Flexibly deployed Pax genes in eye development at the early evolution of animals demonstrated by studies on a hydrozoan jellyfish - PubMed (original) (raw)

Flexibly deployed Pax genes in eye development at the early evolution of animals demonstrated by studies on a hydrozoan jellyfish

Hiroshi Suga et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Pax transcription factors are involved in a variety of developmental processes in bilaterians, including eye development, a role typically assigned to Pax-6. Although no true Pax-6 gene has been found in nonbilateral animals, some jellyfish have eyes with complex structures. In the cubozoan jellyfish Tripedalia, Pax-B, an ortholog of vertebrate Pax-2/5/8, had been proposed as a regulator of eye development. Here we have isolated three Pax genes (Pax-A, Pax-B, and Pax-E) from Cladonema radiatum, a hydrozoan jellyfish with elaborate eyes. Cladonema Pax-A is strongly expressed in the retina, whereas Pax-B and Pax-E are highly expressed in the manubrium, the feeding and reproductive organ. Misexpression of Cladonema Pax-A induces ectopic eyes in Drosophila imaginal discs, whereas Pax-B and Pax-E do not. Furthermore, Cladonema Pax-A paired domain protein directly binds to the 5' upstream region of eye-specific Cladonema opsin genes, whereas Pax-B does not. Our data suggest that Pax-A, but not Pax-B or Pax-E, is involved in eye development and/or maintenance in Cladonema. Phylogenetic analysis indicates that Pax-6, Pax-B, and Pax-A belong to different Pax subfamilies, which diverged at the latest before the Cnidaria-Bilateria separation. We argue that our data, showing the involvement of Pax genes in hydrozoan eye development as in bilaterians, supports the monophyletic evolutionary origin of all animal eyes. We then propose that during the early evolution of animals, distinct classes of Pax genes, which may have played redundant roles at that time, were flexibly deployed for eye development in different animal lineages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Pax genes cloned from Cladonema and Microciona, and molecular phylogenetic tree of the Pax family. (A) Medusa of C. radiatum. Arrowhead indicates an eye. b, tentacle bulb; m, manubrium; t, tentacle; u, umbrella. Photo courtesy of Claudia List. (B) Structures of Pax proteins. Those cloned in this study are marked by asterisks. Red box, green ellipse, and blue box represent the PD, octapeptide (OP), and HD, respectively. Octapeptide-like motifs of the poxn subfamily are found at amino acid positions 360–367 for Drosophila Poxn, 435–442 for Cladonema Pax-A, 324–331 for Acropora Pax-A, and 153–160 for Acropora Pax-C. Symbols with dashed line and pale color indicate highly divergent sequences. (C) The ML tree inferred from a comparison of the whole PD amino acid sequences. See

Fig. S1

for details. Two possible root positions are indicated according to previous publications (4, 5, 22) (white arrowheads). The eyg subtree derived from an additional ML analysis (

Fig. S2_B_

), which is performed on the basis of comparison of the latter halves (RED subdomains) of the PD sequences, is shown in a box. Black arrowheads indicate two possible ML positions of the eyg subtree suggested by further ML analyses based either on a comparison of the RED subdomain sequences (arrowhead A;

Fig. S3_A_

) or on that of the RED subdomain plus the whole HD sequences (arrowhead B;

Fig. S3_B_

). Because complete HDs are present only in Pax-C among the members of the poxn subfamily, Pax-A and poxn were excluded from the latter analysis. The position of the eyg subtree is therefore ambiguously shown at the root of poxn subtree (gray ellipse). Subtrees corresponding to distinct subfamilies are shaded gray and the subfamily names are shown on the right, with the names of cnidarian members in parentheses. Cnidarian sequences and poriferan sequences are shown in red and blue, respectively. Red and blue circles indicate the cnidarians–bilaterians and the poriferans–eumetazoans splits, respectively. Filled rhombi indicate the gene duplications that gave rise to the subfamilies. The extended local bootstrap probability is shown at each branch that separates subfamilies.

Fig. 2.

