Drosophila spalt/spalt-related mutants exhibit Townes-Brocks' syndrome phenotypes

Abstract

Mutations in SALL1, the human homolog of the Drosophila spalt gene, result in Townes-Brocks' syndrome, which is characterized by hand/foot, anogenital, renal, and ear anomalies, including sensorineural deafness. spalt genes encode zinc finger transcription factors that are found in animals as diverse as worms, insects, and vertebrates. Here, we examine the effect of losing both of the spalt genes, spalt and spalt-related, in the fruit fly Drosophila melanogaster, and report defects similar to those in humans with Townes-Brocks' syndrome. Loss of both spalt and spalt-related function in flies yields morphological defects in the testes, genitalia, and the antenna. Furthermore, spalt/spalt-related mutant antennae show severe reductions in Johnston's organ, the major auditory organ in Drosophila. Electrophysiological analyses confirm that spalt/spalt-related mutant flies are deaf. These commonalities suggest that there is functional conservation for spalt genes between vertebrates and insects.

Fig. 1.

sal expression in the developing second antennal segment. (A) Sal protein (red) and GFP (green) are coexpressed (yellow) in the presumptive second antennal segment (a2) in a third-instar larva of genotype sal-GAL4; UAS-GFP-nls. This indicates that the sal-GAL4 line approximately reproduces the sal expression pattern in the antenna. (B) In a late pupal antenna of the same genotype, GFP (green) is expressed in both epidermal cells and JO cells in a2. (C, C′, D, and D′) At earlier pupal stages, sal-GAL4 drives expression of nuclear GFP (green) strongly in epidermal cells (e) of distal a2 and proximal a3. The actin bundles of the scolopale cells have been stained with rhodamine-phalloidin (pink). The a2/a3 joint is labeled. C and C′ are lateral views of distal a2 and proximal a3. D and D′ are highe-magnification views looking distally toward the a2/a3 joint from proximal a2. GFP is weakly expressed in some of the scolopale cell nuclei (arrowheads).

Fig. 2.

sal- and salr-null JOs develop abnormally and degenerate during pupal stages. (A and A′) Wild-type pupal antenna stained with anti-horseradish peroxidase (HRP; blue) and rhodamine-phalloidin (pink). Anti-HRP detects neuronal membranes. Phalloidin binds to actin, which is particularly prominent in the scolopales of the scolopale cells. Note the large anti-HRP-labeled axon bundle exiting a2 proximally (arrow). (B and B′) Similar staining of a sal^FCK-25/Df(2L)32FP-5 FRT40A antenna. Scolopidia are forming, albeit at reduced numbers, and are highly disorganized. The anti-HRP-labeled axon bundle exiting a2 proximally is indicated with an arrow. (C) Internal optical section of part of a wild-type adult antenna. The arrow indicates the a2/a3 joint to which the JO attaches. (D) Similar view of a sal^FCK-25/Df(2L)32FP-5 FRT40A antenna in which the specialized a2/a3 joint is lacking. (E) Histological section of part of a wild-type antenna. The arrows indicate clusters of scolopidia that comprise the JO. (F) Similar histological section of a sal^FCK-25/Df(2L)32FP-5 FRT40A antenna. Arrows indicate the few remaining scolopidia. In some antennae of this genotype, no scolopidia are observed.

Fig. 3.

Drosophila with sal- and salr-null antennae are completely deaf. (A) Strong sound-evoked potentials recorded from the antennal nerve of wild-type flies are completely absent in sal/salr mutants. Shown for the wild type is the averaged response (avg) to 10 presentations of the computer-generated pulse song; this average is almost indistinguishable from the response to individual presentations (not shown). For the mutant [sal^FCK-25/Df(2L)32FP-5 FRT40A], the response to a single presentation of the pulse song indicates the true level of unevoked background activity; averaging (10 presentations) reduces this background to reveal that there is no indication of even a subtle response. (B) Histogram of response amplitudes in sal/salr^- and control flies of the Oregon R wild-type strain. The average amplitude for the control group was 1,243 μV, with a maximum of 1,622 μV, whereas the mutant flies showed an average of 52 μV, with a maximum of 85 μV. The amplitudes in sal/salr^- flies represent solely background noise; there is no detectable evoked response.

Fig. 4.

sal and salr expression and function in the male genitalia and the testes. (A) Sal protein (red) and GFP (green) are coexpressed (yellow) in the male genital disk of a third-instar larva of genotype sal-GAL4;UAS-GFP-nls. This indicates that the sal-GAL4 line reproduces the sal expression pattern in the genital disk. There is GFP in some cells where Sal protein is not detected. This may be caused by perdurance of GFP. (B) Left half of male pupal genitals of the same genotype in which GFP is expressed in the lateral plate, posterior lobe, and genital arch. The arrow points to the nucleus of a GFP-expressing cell. The arrowhead indicates a bristle socket with background autofluorescence. The dotted line indicates the ventral midline. The sal/salr expression domain in the female genital disk (not shown) is much smaller than that in males. This finding suggests that sal and salr may be differentially regulated between the sexes and that this could contribute to Drosophila sexual dimorphism. (C) Wild-type adult male anal plates and genitalia. (D) Adult male anal plates and genitalia from an animal of genotype y hs-FLPase; P[y+] FRT40A/Df(2L)32FP-5 FRT40A in which large sal-null clones were induced. The right lateral plate and posterior lobe are absent, as is a portion of the genital arch. (E) Adult male anal plates and genitalia from another animal of genotype y hs-FLPase; P[y+] FRT40A/Df(2L)32FP-5 FRT40A in which sal/salr null clones were induced. The right genital arch and posterior lobe are absent, as is a portion of the lateral plate. (F) Higher-magnification view of the area boxed in E. Beneath where the right posterior lobe would have been in a wild-type animal, sal/salr null clones can be detected by virtue of their y- bristles (white arrowheads). This tissue appears normal, and thus probably does not require sal or salr function. Adjacent wild-type tissue has dark bristles (black arrowheads). (G) Testes (arrows) from wild-type (Left) and sal mutant [Right; y hs-FLPase; FRT40A/Df(2L)32FP-5 FRT40A] animals in which clones had been induced. The testes from the sal mutant are smaller, lighter in color, and abnormally coiled. ap, anal plate; ga, genital arch; lp, lateral plate; pl, posterior lobe (pl).

Fig. 5.

Structures affected in both Drosophila and humans with sal mutations and the genetic hierarchies governing ear development. (A) Dll regulates both sal and ato during development of the Drosophila auditory apparatus (38, 48). (B) Dll, sal, and ato homologs, the Dlx, Sall, and Ath genes, respectively, are involved in vertebrate ear development (8, 11-13, 54-57). If their genetic relationships prove similar, it would support the idea that a common vertebrate/invertebrate ancestor possessed a primitive ear whose development was governed by these genes.