The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye

Abstract

A new Drosophila Pax gene, sparkling (spa), implicated in eye development, was isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system and in cone, primary pigment, and bristle cells of larval and pupal eye discs. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. We suggest close functional analogies between Spa and Pax2 in the development of the insect and vertebrate eye.

Figure 1

Cross-reactivity of anti-Poxm antiserum. A heterozygous Df(3R)dsx^D+R5 embryo (A), a homozygous Df(3R)dsx^D+R5 embryo (B), and a homozygous Df(4)G embryo (C) were probed with the anti-Poxm antiserum to demonstrate the cross-reactivity of the antiserum in embryos lacking the poxm locus (B) or the locus of the cross-reacting antigen (C). A wild-type embryo (D) was hybridized in situ with a digoxigenin-labeled cpx1 cDNA, which encodes the cross-reacting antigen and was isolated by immunoscreening a cDNA expression library with the anti-Poxm antiserum. Lateral views of stage 13 embryos are shown with their anterior to the left and their dorsal sides up.

Figure 2

Expression patterns of cpx1 in wild type and spa mutants. A wild-type third instar larval eye–antennal disc (A), leg disc (B), and ventral ganglion with brain (C; ventral view) were hybridized with a digoxigenin-labeled cpx1 cDNA. The expression pattern of cpx1 in a wild-type third instar larval eye–antennal disc was compared to that in corresponding discs of homozygous spa¹ (D), heterozygous spa^A (E), and homozygous spa^pol mutants (F), using the same DNA probe. (br) Brain; (MF) morphogenetic furrow; (vg) ventral ganglion.

Figure 3

Structural organization of the spa locus and deduced sequence and conserved domains of the Spa protein. (A) The spa locus is shown with respect to a genomic EcoRI map at the top and three overlapping inserts from a λ DASH II genomic library below. Underneath, an enlarged detailed restriction map of the genomic region spanning the spa transcript is shown, above which two spa mutations, the spa¹ insertion and the spa^pol deficiency, and an insertion polymorphism in intron 12 are indicated. Below the restriction map, the intron–exon structure and the open reading frame (in black; with paired domain P and octapeptide O) corresponding to the longest spa cDNAs from embryos and third instar larvae are depicted, and different splice variants are indicated. In third instar larvae, only two splice variants, which differed with respect to the presence or absence of exon 11, were detected by reverse transcriptase PCR amplification of total disc RNA. Both used the poly(A) addition site in intron 12 and therefore, lacked the putative inhibitory domain encoded by exon 13. Occasionally, an alternative 3′ splice acceptor site of intron 9 is used, resulting in the in-frame deletion of the first two amino acids encoded by exon 10. Abbreviations of restriction sites: (H) HindIII; (K) KpnI; (R) EcoRI; (RV) EcoRV; (S) SpeI; (Sal) SalI. (B) The deduced amino acid sequence of the longest open reading frame encoded by spa cDNAs with encircled methionines indicating the positions of the five potential initiators preceding the paired domain. Underlined are the paired domain and octapeptide by solid lines, a conserved highly charged dodekapeptide by a dotted line, and a peptide homologous to the amino terminus of a homeodomain by a dashed line. (C–E) The conservation of the octapeptide and highly charged dodekapeptide sequences (C), of the fractional homeodomain sequences (D), and of the carboxy-terminal transactivation and inhibitory domains of Pax2, Pax5, and Pax8 and Spa (E). (F) The conservation of domains and positions of introns in Spa and Pax2, Pax5, and Pax8 proteins. The position of introns are indicated by filled triangles, alternative splice sites by open triangles in Spa and human Pax8. In addition to the paired domain (P), octapeptide (O), amino-terminal portion of a homeodomain (H), transactivation domain (A), and inhibitory domain (I), Ser/Thr-rich domains (S/T), Gln-rich domains (N), and highly charged regions (+/−) are indicated.

Figure 4

Localization of Spa protein in nuclei of cone, primary pigment, and bristle cells of the developing eye. Spa protein is observed in cone cells of a third instar larval eye disc (A). In a 24-hr pupal eye disc (B,C; dorsal side up), Spa remains detectable in nuclei of cone cells and appears in nuclei of primary pigment cells (B) and bristle cells (C). Horizontal marks in A indicate the regular spacing of the centers of stained hexagonal ommatidial rows 7–16 behind the morphogenetic furrow (MF). The number of ommatidial rows behind the morphogenetic furrow was assessed independently by double-staining for Armadillo, which outlines the growing ommatidial clusters, and Spa and subsequent confocal microscopy (not shown). (B) A more apical optical section than the basal section shown in C. (c) Cone cell; (1) primary pigment cell.

Figure 5

Disrupted surface structures of spa mutant eyes caused by a disorganized underlying cellular pattern. Scanning electron micrographs of left eyes (A–D) and corresponding histological sections displaying the underlying ommatidial patterns (E–H) of 3-day-old wild-type flies (A,E) are compared to those of 3-day-old homozygous spa¹ (B,F), spa^A/+ (C,G), and homozygous spa^pol flies (D,H). Each eye (whole mount or section) is shown with its anterior to the left and dorsal side up.

Figure 6

Abnormal development of cone and pigment cells in spa mutants. Early (24 hr APF at 25°C; A,C,E,G) and mid-pupal (45 hr APF at 25°C; B,D,F,H) eye discs of wild-type (A,B), homozygous spa¹ (C,D), spa^A/+ (E,F), and homozygous spa^pol (G,H) flies were stained with cobalt sulfide to visualize their cone and pigment cells at the apical surface of the retina. Examples of primary (1), secondary (2), and tertiary (3) pigment cells and of bristle cells (arrows) are marked in wild-type discs. Discs are shown with their anterior to the left and dorsal side up.

Figure 7

Loss of Spa function affects expression of Cut and Bar proteins in cone and primary pigment cells of the eye disc. Cut expression in cone cells of a spa^pol (B), as compared to a wild-type (A), early pupal eye disc (24 hr APF at 25°C) is reduced. However, Cut expression in cone cells of a spa^pol mid-pupal eye disc (45 hr APF at 25°C; D) recovers and increases above the level observed in a wild-type mid-pupal eye disc (C). Bar expression in primary pigment cells of a wild-type (E) was also compared to that of a spa^pol (F) mid-pupal eye disc. Unlike the loss of its expression in primary pigment cells, Bar protein levels appear unaffected in bristle cells of a spa^pol (H) when compared to that of a wild-type mid-pupal eye disc (G). Note that the S12 anti-BarH1 antiserum used recognizes both BarH1 and BarH2 proteins (Higashijima et al. 1992b).