Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis Anastasios Pavlopoulos1 and Michael Akam1 Laboratory for Development and Evolution, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom Edited* by Sean B. Carroll, University of Wisconsin, Madison, WI, and approved January 7, 2011 (received for review October 7, 2010) Hox genes encode highly conserved transcription factors that expressed in the hind wings of all winged insects studied (10–13), regionalize the animal body axis by controlling complex develop- but it is only in Dipterans that Ubx brings about the striking mental processes. Although they are known to operate in multiple morphological transformation of hind wings into halteres. cell types and at different stages, we are still missing the batte- Compared with the wing blade, the haltere capitellum is reduced ries of genes targeted by any one Hox gene over the course of in size by a fivefold reduction in cell number and an eightfold a single developmental process to achieve a particular cell and reduction in apical cell surface area (14, 15). It forms a balloon organ morphology. The transformation of wings into halteres by shape rather than a flat bilayer because of changes in cell ad- the Hox gene Ultrabithorax (Ubx)inDrosophila melanogaster hesion, it shows no differentiation between vein and intervein presents an excellent model system to study the Hox control of cells, and it lacks marginal bristle rows (15). transcriptional networks during successive stages of appendage The wing epithelium develops in the absence of any Hox input morphogenesis and cell differentiation. We have used an inducible (16) and represents one of the best-studied model systems in misexpression system to switch on Ubx in the wing epithelium at terms of genetic networks underlying pattern formation, growth, — successive stages during metamorphosis in the larva, prepupa, and differentiation. Ubx modifies this wing developmental net- and pupa. We have then used extensive microarray expression pro- work by regulating the expression of target genes to generate filing and quantitative RT-PCR to identify the primary transcrip- fi halteres (17, 18). tional responses to Ubx. We nd that Ubx targets range from Molecular genetic studies have shown that many key patterning regulatory genes like transcription factors and signaling compo- genes in the larval wing primordium are indeed regulated by Ubx, nents to terminal differentiation genes affecting a broad reper- directly or indirectly, in the haltere primordium (14, 17, 19–25). toire of cell behaviors and metabolic reactions. Ubx up- and down- This finding has prompted the use of microarray expression regulates hundreds of downstream genes at each stage, mostly in fi a subtle manner. Strikingly, our analysis reveals that Ubx target pro ling to systematically identify differentially expressed genes genes are largely distinct at different stages of appendage morpho- between wing and haltere imaginal discs (26, 27). These genome- GENETICS genesis, suggesting extensive interactions between Hox genes and wide approaches, however, were not designed to reveal the pri- hormone-controlled regulatory networks to orchestrate complex mary transcriptional responses to Ubx and have uncovered the genetic programs during metamorphosis. cumulative effects from Ubx function throughout development. Moreover, most studies have focused disproportionately on the appendage specialization | homeotic genes | serial homology role of Ubx in appendage patterning and growth in larval stages, whereas the actual transformation in cell and organ morphology tudies of Hox genes first prompted the realization that bila- elicited by Ubx occurs later during metamorphosis (15). Sterians share an extensive genetic makeup for body patterning Elucidation of the architecture of Hox-controlled gene regu- (1) and have since spearheaded the quest for the genetic changes latory networks is fundamental to understanding morphogenesis that drive morphological evolution (2). Hox genes encode and cell differentiation and how these processes underlie di- homeodomain transcription factors that operate in many tissues versification of serially homologous structures like wings and and cell types and modulate a wide range of cell responses by halteres. To identify the genes directly regulated by Ubx to dis- controlling expression of subordinate target genes. Despite their tinguish halteres from wings, we used the TARGET version of the highly conserved roles in body regionalization, only a limited GAL4/UAS system (28) coupled with microarrays to profile number of Hox-controlled morphogenetic and differentiation transcriptional changes in wings shortly after Ubx misexpression programs have been studied in detail. For all of these processes, (unlike previous studies that compared wings with halteres that we do not understand the batteries of genes modulated by Hox express Ubx throughout development). Altering the temperature, proteins to bring about complex developmental