1 Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain
2 Laboratorio di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e Biochimica, Università di Pisa, Via Carducci 13, 56010 Ghezzano, Pisa, Italy
3 Dipartimento di Morfologia Umana e Biologia Applicata, Università di Pisa, Via A. Volta 4, Pisa, Italy
* These authors contributed equally to this work
Author for correspondence (e-mail: esalo{at}porthos.bio.ub.es)
Accepted 26 December 2001
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SUMMARY |
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Key words: Planarians, Regeneration, Eye, Central nervous system, Paired domain, Pax6, RNAi, Ultrastructure, In situ hybridization
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INTRODUCTION |
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Planarians are flatworms well known for their exceptional regenerative capabilities. They are free-living members of the Platyhelminthes, considered a basal phylum of the Lophotrocozoa clade (Aguinaldo et al., 1997; Carranza et al., 1997
; Bayascas et al., 1998
). The study of the role of Pax6 genes during eye and central nervous system (CNS) regeneration and maintenance will be of interest for understanding the different ways that this genetic network evolved in the animal kingdom. Planarians are one of the simplest organisms with cephalization, defined by the presence of two dominant cephalic ganglia connected by commissural connections and followed by two ventral nerve cords. The nerve cords run the entire length of the body and are regularly connected by commissures producing a small concentration of neurons at the crossing points (Baguñá and Ballester, 1978
; Rieger et al., 1991
). Light perception by special photosensitive cells occurs in planarians. Photoreceptor structures, which are capable of detecting light and shadow, are grouped into eyespots defining a simple ancestral visual system that consists of bipolar retinal neurons whose dendrites project into a cup-shaped structure composed of pigment cells (Kishida, 1967
; Sakai et al., 2000
). Although the planarian eyes cannot focus the light pattern into images, as they have no focusing lens, they serve essentially the same function, receiving and transducing light into neuronal signals, as eyes do in all metazoans.
We report an extensive search for Pax6 genes in Platyhelminthes that has led to the characterization of two related Pax6 genes in the planarian species Dugesia japonica (DjPax6A and DjPax6B) and Girardia tigrina [GtPax6A and GtPax6B, the latter previously named DtPax6 (Callaerts et al., 1999)]. The Pax6A and Pax6B sequences share high similarity and comparable expression patterns in both species, suggesting that they originated by duplication in a triclad or, possibly, in a platyhelminth ancestor. Both genes are detected in the adult CNS a pattern of expression shared with all Pax6 genes described so far and are activated during regeneration, Pax6A being more expressed than Pax6B. Although no specific transcripts were detectable in the photoreceptors by whole-mount in situ hybridization, the presence of low levels of Pax6A and Pax6B mRNA could be demonstrated in the eye cells by electron microscope in situ hybridization. Inactivation of both Pax6 genes by RNAi neither prevents eye formation during regeneration nor inhibits the eye expression pattern of the planarian genes Gtsix-1 or Gtops, supporting the hypothesis that the function of Pax6A and Pax6B is not essential in planarian eye regeneration.
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MATERIALS AND METHODS |
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Isolation and characterization of planarian genes related to Pax6
A GtPax6A PCR fragment of 111 bp was amplified with two degenerate primers. The primers correspond to the amino acid sequences LEKEFER (sense strand) and QVWFSNR (antisense strand) of the homeodomain. A Gt10 cDNA library from regenerating G. tigrina was screened according to Garcia-Fernandez et al. (Garcia-Fernandez et al., 1991
), with the 111 bp PCR fragment as a probe. One positive clone was found corresponding to the 3' Pax6A gene end (Pax6A3'). A 5' RACE strategy was used to elongate the sequence at the 5' end, obtaining a second clone (GtPax6A790) that contained 790 bp from the paired box to the homeobox of the Pax6A sequence. The GtPax6A sequence has been deposited to the EMBL/GenBank with the Accession Number AY028904. DjPax6A was directly amplified from the sequence deposited in the EMBL/Genbank (Accession Number, AB017632), as described by Rossi et al. (Rossi et al., 2001
). A fragment (clone DjPax6B-520 bp) of the second Pax6 gene, DjPax6B, was amplified in D. japonica (Rossi et al., 2001
). A sense-strand degenerate primer was designed, taking advantage of the high sequence similarity in the paired domain between DjPax6A and the Pax6 gene, here referred to as GtPax6B, previously characterized in G. tigrina (Callaerts et al., 1999
). The antisense primer corresponded to the GtPax6B amino acid region TLFGYN. A 5'/3'RACE strategy was used to further characterize the DjPax6B sequence, deposited at the EMBL/Genbank with the Accession Number AJ311310. The sequence-specific antisense primer, corresponding to the amino acid region SKPRVATN was used to amplify selectively the 5' region. The DjPax6B 3' region was obtained with the sequence-specific sense primer, corresponding to INTWPPTS. The PCR products were TA-cloned using the pGEM-T easy vector (Promega). All clones were sequenced by automated fluorescent cycle sequencing (ABI).