Expression of Cladonema Pax genes. Cladonema medusae were dissected into four body parts, tentacles (t), tentacle bulbs with eyes (b), manubrium (m), and umbrella (u) (Fig. 1_A_) and expression levels of CrPax-A (A), CrPax-B (B), and CrPax-E (C) were quantified by real-time RT-PCR analysis. The expression level in each body part relative to the whole body (1.0) was calculated and subsequently normalized to the expression level of elongation factor 1α (EF1α). The quantifications were performed three times on different cDNAs generated independently, and geometric means were calculated. The y axes are arbitrary. Error bars represent SDs. (D–J) Whole-mount in situ hybridization analyses with anti-sense RNA probes for CrPax-A (D–G) and CrPax-B (H–J). Cross-sections of the eye (F) and the whole tentacle (G) of the CrPaxA probe-hybridized jellyfish are shown. The cutting planes are indicated by the dashed lines in E. Note that the natural eye pigment disappears during the hybridization procedure. (G) A phase contrast microscopic picture of the ectoderm tissue, which corresponds to the box, is shown in the Inset. Dashed line indicates the border between endoderm and ectoderm. Arrowheads indicate the frontal side of a tentacle. (H and I) Excised manubriums. The original position of the umbrella is indicated by a dashed line in I. ec, ectoderm; en, endoderm; g, gonads; i, immature oocyte; l, lens; m, manubrium; mo, mouth; n, nematoblast; nu, nucleus; o, mature oocyte; r, retina; t, tentacle; u, umbrella. (Scale bars, 500 μm in D, 100 μm in E and G–I, 10 μm in F.) (K) CrPax-B expression levels quantified independently for five just-detached and five full-grown medusae by real-time RT-PCR. Their expression levels relative to the average (red line) of the just-detached medusae are presented. Data normalized to EF1α.

Fig. 3.

Ectopic eye induction in Drosophila and rescue of the spapol mutant phenotype by Cladonema Pax genes. (A) CrPax-A was expressed under the control of _dppblink_-Gal4 driver with UAS-Gal4. Arrowhead indicates the induced eye. (B) SEM picture of the induced eye. (C) Misexpression of CrPax-A under the control of _dppblink_-Gal4 driver in a homozygous ey null mutant (eyJ5.71) background induced ectopic eyes (arrowhead). The anteroposterior (a-p) and dorsoventral (d-v) axes are shown. Note that the natural compound eye is absent. The genotype of ey mutant was confirmed by the absence of the second exon of ey (30) by PCR. (D) SEM picture of the induced eye. (E) Eye phenotype of the spapol homozygous fly. (F–I) Rescue experiments of the spapol mutant phenotype. UAS-D-Pax2 (F; positive control), UAS-CrPax-A (G), UAS-CrPax-B (H), and UAS-CrPax-E (I) transgenic lines were crossed with the _spa_-Gal4 driver line in a spapol homozygous background. (Scale bars, 30 μm.)

Fig. 4.

CrPax-A PD binding sites in the upstream region of eye-specific Cladonema opsins. (A) Schematic drawing showing the positions of the probes generated for EMSA. Probes to which CrPax-A PD bound are shown in red. Red vertical bars indicate the positions of the CrPax-A binding sites, which were precisely identified by the use of mutated probes. Black triangles, arrows, and gray boxes represent TATA boxes, transcription starting sites, and protein coding regions, respectively. (B) EMSA for the three positive probes: probe 5.1 of CropG1, and probe 4.3 and 3.2 of CropN1. +, wild-type probes; m, mutated probe. The size (bp) of the probe is shown at left. Arrowheads indicate the band shifts caused by the binding of proteins. Note that the bindings of CrPax-B PD are very faint or undetectable (white arrowhead), even though the same amount of the proteins as CrPax-A PD were used. CrPax-B PD was separately proven to be active by the use of a control probe carrying D-Pax2 binding sites (Materials and Methods).

Fig. 5.

Evolution of Pax genes deployed for animal eye development. 1: Gene duplications that gave rise to distinct Pax classes (corresponding to subfamilies) occurred. 2: The ancestral animal eye evolved and different classes of Pax genes were redundantly recruited for eye development. 3: In each of three different animal lineages, a specific Pax gene was selected for the eye development. 4: Pax genes responsible for eye development were altered in some bilaterians. See text for detailed description.

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Flexibly deployed Pax genes in eye development at the early evolution of animals demonstrated by studies on a hydrozoan jellyfish - PubMed (original) (raw)