transformations. fi The systematic elucidation of Hox targets has been hampered we were able to switch on ectopic Ubx speci cally in developing by the complexity and multiplicity of the regulatory networks that wing blades at levels similar to those observed in normal halteres Hox genes control and the short and degenerate binding sites of and measure the immediate transcriptional responses to Ubx at Hox proteins. It has been further complicated by the functional successive stages during larval, prepupal, and pupal development. redundancy of the Hox proteins and the cross-regulatory inter- actions between them. These factors have made it difficult to elucidate the complete set of target genes modulated by any one Author contributions: A.P. and M.A. designed research; A.P. performed research; A.P. Hox gene over the course of a single developmental process (3– analyzed data; and A.P. and M.A. wrote the paper. 8). Thus, it remains an open question of how these widely con- The authors declare no conflict of interest. served patterning genes link to the actual effectors of cell and *This Direct Submission article had a prearranged editor. organ morphology at a genome-wide level over time. Data deposition: The data reported in this paper have been deposited in the Gene Ex- The dorsal flight appendages of Drosophila melanogaster pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22354). present an excellent system to study Hox-controlled morpho- 1To whom correspondence may be addressed. E-mail: [email protected] or m.akam@ genesis. Drosophila and flies in general have evolved from four- zoo.cam.ac.uk. winged ancestors by modifying their hind wings into drumstick- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. like balancing organs called halteres (9). Ultrabithorax (Ubx)is 1073/pnas.1015077108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1015077108 PNAS | February 15, 2011 | vol. 108 | no. 7 | 2855–2860 Downloaded by guest on October 1, 2021 Results Genetic System for Precisely Controlled Ubx Misexpression in Developing Drosophila Wings. We generated an experimental fly line that produces a complete transformation of the wing blade to haltere capitellum (Fig. 1 A and D). This line combined the nab-Gal4NP3537 driver, a tub-GAL80ts transgene, and a UAS- UbxIa transgene (or a UAS-eGFP control), and it satisfied a number of essential criteria to study Hox gene function on a genome-wide scale. Fig. 2. Effects of Ubx misexpression in the wing epithelium. (A, A′, B, B′, C, With this system, Ubx (or eGFP) was misexpressed specifically and C′) Late pupal phalloidin (F-actin) staining of (A) WT wing, (B) WT hal- in the developing wing blade, a tissue of low complexity composed tere, and (C) experimental wing with a complete homeotic transformation of wing blade into haltere capitellum. High magnification of the same primarily of the vein and intervein cells, the marginal bristle cells, ′ ′ and a few sensilla (Fig. 1 C, F, and G). By measuring gene ex- appendages showing the characteristics of (A ) wing cells, (B ) haltere cells, and (C′) transformed wing cells that have acquired the properties of haltere pression in the fairly homogeneous wing epithelium, we ensured cells. (Scale bar, 25 μm.) the maximum sensitivity to detect transcriptional responses, be- cause each cell type made a relatively big contribution to the total transcript population. our microarray samples 16 h ATS when ectopic Ubx levels in the The experimental line used did not interfere with normal wing wing blade reached about 90% of the levels normally observed in development at 19 °C; a homeotic transformation of wings into the homologous region of the haltere (Fig. 3 F and J). This is halteres was only observed after a temperature shift (ATS) from about 10 h after the first detection of ectopic Ubx protein and 19 °C to 29 °C. The transformation was evident both at the organ about 5 h after ectopic Ubx levels reached 50% of WT haltere (Figs. 1 B and E and 2 A–C) and cell level (Fig. 2 A′–C′). The levels (Fig. 3I). Thus, we presumed that assaying gene expression wing blade turned into a hollow balloon composed of fewer cells 16 h ATS would preferentially identify primary rather than sec- that resembled haltere cells. Normal wing cells are squamous ondary transcriptional responses to Ubx. This assumption was stellate polygons that project one long hair. After misexpressing confirmed by all our subsequent analyses. Ubx, wing cells were transformed into haltere-like cuboidal cells We confirmed by in situ hybridization that ectopic Ubx in the that have a smaller rectangular surface area and produce mul- wing was capable of regulating two enhancers controlling the spalt tiple short hairs. and knot genes targeted by Ubx in WT halteres (19, 21). This We quantified the levels of ectopic Ubx protein in the wing analysis also suggested that samples collected 16 h ATS were epithelium of the experimental line ATS from 19 °C to 29 °C (Fig. appropriate to capture the primary transcriptional responses to – 3 A–J).