Conservation of the expression and function of apterous orthologs in Drosophila and mammals

Abstract

The Drosophila apterous (ap) gene encodes a protein of the LIM-homeodomain family. Many transcription factors of this class have been conserved during evolution; however, the functional significance of their structural conservation is generally not known. ap is best known for its fundamental role as a dorsal selector gene required for patterning and growth of the wing, but it also has other important functions required for neuronal fasciculation, fertility, and normal viability. We isolated mouse (mLhx2) and human (hLhx2) ap orthologs, and we used transgenic animals and rescue assays to investigate the conservation of the Ap protein during evolution. We found that the human protein LHX2 is able to regulate correctly ap target genes in the fly, causes the same phenotypes as Ap when ectopically produced, and most importantly rescues ap mutant phenotypes as efficiently as the fly protein. In addition, we found striking similarities in the expression patterns of the Drosophila and murine genes. Both mLhx2 and ap are expressed in the respective nerve cords, eyes, olfactory organs, brain, and limbs. These results demonstrate the conservation of Ap protein function across phyla and argue that aspects of its expression pattern have also been conserved from a common ancestor of insects and vertebrates.

Figure 1

Amino acid sequence comparison of Drosophila Ap and its mouse (MLHX2) and human (HLHX2) orthologs. (A) Sequence alignment. Identical amino acids between the three proteins are displayed in reverse type with capital letters, whereas conservative substitions are displayed in lower case letters. Asterisks indicate the only four different residues between the mouse and human proteins in the overlapping region. The two tandem LIM domains, the putative nuclear localization signal (NLS), and the homeodomain (HD) are underlined. The consensus residues of the LIM domains are highlighted with black circles. Gaps denoted by dots have been inserted to maximize sequence alignment. (B) Domain comparisons between Drosophila Ap, its mammalian orthologs, and other LIM-homeodomain proteins. These are: ISL-1 from rat (60), LIN-11 and MEC-3 from C. elegans (20, 21). Percentage of amino acid sequence identity is indicated within the LIM domains and homeodomain.

Figure 2

Comparison of ap and mLhx2 expression patterns. (A) mLhx2 expression in the forebrain and limbs of an E11.5 mouse embryo. (B) At this stage, mLhx2 is expressed in the walls of the lateral ventricles (Lv) and third ventricle (III) of the brain. In the eyes, mLhx2 is expressed in the future nervous layer of the retina (arrow) and in the optic stalk (not shown). (C) ap expression in the brain hemispheres (arrow) of a stage 15 fly embryo. (D) In the adult fly, Ap is immunodetected in the lamina (La) and medulla (Me) of the optic lobe and in the central brain (arrow). (E and F) At E11.5, mLhx2 is expressed along the neural tube (E) in a group of dorsal commissural interneurons (arrow in F). (G) ap expression in the VNC (arrows) of a stage 15 fly embryo. Out of focus, expression is also evident in the brain hemispheres and muscles of the body wall and pharynx. (H) Drosophila larval central nervous system showing expression of a UAS:tau-GFP responder driven by the ap-VNC enhancer. Note the axonal projections of ap-expressing interneurons along ascending longitudinal tracts. (I and J) mLhx2 expression in E11.5 mouse limbs. Label is detected in the mesenchyme, in a region roughly corresponding to the progress zone (I). In cross-sections, mLhx2 is observed in both dorsal (up) and ventral (down) regions of the limb and is excluded from the apical ectodermal ridge (arrow in J). (K) Ap immunodetection in the dorsal compartment of a Drosophila wing imaginal disc. (L) Section from an E11.5 mouse embryo showing mLhx2 expression in the olfactory epithelium surrounding the nasal pits (arrows). (M) ap expression in the center of a Drosophila antennal disc. (N) X-Gal stain of a Drosophila adult head carrying the enhancer detector ap^rk568, which expresses lacZ in an ap-like fashion. Note lacZ expression in the fly olfactory organs: the antenna (a) and the palpus (p).

Figure 3

Ectopic expression of ap and hLhx2 in wing imaginal discs produce similar wing phenotypes. (A and D) UAS:lacZ expression from the ptc-GAL4 (A) and 32B-GAL4 (D) drivers. (B and C) Wing phenotypes caused by ectopic expression of ap (B) and hLhx2 (C) by using the ptc-GAL4 driver. (E and F) Wing phenotypes caused by ectopic expression of ap (E) and hLhx2 (F) by using the 32B-GAL4 driver. (G) Wild-type wing.

Figure 4

hLhx2 correctly regulates ap target genes in Drosophila wing imaginal discs. Panels show β-galactosidase or Ser immunodetections following ectopic ap or hLhx2 expression in third instar larvae wing discs. The wild-type pattern of the fng-lacZ (A), Ser (D), vg-lacZ (G), and wg-lacZ (J) markers are depicted at the top. The dpp-GAL4 driver (M) was used to direct expression of the indicated UAS transgenes along the antero-posterior axis. Note that in all cases wing discs coexpressing dpp-GAL4 and either UAS:ap (B, E, H, K) or UAS:hLhx2 (C, F, I, L) exhibit ectopic activation of the molecular markers within the ventral compartment. Arrowhead indicates the wild-type expression of fng in the ventral compartment. Arrows point to the sites of ectopic expression.

Figure 5

hLhx2 rescues the wing phenotype of ap mutants. (A) Wing imaginal disc expressing UAS:lacZ from the ap-GAL4^MD544 driver. This GAL4 P-element insertion in ap inactivates the gene and recapitulates its expression pattern. (B) ap-GAL4^MD544/ap^UGO35 mutant fly. Note the lack of wings, halteres, and the scutellum region of the notum. (C and D) ap-GAL4^MD544/ap^UGO35 mutant flies carrying the UAS:ap and UAS:hLhx2 transgenes, respectively. Note that, in both cases, the wing, notum (white arrow), and haltere (black arrow) phenotypes are rescued.