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First published online 16 March 2005
doi: 10.1242/dev.01785

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DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance

Alessandra Salvetti^1,*, Leonardo Rossi^1,*, Annalisa Lena¹, Renata Batistoni², Paolo Deri², Giuseppe Rainaldi³, Maria Teresa Locci⁴, Monica Evangelista³ and Vittorio Gremigni^1,

¹ Dipartimento di Morfologia Umana e Biologia Applicata, Sezione di Biologia e Genetica, Università di Pisa, Pisa, Italy
² Dipartimento di Fisiologia e Biochimica, Laboratorio di Biologia Cellulare e dello Sviluppo, Università di Pisa, Pisa, Italy
³ Istituto di Fisiologia Clinica, Laboratorio di Terapia Genica e Molecolare, CNR, Pisa, Italy
⁴ Dipartimento di Patologia Sperimentale Biotecnologie Mediche, Infettivologia e Epidemiologia Università di Pisa, Pisa, Italy

View larger version (132K): [in a new window]   Fig. 1. DjPum encodes a homologue of Drosophila Pumilio. (A) Nucleotide sequence and predicted protein product of the planarian DjPum cDNA. Grey background indicates the highly conserved PUF repeat domain and the flanking regions. DjPum Accession Number is AJ639658. (B) Comparison of PUF repeats and flanking regions of DjPum (Dj) with the corresponding regions of Drosophila Pumilio (Dm) and human Pumilio 2 (Hs2). Amino acid sequences are aligned using CLUSTAL W and visualized with BOXSHADE. Black backgrounds indicate identical residues and grey backgrounds indicate conserved residues. (C) Phylogenetic tree based on the PUF repeats and the flanking regions of PUF proteins of different species. DjPum (DJ) is circled. Accession numbers: Caenorhabditis elegans Ce1 (FBF-1), AAF39879 Ce2 (FBF-2), Q09312; Ce3 (PUF-3), CAB63369 Ce4 (PUF-4), T33752; Ce5 (PUF-5), T22634; Ce6 (PUF-6), T21080; Ce7 (PUF-7), T32528; Ce8 (PUF-8), T15717; Ce9 (PUF-9), T26218; Ce10 (PUF-10), AAF60691 Ce11 (PUF-11), AAK68592 Drosophila melanogaster Dm (Pumilio), A46221; Leishmania major Lm, CAB62815 Trypanosoma brucei Tb, AAK62674 Saccharomyces cerevisiae Sc1 (JSN1), P47135; Sc2 (PUF2), NP-015367; Sc3 (PUF3), NP-013088; Sc4 (PUF4), P25339; Sc5 (PUF5/MPT5/UTH4), P39016; Sc6 (PUF6), S69554; Schizosaccharomyces pombe Sp1, CAB60694 Sp2, Q09829; Sp3, Q92359; Sp4, Q10238; Sp5, CAA20674 Sp6, CAA18887 Sp7, CAB54870 Neurospora crassa Nc, T49434; Dictyostelium discoideum Dd (PufA), AAD39751 Homo sapiens Hs1, AAG31807 Hs2, AAG31806 Mus musculus Mm1, AAG42319 Mm2, AAG31805 Xenopus laevis Xl, BAB20864 Arabidopsis thaliana At1, AAC95220 At2, AAC95216 At3, AAF02808 At4, AL049480; At5, BAA97177 At6, AAF87849 At7, CAB82120 At8, AAC28191 At9, AC007727; Oryza sativa Os, AAK73144 Populus tremula x Populus tremuloides Pt, AAF71823

View larger version (70K): [in a new window]   Fig. 2. Analysis of DjPum expression in intact and regenerating D. japonica by in situ hybridization. Broken blue lines indicate the blastema region, which does not contain proliferating cells. (A) Schematic drawing of a planarian (anterior towards the top). cg, cephalic ganglia; e, eyes; nc, nerve cords; ph, pharynx. (B-G) Dorsal view of whole-mount in situ hybridized planarians. (B,C) Intact specimens. (D,E) Head fragments and (F,G) lateral fragments, 3 days after transection. (B) DjPum expression. (C) DjPum expression, 10 days after X-ray irradiation. (D) DjPum expression. (E) Expression of DjMCM2 is shown for comparison with the DjPum expression depicted in D. (F) DjPum expression. (G) Expression of DjMCM2 is shown for comparison with the DjPum expression depicted in F. (H) Schematic drawing of a regenerating lateral fragment showing the distribution of DjPum- and DjMCM2-positive cells (blue lines). Scale bar: 400 µm. (I) Visualization of two neoblast-like cells expressing DjPum mRNA after in situ hybridization on dissociated cells. (L-N) Double fluorescent in situ hybridization on dissociated cells. (L) Expression of DjMCM2 mRNA in two neoblast-like cells. (M) DjPum mRNA expression is detectable in one of the two cells depicted in L. (N) L and M images were combined using Adobe Photoshop 7.0. Scale bar: 10 µm.

