The genetic network of prototypic planarian eye regeneration is Pax6 independent
David Pineda1,*,
Leonardo Rossi3,*,
Renata Batistoni2,
Alessandra Salvetti3,
Maria Marsal1,
Vittorio Gremigni3,
Alessandra Falleni3,
Javier Gonzalez-Linares1,
Paolo Deri2 and
Emili Saló1,
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
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Fig. 1. Amino acid sequence comparison of Pax6A and Pax6B from D. japonica and G. tigrina. (A) Multiple alignment of DjPax6A, GtPax6A, and DjPax6B, GtPax6B sequences. Amino acids that are identical both in Pax6A and Pax6B are shown in blue; amino acids conserved between DjPax6A and GtPax6A are indicated in green; amino acids conserved between DjPax6B and GtPax6B are indicated in red; differing amino acids are shown in black. The paired domain, the conserved motif found in the linker region, and the homeodomain are boxed. The missing sequence in the 5' end of GtPax6A is indicated by dots; the introduced gaps are indicated by dashes. (B,C) Sequence comparison of the paired domain (B), and the homeodomain (C) of the planarian Pax6 proteins to the Pax6 paired domain and homeodomain of different species. Only Homo sapiens and Mus musculus Pax6 are shown for vertebrates. Drosophila eyless and toy are included as invertebrate Pax6. The shaded bars indicate Pax6-specific amino acids. Percentages of sequence identity (%I), determined by comparison with Homo sapiens and Mus musculus Pax6, are indicated at the end of each line. The structure of paired domain and homeodomain are shown on the top in B and C, respectively. Identical amino acids are indicated by dots. The incomplete sequence of GtPax6A is indicated by a dashed line. Non conserved amino acid residues are indicated in bold. The homeodomain flanking regions are shown in C.
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Fig. 2. Phylogenetic tree of Pax genes. The tree was derived from the homeodomain and flanking regions of representatives from different Pax classes (indicated on the right), using the Neighbor-joining method. Sequences reported in this paper are in bold. Bootstrap percentage values (1000 replicates) are shown over the corresponding nodes. All branch lengths are proportional to the distances between sequences. The tree was rooted using Hydra PaxB as an outgroup. The analysis includes: human Pax 3, 4, 6 (Aniridia) and 7; mouse Pax3, 4, 6 and 7; zebrafish Pax6; sea urchin suPax6; chordate PmPax6 and Amphi-Pax6; mollusc Lo-Pax6; Drosophila ey and toy; C. elegans Cevab-3; nemertine LsPax6; planarian GtPax6A and GtPax6B, DjPax6A and DjPax6B; and cnidarian PaxC and Hl PaxB.
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Fig. 3. Expression of Pax6A mRNA in an intact planarian, as detected by whole-mount in situ hybridization. (A) Dorsal view of GtPax6A expression in G. tigrina. (B-G) Anteroposterior sequence of some representative transverse cryosections of whole-mount depicted in A. GtPax6A is expressed in the cephalic ganglia and the nerve cords. Presence of GtPax6A transcripts can also be observed in the lateroposterior region, close to the dorsoventral border. (H) Nomarski view of a higher magnification of a transverse cryosection of the whole-mount in A, showing the localization of GtPax6A-labelled cells (arrowheads) in the lateroventral marginal region, close to the eosinophilic secretory cells (arrows). Scale bars: 0.5 mm.
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Fig. 4. In situ hybridization and camera lucida drawings of transverse paraffin sections from the cephalic region of D. japonica. (A) The CNS in an intact planarian, which is composed of a mass of nerve cells, the cephalic ganglia (cg) and a pair of ventral nerve cords (nc). I, II and III indicate paraffin section levels. (B) The planarian eye. pc, pigment cell; phc, photoreceptor cell. (C-E) Anteroposterior sequence of transverse paraffin sections, visualized after in situ hybridization with Djsyt. (F-H) Camera lucida drawings of sections in C-E, illustrating the various morphological structures. (I-M) Anteroposterior sequence of transverse paraffin sections, visualized after in situ hybridization with DjPax6A. (N-P) Camera lucida drawings of sections in I-M, illustrating the various morphological structures. The nerve cell marker Djsyt labels all nerve cells, including the photoreceptors, while DjPax6A is expressed only in a subset of nerve cells. No detectable DjPax6A expression is observed in visual cells. Scale bars: 0.05 mm.
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Fig. 5. TEM in situ hybridization on D. japonica. (A-D) DjPax6A antisense-strand RNA; (F) DjPax6A sense-strand RNA; (E,G) Dj18S antisense-strand RNA; (H) DjSyt antisense-strand RNA. (A) Low magnification of the pigment cup ocellus showing some nuclei of photosensitive cells. The box indicates the figure shown in B. (B) Enlargement of A with clusters of gold particles (arrows) on the cytoplasm. (C) Clusters of gold particles (arrows) on the dendrite and the rhabdomeric region of a photosensitive cell. (D) A cluster of gold particles on the perinuclear cytoplasm of a pigment cell. (E) Clusters of gold particles (arrows) on the nucleolus and the endoplasmic reticulum of a cell. (F) No cluster of gold particles is visible on the cytoplasm of a pigment cell or in the rhabdomeric region of a photoreceptor cell. (G) A large cluster of gold particles (arrows) on the rhabdomeric region of a photoreceptor cell. (H) A cluster of gold particles (arrow) on the perikaryon of a photoreceptor cell. d, dendritic region of the photosensitive cell; n, nucleus; nu, nucleolus; p, pigment cup; pc, pigment cell; r, rhabdomeres. Scale bars: 5 µm in A; 0.5 µm in B-H).