Phylogenetic analysis of the Pax family homeodomains
The phylogenetic tree of the homeodomains and their flanking regions sequences was inferred using the CLUSTALX package. Sequences were aligned with CLUSTALX software. Evolutionary distances were calculated using Kimuras equation (Nei and Koehn, 1983), and used for phylogenetic tree construction by the Neighbor-joining method. Sequences were obtained from the EMBL/ GenBank.
In situ hybridization experiments
All sense and antisense digoxigenin-labeled RNA probes used for in situ hybridization experiments were made using the RNA in vitro labeling kit (Roche). Whole-mount in situ hybridization was carried out on intact and regenerating planarians according to Agata et al. (Agata et al., 1998). Cryosections after whole-mount in situ hybridization were performed as described by Pineda et al. (Pineda et al., 2000
). In situ hybridization on sections was carried out as described by Kobayashi et al. (Kobayashi et al., 1998
). Hybridization took place at 55°C for 24-60 hours. The final probe concentration was 0.1 ng/µl. In some hybridized sections, 20 µg/ml DAPI was added to detect nuclei. Transmission electron microscope (TEM) in situ hybridization was performed on specimens of D. japonica, fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour at 4°C. After dehydration in a graded series of methanol (each step for 30 minutes at 20°C) specimens were embedded in Unicryl resin and polymerized for 72 hours at 4°C under u.v. light. Ultrathin sections, obtained with a diamond knife on Ultracut Reichert-Jung microtome, were placed on Formvar-carbon coated nickel grids and were incubated face down on a drop containing the hybridization buffer (50% formamide, 10% dextran sulphate, 4xsaline sodium citrate, 400 µg/ml salmon testis DNA and 8 ng/µl of antisense or sense probes) for 4 hours at 37°C. After hybridization, ultrathin sections were washed in phosphate buffer, pre-incubated in 1% bovine serum albumin and then incubated in anti-digoxigenin antibody (1:40 in phosphate buffer) conjugated to gold particles of 10 nm in diameter, for 30 minutes. Grids were stained with uranyl acetate and lead citrate and observed with a Jeol 100 SX transmission electron microscope. Controls were performed using both sense probes and RNAse treatment (100 µg/ml of RNAse A for 90 minutes at 37°C) (Le Guellec et al., 1991
) before the hybridization step.
The following clones were used as probes for in situ hybridization experiments.
D. japonica: DjPax6A, DjPax6B 520 bp, Dj18S (central region of 18S rDNA, about 1.1 kb) Djops 480 bp (Accession number, AJ421264) and Djsyt (Tazaki et al., 1999).
G. tigrina: GtPax6A3' and GtPax6A790 were used in non-injected animals, GtPax6A790, Gtsix-1 so-5' and so-3'.2 (Accession number, AJ251661), and Gtops p-250 (accession no. AJ251660) were used in dsRNA-injected animals.
Microinjection of double-strand RNA (dsRNA) and analysis of endogenous transcripts
Double-stranded RNA was synthesized as described by Sanchèz-Alvarado and Newmark (Sanchèz-Alvarado and Newmark, 1999). In G. tigrina Pax6A3' clone and a ClaI-HindIII fragment of 400 bp (GtPax6B3'clone) of GtPax6B were used for dsRNA synthesis. In D. japonica dsRNA was synthesized from DjPax6A 1600 bp and DjPax6B 520 bp clones. Planarians were injected with 1010-1011 molecules of each dsRNA preparation or with an equimolar mixture of both Pax6A and Pax6B dsRNA. After the first injection, performed just after amputation, further injections were performed every 1 or 3 days, using a Drummond Scientific (Broomall, PA) Nanoject injector. Control injections were performed with water or ß-Gal dsRNA. Injected planarians were kept at 17°C. Injected G. tigrina specimens were fixed at different regeneration times and processed for whole-mount in situ hybridization.
Total RNA was isolated from injected planarians after 1 to 3 days of regeneration and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed according to Bayascas et al. (Bayascas et al., 1998). Control reactions were performed identically in the absence of reverse transcriptase. Specific oligonucleotides from Pax6A and Pax6B were used to ascertain the reduction of Pax6A and Pax6B endogenous transcripts. Each couple of primers was designed from two regions, one internal and the other external to the sequence used for dsRNA synthesis. As an internal control, the ubiquitous transcripts of the homeobox gene Dth2 (Garcia-Fernandez et al., 1993
) and the eye-specific Gtsix1 and Gtops transcripts (Pineda et al., 2000
; Pineda et al., 2001
), were amplified in G. tigrina. Two specific primers for the constitutively transcribed elongation factor gene DjEF2 were used for control amplifications in D. japonica.
