Distinct functions of homeodomain-containing and homeodomain-less isoforms encoded by homothorax

Abstract

The homothorax (hth) gene of Drosophila melanogaster is required for executing Hox functions, for head development, and for forming the proximodistal (PD) axis of the appendages. We show that alternative splicing of hth generates two types of protein isoforms, one that contains a DNA-binding homeodomain (HthFL) and one that does not contain a homeodomain (HDless). Both types of Hth isoforms include the evolutionarily conserved HM domain, which mediates a direct interaction with Extradenticle (Exd), another homeodomain protein. We show that although both HthFL and HDless isoforms of Hth can induce the nuclear localization of Exd, they carry out distinct sets of functions during development. Surprisingly, we find that many of hth's functions, including PD patterning and most Hox-related activities, can be executed by the HDless isoforms. In contrast, antennal development shows an absolute dependency on the HthFL isoform. Thus, alternative splicing of hth results in the generation of multiple transcription factors that execute unique functions in vivo. We further demonstrate that the mouse ortholog of hth, Meis1, also encodes a HDless isoform, suggesting that homeodomain-less variants of this gene family are evolutionarily ancient.

Figure 1.

The hth locus encodes for evolutionary conserved HDless variants. (A) Genomic organization of hth (not shown to scale). White and gray boxes denote noncoding and protein-coding exons, respectively, while angled lines represent sites of alternative splicing. The 6′- and 7′-specific ORFs are indicated by the dashed and black boxes, respectively. (B) In situ hybridization using probes for specific hth isoforms shows very similar expression patterns during both embryonic (whole embryo, top panels) and larval (e.g., eye-antennal imaginal disc, bottom panels) stages. White and black arrows indicate high levels of hth expression in the gastric caeca and the thoracic ectoderm, respectively. White arrow heads point to hth staining in the visceral mesoderm. The positions of the probes used are indicated in A. See also Supplementary Figure 1. (C) Comparison of the predicted protein sequences of the 6′ and 7′ isoforms. The species indicated are D. melanogaster (D. mel), D. pseudoobscura (D. pse), D. mojavensis (D. moj), D. virilis (D. vir), A. gambiae (A. gam), M. musculus (Mm), and Homo sapiens (Hs). The protein sequences of exon 6 and alternative exon 6 and 7 are compared. Amino acids conserved across all species are outlined by the gray shading. Sequence conservation for the 6′ and 7′ exons within the insect or mammal subgroups are boxed. The two black triangles demarcate the bypassed splice junction at the end of exon 6. Underlined in black are two small motifs common to the 6′ and 7′ isoforms.

Figure 2.

Hth HDless isoforms are capable of interacting with Exd. (A) Immunoblot of wild-type (wt), hth^P2, and hth^100-1 embryos stained with an anti-Hth antibody that detects all HM-containing isoforms (α-Hth). No Hth isoforms are detected in hth^P2 embryos, while only putative HDless isoforms are present in hth^100-1 embryos. (Red asterisk) HthFL; (black asterisk) HDless; (gray asterisks) additional HDless isoforms are observed in hth^100-1 extracts, probably as a result of the premature stop codon in exon 9. (Bottom) Blue Coomassie staining of a portion of the same membrane showing equivalent loading. (B) Immunoprecipitation of total embryonic extracts with either preimmune serum (mock IP) or anti-Exd antibody (Exd IP). The blot was probed with either an α-Hth (left panel) or an antibody raised specifically against the HD and C-tail of Hth (α-HthHD; right panel). Both full-length and HDless variants of Hth are coimmunoprecipitated with Exd, but only the larger isoform is detected with the anti-HthHD antibody. Equivalent amounts of extracts were loaded (data not shown). The additional band observed in both mock and Exd IPs in the right panel is likely due to cross-reactivity with the antibody used for the IP. (C) Wing imaginal discs with mitotic clones of hth^P2 and hth^100-1, marked by the absence of GFP (green), stained with anti-Hth and anti-Exd as indicated. hth^P2 clones have no detectable Hth staining and no nuclear Exd, while Hth and nuclear Exd are still detected in hth^100-1 clones. Arrows point to mutant tissue. (D) Wing or haltere imaginal discs with mitotic clones of hth^P2 and hth^100-1, marked by the absence of GFP (green), stained with anti-HthHD and anti-Hth6′ as indicated. hth^100-1 mutant clones are devoid of the HD-containing isoforms, but still stain for the 6′ HDless variant. Neither form was detected in hth^P2 mutant tissue. Arrows point to mutant tissue.

Figure 3.