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Fig. 6. Expression of GtPax6A mRNA in regenerating G. tigrina, as detected by whole-mount in situ hybridization. (A-F) Dorsal view of fragments regenerating a head. Anterior is towards the top. (A) Activation of GtPax6A after 2 days of regeneration is detected as two hybridization spots located in the region close to the sectioned old nerve cords, where new cephalic ganglia are forming. (B) After 3 days of regeneration, GtPax6A-positive spots merge and follow the fibers emerging from the old nerve cords. (C-F) After 6, 8, 11 and 15 days of regeneration, GtPax6A expression becomes broader and follows the regenerating cephalic ganglia. (G) Dorsal view of a posterior regeneration. Six days after cutting, two labeling spots in the area corresponding to the regenerating nerve cords are observed. (H,I) Dorsal view of a lateral regeneration. An increased level of GtPax6A expression is observed where a cephalic ganglion is regenerating. A basal expression is detected in the corresponding non-regenerating ganglion. Scale bars: 0.5 mm.
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Fig. 7. Expression of Djops in regenerating D. japonica after injection with Pax6A/Pax6B dsRNA mixture, visualized by in situ hybridization on transverse paraffin sections at the eye level. (A,B) Nomarski images of Djops-expressing photoreceptors in a dsRNA-injected animal (A) and in a water-injected control (B), after 15 days of regeneration. Scale bar: 0.015 mm.
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Fig. 8. Effects of GtPax6A and GtPax6B dsRNA injection in regenerating G. tigrina. (A) Visualization of comparative RT-PCR experiments. Relative levels of endogenous transcripts in water-injected controls and in GtPax6A, GtPax6B dsRNA-injected animals are shown. Reduction of the Gtops mRNA level after Gtops dsRNA injection is shown as a comparison. Expression of the homeobox gene Dth2 is unaffected by GtPax6A and GtPax6B RNAi experiments. (B-E) Dorsal view of regenerating heads visualized by GtPax6A whole-mount in situ hybridization. (B-C) Control animals injected with water. The typical arch-shaped GtPax6A expression is observed in cephalic regeneration after 6 (B) or 9 (C) days from cutting. (D,E) After injection with GtPax6A/GtPax6B dsRNA mixture, no GtPax6A hybridization signal is detected in regenerating animals after 6 (D) and 9 (E) days from cutting. Regenerating eyespots can be observed both in the controls and in planarians injected with GtPax6A/GtPax6B dsRNA mixture. Scale bars: 0.5 mm.
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Fig. 9. Dorsal view of cephalic ganglia regeneration in G. tigrina after injection of GtPax6A/GtPax6B dsRNA mixture, visualized by whole-mount neuropeptide FMRFamide immunoreactivity pattern at different regenerative stages. (A,C) After 5 (A) and 9 days (C) of regeneration, new transversal commissures produced from the amputated old nerve cords and differentiating the proximal cephalic ganglia (arrows) can be observed in water-injected controls. The distal part of the new cephalic ganglia is organizing an arch-shaped structure that connects the nerve cords (arrowhead). (B,D) No abnormal cephalic ganglia regeneration is apparent in corresponding head-regenerating fragments subsequent to injection of GtPax6A/GtPax6B dsRNA mixture. Regenerating eyes appear as small black spots of similar size in both C and D. (E,F) After 20 days from cutting, a complete regeneration of the cephalic ganglia can be observed. No differences in the CNS pattern can be seen between a water-injected control (E) and an dsRNA-injected planarian (F). Scale bars: 0.5 mm.
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Fig. 10. Expression of GtSix-1 and Gtops in regenerating G. tigrina after injection with Pax6A/Pax6B dsRNA mixture, visualized by whole-mount in situ hybridization. Dorsal view of head regenerating fragments after 3, 6 and 9 days from cutting. (A-C) Water-injected controls hybridized with Gtsix-1. (D-F) GtPax6A/GtPax6B dsRNA mixture-injected organisms hybridized with GtSix1. (G-I) GtPax6A/GtPax6B dsRNA mixture-injected organisms hybridized with Gtops. (A,D) A faint GtSix1 hybridization signal is visible after 3 days of regeneration both in the controls and in injected animals. Later on, GtSix1 mRNA is clearly visualized at the eye level. No differences are detected between controls (B,C) and injected animals (E,F). (G-I) A normal expression pattern of Gtops mRNA can also be detected in animals injected with GtPax6A/GtPax6B dsRNA. Scale bars: 0.5 mm.
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© The Company of Biologists Ltd 2002
