ hybridization against hng1 and hng2 transcripts confirms that hng1 and hng2 are expressed in wing imaginal discs. The brackets mark the wing pouch with the red arrows marking part of the hinge-forming region. (J) Expression pattern of hng3 as shown by anti-GFP staining of an enhancer trap line (YB0086DE) in wing imaginal discs. The bracket marks the wing pouch with the yellow arrows marking part of the hinge-forming region. (K) Expression pattern of Coop (CG1621) as shown by anti-GFP staining protein trap line in wing imaginal discs. The bracket marks the wing pouch with the yellow arrows marking part of the hinge-forming region. (L) The hng1i animal has a bent wing hinge, cannot fold its wings over the abdomen normally, and holds out its wings. The animals can flap their wings but cannot fly.

Figure 4

hinge genes are part of the GRN that patterns the wing hinge. (A) A knockdown of tsh and hth in the MS1096/+; hng1i/+ animal rescues the hng1 phenotype, whereas overexpression of hth enhances the hng1 phenotype severely. The RNAi and the UAS lines used to alter transcript levels for hth and tsh, by themselves, have mild hypomorphic effects. (B) Hth is broadened/derepressed in and around the gap region when hng1 expression is reduced in the MS1096-Gal4 expression domain in the wing imaginal discs. (C) A knockdown of jing in the MS1096/+; hng1i/+ animal leads to a rescue of the hng1 phenotype while co-expression of UAS-jing leads to an enhancement of the proximal wing phenotype. (D) Wg is derepressed in and around the gap region between the IR and the OR in the MS1096/+; UAS-hng1i/UAS-hng1i wing imaginal disc. (E) Wg is derepressed in a small stripe in the gap region when hng1 expression is reduced in the ptc-Gal4 expression domain. (F) hng1 knockdown in heterozygous Wg^spadeflag background in the MS1096-Gal4 domain mildly rescues the hinge defect. (G) Wing size (mm²) and alula size (mm²) are measured for genetics interactors of hng1 with known wing-hinge GRN genes. Arrowhead indicates the MS1096/+; UAS-hng1/+ line used as a control for statistical analyses. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

hng1 phenotype is enhanced by knockdown of other MADF-BESS genes. (A) CG6854, stwl, coop, CG3838, CG4404, and CG11723 knockdown in the hng1 background enhance the MS1096/+; UAS-hng1i/+ phenotype. The knockdown of these genes by themselves, with the exception of stwl, using RNAi lines does not affect the hinge significantly. The enhancement indicates that these genes are expressed in the cells that pattern the wing hinge and may have partially redundant roles in the hinge-mediated development of the wing hinge. (B) Wing size (mm²) and alula size (mm²) are measured for genetic interactions of hng1 with other MADF-BESS family genes. Arrowhead indicates the MS1096/+; UAS-hng1/+ line used as a control for statistical analyses. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

hng1 phenotype is rescued by expression of other MADF-BESS genes. (A) CG13204 and CG11723 expression rescue the MS1096/+;UAS-hng1i/+ phenotype. This indicates that MADF-BESS proteins retain similar biological activity and equivalence in terms of their protein function. (B) Wing size (mm²) and alula size (mm²) are measured for rescue of hng1 phenotype. Arrowhead indicates the MS1096/+; UAS-hng1/+ line used as a control for statistical analyses. *P < 0.05 and **P < 0.01.

Figure 7

Model for MADF-BESS function in the wing-hinge. (A) The GRN for wing-hinge development includes wg, tsh, hth, and exd as major patterning genes. In the hng1 loss of function, our data indicate an increase in activity of Tsh/Hth/Exd. hng1 appears to negatively regulate the Wg/Hth-positive autoregulatory loop. hng1 also negatively regulates tsh, possibly acting downstream of jing. (B) The three hng genes along with stwl appear to be functionally equivalent and are part of the GRN that patterns the wing hinge. Five additional genes retain, at least partially, functions of the hng family of genes and can replace, to an extent, hng function. The four hinge genes code for proteins (blue circles), which we hypothesize may be part of a dimer/tetramer that is the active transcriptional regulator. Function could be regulated by increasing/decreasing the concentration of the Hng proteins, with the concentration of the functional polymer dependent on spatiotemporal expression and also the levels of the hng genes.