Cloning and Functional Analysis of Pax6 from the Hydrothermal Vent Tubeworm Ridgeia piscesae

Abstract

The paired box 6 (Pax6) gene encodes a transcription factor essential for eye development in a wide range of animal lineages. Here we describe the cloning and characterization of Pax6 gene from the blind hydrothermal vent tubeworm Ridgeia piscesae (RpPax6). The deduced RpPax6 protein shares extensive sequence identity with Pax6 proteins from other species and contains both the paired domain and a complete homeodomain. Phylogenetic analysis indicates that it clusters with the corresponding sequence from the closely related species Platynereis dumerilii (P. dumerilii) of Annelida. Luciferase reporter assay indicate that RpPax6 protein suppresses the transcription of sine oculis (so) in D. melanogaster, interfering with the C-terminal of RpPax6. Taking advantage of Drosophila model, we show that RpPax6 expression is not able to rescue small eye phenotype of ey2 mutant, only to cause a more severe headless phenotype. In addition, RpPax6 expression induced apoptosis and inhibition of apoptosis can partially rescue RpPax6-induced headless phenotype. We provide evidence RpPax6 plays at least two roles: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell apoptosis. Our results indicate alternation of the Pax6 function may be one of the possible causes that lead the eye absence in vestimentiferan tubeworms.

Fig 1. Comparison of amino acid sequences of Pax6 genes.

Identical amino acids are indicated by dots. The introduced gaps are indicated by dashes. The paired domain, homeodomain and conserved C-terminal motif are boxed. Pax6-specific amino acids [5] are shaded. Arrowhead indicates a single amino acid change of the Pax6-specific amino acids.

Fig 2. Phylogenetic position of RpPax6.

A neighbor-joining tree based on a comparison of the deduced amino acid sequences of full-length clones of Pax6 with mouse Pax2 included as outgroup. Numbers at nodes indicate the levels of bootstrap support based on data for 1,000 replicates; only values greater than 50% are shown. Bar 5% estimated sequenced divergence.

Fig 3. Structural-functional analysis of ey and RpPax6.

Schematic summaries of the original and chimeric constructs are listed in the left column. The right column shows the effect of ey and RpPax6 proteins with or without mutation on promoter activity of pGL3-so and pGL3-eya. The assay was carried out in 293T cells as described in Materials and Methods. Luciferase activities are shown relative to those of pCMV-myc. pSV-β-galactosidase was included in transfection as an internal control of the transfection efficiency. The values are the means from three independent experiments ±SE. **P < 0.01.

Fig 4. Eye phenotypes caused by misexpression of RpPax6 and ey genes.

(A, B) head of y w and UAS-RpPax6 pharate. (C) Rough eye in a UAS-RpPax6/GMR-GAL4 pharate. (D) UAS-RpPax6/ey-GAL4 pharate with most head structures and both eyes absent. (E) Ectopic eyes in UAS-ey/dpp-GAL4 fly (arrows). (F) Head of UAS-ey pharate. (G, H) Right and left eye of UAS-ey/ey-GAL4 pharate. The left eye was significantly reduced in size and the right eye was slightly reduced.

Fig 5. Third instar eye-antenna discs.

(A): y w (B): UAS-RpPax6 (C): UAS-RpPax6/ey-GAL4 flies. The boxed areas show growth defects in the eye disc of UAS-RpPax6/ey-GAL4 larvae. Scale bars = 100 μm.

Fig 6. Expression of RpPax6 promotes apoptotic cells were identified by acridine orange staining.

Arrow points to a cluster of apoptotic cells. All discs are reproduced at the same magnification. Scale bars = 100 μm. (A) Third instar wing disc of UAS-RpPax6/da-GAL4,tub-GAL80^ts reared continuously at 18°C exhibit no cell death as indicated by a lack of nuclear acridine orange staining. (B) Expression of RpPax6 for 24 h (UAS-RpPax6/da-GAL4,tub-GAL80^ts, shifted to 29°C) lead to cell death in third instar wing disc as indicated by nuclear acridine orange staining (green spots). (C) Third instar of eye-antenna discs of UAS-RpPax6/da-GAL4,tub-GAL80^ts reared continuously at 18°C show a few cell death. (D) Expression of RpPax6 for 24 h (UAS-RpPax6/da-GAL4,tub-GAL80^ts, shifted to 29°C) lead to high frequency of apoptotic cells in third instar eye-antenna discs.

Fig 7. Mutant rescue by targeted expression of ey and mPax6.

(A) eye of y w fly. The ey² mutant phenotype (small eye, see Fig B) can be completely rescued by targeted expression of ey (C) and mPax6 (D) driven by ey-GAL4.

Fig 8

Eye phenotype caused by expression of RpPax6 is partially rescued by inhibition of apoptosis (A, B) head of y w and UAS-RpPax6 pharate. (C) UAS-RpPax6/ey-GAL4 pharate with most head structures and both eyes absent. (D) UAS-RpPax6/ey-GAL4 pharate headless phenotype can be largely rescued by coexpression of p35, an inhibitor of apoptosis.