Primer sequences used for PCR were as follows:
GtPax6A reverse, 5'-GAAGCTTCTGTTTCTGTTTTAGAG-3';
GtPax6A forward, 5'-CGTACTTCGTTTTCGACAGATCAA-3';
GtPax6B reverse, 5'-TCGCTTCTTTTGTTGTACAGTTTG-3';
GtPax6B forward, 5'-CGGACTTCATTTACAAATGATCAG-3';
Dth-2 reverse, 5'-TGGGAGACCGTTCTTTATCGTCAAC-3';
Dth-2 forward, 5'-CCAATGCTAGTAATGATCCGCGTAT-3';
Gtops reverse, 5'-GGACAGATACTTTGTTATCGCTCA-3';
Gtops forward, 5'-TAACAAAATTCCCGATGTACATTC-3';
Gtsix-1 reverse, 5'-AACGGCTCGGGATTTTTCTTTAAA-3';
Gstix-1 forward, 5'-ATATGGTCTCTTCCACCTTGCCAA-3';
DjPax6A forward, 5'-CCAAATCTTTCGCAATCTTC-3';
DjPax6A reverse, 5'-CAATAAGTATCAAATACGTTACA-3';
DjPax6B forward, 5'-CATCAATACATGGCCGCCTACAA-3';
DjPax6B reverse, 5'CGTCTCCATTTTGCTCTGCGATT-3';
DjEF2 forward, 5'-TTAATGATGGGAAGATATGTTG-3'; and
DjEF2 reverse 5'- GTACCATAGGATCTGATTTTGC-3'
For each PCR reaction the concentration of cDNA, primers and the number of cycles used were optimized with the aim of observing a quantifiable signal within the linear range of the amplification, according to both the putative abundance of each mRNA amplified and to the size of the corresponding PCR product.
Immunohistochemistry
Whole-mount immunohistochemistry of regenerating G. tigrina was carried out as described by Cebrià et al. (Cebrià et al., 1997). Rabbit anti-FMRFamide (Diasorin) was used as a primary antibody at a 1:100 dilution, and a fluorescein-conjugated goat anti-rabbit (IgG; Sigma) was used as a secondary antibody at a 1:50 dilution. The samples were examined with a epifluorescence microscope (Axiophot, Zeiss) and with a confocal laser scanning microscope (Leica Lasertechnik, Heidelberg).
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RESULTS |
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The evolutionary relationships between the planarian Pax6A and Pax6B and Pax proteins of other organisms were inferred from the homeodomain and their flanking regions (Fig. 2). We can observe the closest similarity to Pax6 protostomate representatives, clustering the planarian Pax6A with Drosophila ey and toy, although with low bootstrap values. Pax6B shows the lowest similarity and is at the base of the Pax6 cluster. Such results indicate that Pax6A and Pax6B are bona fide Pax6 members.
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DISCUSSION |
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Pax6A and Pax6B are expressed in intact planarians and activated during regeneration
Both Pax6 planarian genes are expressed in the CNS of adult organisms, Pax6A being more strongly expressed than Pax6B. The presence of Pax6B transcripts along the anteroposterior planarian axis, which was barely detected by in situ hybridization, was confirmed by RT-PCR experiments (Callaerts et al., 1999). Both genes are expressed in a subset of cells located along the entire CNS. The time course of Pax6A expression in cephalic regeneration clearly demonstrated activation of this gene during cephalic ganglia formation. Similarly, increased production of Pax6B transcripts during regeneration was demonstrated in G. tigrina (Callaerts et al., 1999
). Regeneration of the new cephalic ganglia requires the presence of the old nerve cords. This process has been followed by immunoreactivity to the molluscan cardioactive peptide (FMRFamide). New neural fibers emerge very early from the sectioned old nerve cords and reach the blastema, then bend transversally and fuse, producing a commissure, where cephalic ganglia will differentiate (Reuter et al., 1996
) (Fig. 9A). Early activation of Pax6 at the level of cells located near the old nerve cords that reach the blastema suggests pivotal functions of these genes in the formation of neural structures in these organisms. A role for Drosophila ey in axon pathway selection during embryogenesis has recently been proposed by Noveen et al. (Noveen et al., 2000
). In addition, it has been reported that severe defects in adult brain structures that are essential for vision, olfaction and the coordination of locomotion, are detectable in eyeless mutant Drosophila (Callaerts et al., 2001
). As planarians are considered to be close relatives of primitive animals that acquired the CNS, further study of the role of Pax6 genes during CNS regeneration will be of interest for understanding the evolution of the genetic program which triggers brain formation in higher organisms.