HDless isoforms are necessary for embryonic patterning. (A) RT–PCR from embryos injected either with buffer or siRNAs directed against the 6′ and 7′ isoforms. Specific reduction of both the 6′ and 7′ transcripts, but not the HD-containing mRNAs was observed. RT–PCR for ribosomal protein 49 transcripts (RP) was used as a control. (B) Cuticle preparations of wild-type, hth^P2, hth^100-1, and 6′ + 7′ siRNA-injected embryos. (Top panels) Head involution defects can be observed in all mutant and siRNA-injected embryos. Strong posterior-directed transformations of the A1 and A2 segments were observed in hth^P2 and 6′ + 7′ siRNA-injected embryos and weaker transformations of the A1 segment were observed in hth^100-1 embryos (arrows). Arrowheads identify the KOs. (Bottom panel) Segmental fusions were observed in hth^P2 and 6′ + 7′ siRNA-injected embryos but not in hth^100-1 cuticles.

Figure 4.

Differential requirement of the Hth HD for the transcriptional regulation of fkh and lab. (A) Embryos carrying fkh[250]-lacZ (top panels), lab48/95-lacZ (middle panels), and lab550-lacZ (bottom panels), stained for β-galactosidase (β-gal). fkh250-lacZ is expressed in wild-type and hth^100-1 embryos, but not in hth^P2 embryos. Wild-type embryos carrying the lab48/95-lacZ or lab550-lacZ transgenes show a very similar pattern of nuclear β-gal in a central band of endodermal cells. In hth^100-1 embryos, lab 48/95-lacZ expression is lost, while lab550-lacZ staining is still present, underlying a differential requirement for the Hth HD by the two enhancers. In hth^P2 embryos, both lab 48/95-lacZ and lab550-lacZ are lost. (Arrows) LacZ positive staining; (asterisks) no lacZ expression, although some background fluorescence is observed in these midguts. (B) HDless/Exd and Lab can form a complex on the lab48/95 element in vitro, as attested by EMSA analysis. Binding is cooperative, but weaker than that observed for HthFL/Exd/Lab.

Figure 5.

The Hth HD is required for the specification of antennal fates. (A) Adult wild-type, hth^P2, and hth^100-1 mutant antennae. Transformation of the antenna toward leg is observed in both hth^P2 and hth^100-1 appendages, suggesting a strict requirement for the HD in the instruction of antennal identity. (B) hth^P2 and hth^100-1 mitotic clones in antennal imaginal discs, marked by the absence of GFP. (Top panels) hth^P2 and hth^100-1 clones are devoid of Sal staining, a marker of antennal fates. Asterisks mark the mutant tissue. (Bottom panels) The Hth HD is also necessary for the repression of leg identity in the antenna, as shown by the expansion of dac expression into a leg-like pattern in both hth^P2 and hth^100-1 mutant clones (arrowheads).

Figure 6.

The Hth HD is dispensable for the formation of a correct PD axis in ventral appendages. (A–C) Adult structures are shown for wild-type (A), hth^P2 (B), and hth^100-1 mutant legs (C). hth^P2 mutant legs are characterized by the loss of proximal elements, while hth^100-1 mutant legs have a normal PD axis. (D,E) High-magnification views of the proximal domains of typical hth^P2 (D) and hth^100-1 (E) legs. Bracted bristles, indicative of more medial/distal identities can be observed in hth^P2, but not hth^100-1, proximal domains (arrowheads). (F,G) Mitotic clones for hth^P2 (F) and hth^100-1 (G) in leg imaginal discs. hth^P2 clones show loss of tsh, a proximal fate marker, and the derepression of dac, a medial marker (arrows). hth^100-1 clones show no loss of tsh, or derepression of dac. All clones are marked by the absence of GFP (green). Although dac expression was usually normal, we observed derepression in some hth^100-1 clones located in the dorsal-most region of the disc (data not shown). This rare dac derepression might account for single bracted bristles in otherwise normal trochanters of some hth^100-1 legs (data not shown).

Figure 7.

Altering the balance of HthFL and HDless isoforms produces unique phenotypes. (A,B) Wing imaginal discs containing flip-out clones that ectopically express HthFL (A) or HDless (B) Hth isoforms. Clones are marked by the presence of GFP (green). Expression of HthFL leads to a reduction in the amount of endogenous HDless isoforms, as visualized by anti-Hth6′ staining (A, arrow), while expression of HDless leads to a reduction in the amount of endogenous HthFL, as visualized by anti-HthHD staining (B, arrows). (C) The range of head phenotypes observed in hth-Gal4; UAS-HthFL adult flies. (Class I) Small eyes (arrow); (class II) no eyes; (class III) no head, only the proboscis remains. (D) Frequencies of phenotypes observed in hth-Gal4; UAS-GFP, hth-Gal4; UAS-HDless, hth-Gal4; UAS-HthFL, hth-Gal4; UAS-HthFL, UAS-HDless adults. “n” refers to the number of flies examined; for class I and II, eyes were scored individually. Because there were 10 class III (headless) flies in the HthFL experiment, only 53 flies were examined for the antenna-to-leg transformation.