Isolation and expression of a Pax-6 gene in the regenerating and intact Planarian Dugesia(G)tigrina

Abstract

The Pax-6 gene encodes a transcription factor containing both a paired and a homeodomain and is highly conserved among Metazoa. In both vertebrates and invertebrates, Pax-6 is required for eye morphogenesis, development of parts of the central nervous system, and, in some phyla, for the development of olfactory sense organs. Ectopic expression of Pax-6 from insects, mammals, cephalopods, and ascidians induces ectopic eyes in Drosophila, suggesting that Pax-6 may be a universal master control gene for eye morphogenesis. Platyhelminthes are an ancient phylum, originating from the base of spiralian protostomes, that bear primitive eyes, consisting of a group of rhabdomeric photoreceptor cells enclosed in a cup of pigment cells. The analysis of Pax-6 and its expression pattern should provide insights into the ancestral function of Pax-6 in eye morphogenesis. We have identified the Pax-6 gene of the planarian Dugesia(G)tigrina (Platyhelminthes; Turbellaria; Tricladida). This gene shares significant sequence identity and conserved genomic organization with Pax-6 proteins from other phyla. Phylogenetic analysis indicates that it clusters with the other Pax-6 genes, but in the most basal position. DtPax-6 is expressed as a single transcript in both regenerating and fully grown eyes, and electron microscopy studies show strong expression in the perykarion of both photoreceptor and pigment cells. Very low levels of expression also are detectable in other body regions. Because a bona fide Pax-6 homolog so far has not been detected in diploblastic animals, we speculate that Pax-6 may be typical for triploblasts and that the appearance of additional Pax genes may have coincided with increasingly complex body plans.

Figure 1

Alignments of the amino acid sequences of the paired domains (A) and homeodomains (B) of Pax-6 homologs of some vertebrates and invertebrates. The secondary structure of the domains are shown along the top. The consensus sequence for all genes of the paired family are compared with human PAX6 (Aniridia) and zebrafish as representatives for vertebrate sequences, Amphioxus Branchiostoma floridae (AmphiPax-6) (17), Ascidian Phallusia mammillata (PmPax-6) (11), cephalopod Loligo opalescens (LoPax-6) (13), sea urchin Paracentrotus lividus (PlPax-6) (12), Drosophila (Ey) (10), nemertean Lineus sangineus (LsPax-6) (14), and the nematode C. elegans (vab-3) (15, 16). Solid arrowheads indicate the position of intron–exon boundaries. Shaded bars indicate the Pax-6 specific amino acids (38). Percentages of sequence identity of the paired domains and homeodomains were determined by comparison with human PAX6 and planarian Pax-6, respectively, and are indicated after the sequences.

Figure 2

Phylogenetic trees of Pax gene paired domains (A) and homeodomains (B). The planarian DtPax-6 paired domains and homeodomains clearly cluster with other Pax-6 genes. Human Pax genes: PAX1, 2, 3, 5, 6 (Aniridia), 7, 8, 9; mouse Pax gene: Pax-4; quail Pax gene: Pax-QNR; zebrafish Pax genes: Pax[zf-a], [zf-b], zf-9; sea urchin (Paracentrotus lividus) Pax genes: Su-Pax B, B1, 6, 258; Halocynthia roretzi Pax genes: HrPax-37, 6, 258; Amphioxus Pax genes: Amphi-Pax1, 2, 6; Loligo opalescens Pax gene: Lo-Pax6; Drosophila Pax genes: sparkling, eyeless, poxneuro, poxmeso, paired, gooseberry neuro, gooseberry; C. elegans Pax genes: C. elegans 258, vab-3; Lineus sanguineus Pax gene: Ls-Pax6; Dugesia(G)tigrina Pax gene: Dt-Pax6; seanettle Pax genes: seanettle A, B; Hydra Pax genes: Hydra A, B; Acropora millepora Pax genes: Pax-Aam, Pax-Cam.

Figure 3

DtPax-6 expression in Dugesia(G)tigrina. (A) Northern blot: 10 μg of poly(A)⁺ from intact adults (lane 1) and 7-day-regenerating (lane 2) planarians hybridized at high stringency conditions with 0.55-kb DtPax-6 3′ cDNA fragment. A single band of 2.1 kb was observed in both lanes. Blots were rehybridized with a Drosophila 5C actin gene probe (39) to control for levels of RNA loaded. The transcript size and the positions of 23S and 16S RNAs are indicated. (B) Adult planarian DtPax-6 gene expression at different anteroposterior (–10) regions deduced by RT-PCR amplification using specific oligonucleotides from exons 3 and 4 from the DtPax-6 cDNA sequence (positions 1226–1250 and 1363–1387, respectively) that produce a fragment of 162 nt. As an internal control, we used in the same amplification two new sets of specific oligonucleotides from the Dth-2 cDNA sequence [positions 376–400 and 817–841 (32), respectively] that produce a control fragment of 465 nt. Lane M, 100-bp ladder (Pharmacia) molecular size marker. Lane C, non-retro-transcribed planarian total RNA as a control.

Figure 4

DtPax-6 expression in intact and regenerating planarian adults by in situ hybridization. (A–H) Intact organisms. (I and J) Seven days of regeneration. (K and L) Fourteen days of regeneration. In situ hybridizations were performed in paraffin sections with ³⁵S-labeled RNA antisense (A, C, I–L) and sense (B and D) or by electron microscopy with digoxigenin-labeled RNA antisense (E–G) and sense (H). A and B are transversal sections. C, D, and I–L are sagittal sections; dorsal is in the top and ventral is in the bottom; anterior is to the left and posterior is to the right. A–D, I, and K are dark-field views; J and L are bright-field views. (A and C) Dark-field view of transversal and sagittal paraffin sections at the eye level of an intact planarian. The dorsal position of the eyes is easily observable by the presence of pigmented cells. Clear homogeneous expression can be observed exclusively in the whole eye spot. (B and D) Similar head sections hybridized with a sense probe. Note the absence of silver grains in the eye and the artifactual grains surrounding the pigment cells because of the pigment granules that are refractile in dark-field. (E) Electron microscopy in situ hybridization of the perikaryon of a photoreceptor cell with gold particles (arrows), indicating the presence of Dt-Pax6 transcript in the endoplasmic reticulum. (F) No staining is visible in the rhabdomeric region of the photoreceptor cell. (G) Electron microscopy in situ hybridization of the eye pigmented cell with positive gold particles (arrows), indicating the presence of DtPax-6 transcript in the cytoplasmic region in between the pigment granules. (H) No staining is visible with the sense probe in the eye pigmented cell cytoplasm. (I and J) Dark- and bright-field sagittal section of 7-day head-regenerating planarians. DtPax-6 expression already was observed when the first sign of eye pigment cell differentiation appears at 7 days and is maintained throughout regeneration. (K and L) Fourteen days of regeneration. n, nucleus; nm, nuclear envelope; er, endoplasmic reticulum; p, pigment granules; rh, rhabdomeres.