Owing to the low expression level, Pax6A and Pax6B transcripts were not detected in the eye cells in either intact or regenerating planarians by conventional in situ hybridization on paraffin sections with digoxigenin-labeled riboprobes. However, DtPax6B expression in the eye cells was detected with a more sensitive in situ hybridization method using radioactive riboprobes (Callaerts et al., 1999). TEM in situ hybridization also revealed Pax6A and Pax6B mRNA in eye cells (Fig. 5) (Callaerts et al., 1999
). These transcripts were distributed both in pigment eye cells and in different subcellular compartments of photoreceptors, i.e. throughout the perikaryon, and in the rhabdomeres. The presence of Pax6 transcripts has recently been monitored by competitive RT-PCR in adult human lens epithelium, cornea and monkey retina (Zhang et al., 2001
). Moreover, Pax6 expression persists in amacrine and ganglion cells of the mature retina (Ashery-Padan and Gruss 2001
; Marquardt et al., 2001
). On the whole, these results support a role of Pax6 in the maintenance of eye cells.
Reduction of Pax6A and Pax6B endogenous transcripts by RNAi indicates that both genes are functionally dispensable in eye regeneration
In many organisms, the Pax6 transcription factor is critical for eye formation, as well as in patterning the CNS (Quiring et al., 1994; Kurusu et al., 2000
; Pratt et al., 2000
). As the basic functioning of the eyes in capturing photons and transmitting the information to the brain is similar in all animals, the presence of Pax6 transcripts in light-sensitive cells and pigment cells of planarian eye strongly suggested a conserved role of both Pax6 genes in this structure. Pax6 is considered very ancient and it has been indicated that the ancestral role of this gene was to construct a light-sensitive unit by direct regulation of opsin expression (Sheng et al., 1997
; Pichaud et al., 2001
). The primitive eye of basal metazoans such as planarians is the most suitable model system for studying Pax6 ancient function(s) in visual structures (Gehring and Ikeo, 1999
).
The use of dsRNA to disrupt gene expression is a powerful method of achieving RNA interference in planarians (Sanchèz-Alvarado and Newmark, 1999). Thus, complete loss of eye has been obtained after Gtsix-1 dsRNA injection in planarians regenerating a head (Pineda et al., 2000
). Moreover RNAi-mediated depletion of opsin mRNA also leads to the loss of phototactic behavior in these animals (Pineda et al., 2001
).
Our experiments using dsRNA synthesized by Pax6A and Pax6B provide strong evidence that RNAi acts by decreasing endogenous cognate mRNA levels. The reduction of these gene products was comparable with that obtained for the corresponding RNAs in Gtsix-1 or Gtops RNAi experiments, which give rise to abnormal eye phenotypes. Despite the drastic RNAi-induced reduction in Pax6A and Pax6B transcripts, we did not observe any gross morphological alterations of the CNS in intact planarians or during regeneration. Moreover, eyes formed without any sign of defects during head regeneration and contained several photoreceptors comparable with those found in the eye of water-injected controls. A simple interpretation of these results is that the genetic network that controls eye formation in planarians is not triggered by Pax6 genes. The low level of Pax6 expression in the eye cells opens an alternative hypothesis, that both planarian Pax6 genes control eye cell fate decisions by a dose-independent mechanism, insensitive to the RNAi-induced transcript reduction. We favor the former possibility, as gene dose appears to be a fundamental requirement for the activity of Pax transcription factors, Pax6 being one of the best documented examples (Schedl et al., 1996; Van Raamsdonk and Tilghman, 2000
). An exhaustive search for other Pax6 did not yield any additional Pax6 gene in either planarian species, making an eye-specific Pax6 highly improbable. If Pax6A and Pax6B genes are not crucial for eye induction during regeneration in planarians, we can speculate that other genes play a related role and substitute or compensate Pax6 action in the regulatory eye network. In this respect, the planarian Gtsix-1 gene, which is essential for eye regeneration (Pineda et al., 2000
), could represent a putative candidate. The finding that Pax6 is not expressed during ganglionic photoreceptors (Joseph cells and organs of Hesse) development in Amphioxus (Glardon et al., 1998
), the demonstration that Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice (Zhang et al., 2000
), and our data on a Pax6-independent eye regeneration process in two species of planarians support the hypothesis that more than one molecular pathway can generate functional visual cells. Moreover, the recent hypothesis that other factors acting in parallel to Pax6 during retinal development can compensate Pax6 function (Ashery-Padan and Gruss, 2001
) supports the possibility that several alternative combinations could give rise to the same phenotypic structure. The selection of an alternative combination could be promoted by a peculiar developmental scenario, i.e. blastemal regeneration. In this context, the Pax6-independent eye regeneration can be considered a remarkable example of such flexibility. Further analysis of the role of Pax6 during planarian eye development could contribute to establishing similarities between the processes of eye development and eye regeneration.
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ACKNOWLEDGMENTS |
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