View larger version (74K): [in a new window]   Fig. 3. Effect of DjPum RNAi on regeneration in D. japonica. Brightfield images of the injected organisms. Dorsal view, anterior is towards the top. Broken yellow lines indicate the blastema region. (A) A head fragment injected with DjPum dsRNA. (B) A water-injected head fragment, 4 days after the second transection. (C) A tail fragment injected with DjPum dsRNA, 4 days after the second transection. (D) A water-injected tail fragment, 4 days after the second transection. (E) A head fragment injected with DjPum dsRNA, 14 days after the second transection. (F) A tail fragment injected with DjPum dsRNA, 14 days after the second transection. (G) Visualization of a comparative RT-PCR experiment in planarians injected with DjPum dsRNA, water and Djeya dsRNA. DjEF2 is used as an internal amplification control. Scale bars: 250 µm.

View larger version (192K): [in a new window]   Fig. 4. Ultrastructural analysis of a phenotype lacking the blastema, as a consequence of DjPum RNAi in D. japonica. (A) Wound region of a DjPum dsRNA-injected fragments, 3 days after the second transection. (B) A 3-day-old blastema region from a water-injected control is shown for comparison. uc, unspecialized, neoblast-like cells; we, wound epidermis; bl, basal lamina. Scale bar: 10 µm.

View larger version (95K): [in a new window]   Fig. 5. Expression of representative cell markers after DjPum RNAi in D. japonica. (A-F) DjMCM2 expression visualized by whole-mount in situ hybridization. (A) A regenerating DjPum dsRNA-injected head fragment, 15 days after the first transection. (B) A regenerating water-injected head fragment, 15 days after the first transection. (C) A DjPum dsRNA-injected head fragment lacking a visible blastema, 5 days after the second transection. (D) A regenerating water-injected head fragment, 5 days after the second transection (E) A Djeya dsRNA-injected tail fragment showing a no-eye phenotype, 5 days after the second transection. (F) A water-injected tail, 5 days after the second transection. Regenerating eyes are indicated by arrows. Scale bar: 250 µm. (G) DjMCM2, DjFGFR1, DjMHC-A, DjMHC-B, DjIFb, DjSix-1, Djops and DjClg3 expression visualized by comparative RT-PCR. RNA from DjPum dsRNA-injected planarians and water-injected controls was obtained from intact specimens 4 days after the first injection (1) and from planarian fragments sacrificed 4 days after the first (2) and the second (3) transection. DjEF2 is used as an internal amplification control.

View larger version (100K): [in a new window]   Fig. 6. Distribution of mitoses in D. japonica, as detected by anti-phospho-histone H3. Confocal projections of 40 sections taken at 0.9 µm intervals through 36 µm were obtained from whole-mount preparations. The H3P signal is visualized in red. (A) Schematic drawing of a planarian. Red box indicates the body region corresponding to the confocal images depicted in B-D. (B) H3P immunostaining in a water-injected control, 15 days after the first transection. (C) H3P staining in a DjPum dsRNA-injected planarian 15 days after the first transection. (D) H3P staining of an intact planarian, 15 days after X-ray irradiation. Scale bars: 50 µm.

View larger version (98K): [in a new window]   Fig. 7. Characterization of the cell fraction obtained following the serial filtering procedure in D. japonica. (A) Bright-field image of cells obtained by filtration through the 8 µm pore size mesh. Scale bar: 10 µm. (B) DjMCM2 and DjFGFR1 expression visualized by comparative RT-PCR. RNA was extracted from cell fractions obtained by filtration through nylon meshes of 50 µm (1), 20 µm (2) and 8 µm (3) pore size sequentially. DjEF2 is used as an internal amplification control.

View larger version (27K): [in a new window]   Fig. 8. FACS analysis of neoblasts in DjPum dsRNA-injected D. japonica demonstrates a consistent reduction in the number of proliferating cells. Graphs from a representative experiment are shown. The cell population individuated as proliferating cells is included in the R2 box. (A) Cells from water-injected planarians, stained with PI and analyzed for the DNA content in the fluorescent channel FL2. (B) Analysis of cells included in the R2 box in A, using FSC and SSC. (C) Cells from X-ray-treated planarians sacrificed 15 days after the treatment were stained with PI and analyzed for the DNA content in the fluorescent channel FL2. (D) Analysis of cells included in the R2 box in C, using FSC and the SSC. (E) Cells from DjPum dsRNA-injected planarians were stained with PI and analyzed for the DNA content in the fluorescent channel FL2. (F) FSC and SSC analysis of cells included in the R2 box in E, demonstrates that the cells interpreted as proliferating cells have a similar morphology. The `nuage' at the left in A, C and E is probably due to residual bacterial contamination. Clusters of cells may generate the high values of fluorescence intensity observed at the right of the R2 box in A, C and E. (G) Graphic representation of proliferating cells (in percentage). GraphPad Prism Version 3.0 computer program was used for data analysis. Each bar shows the mean±s.e.m. of three separate experiments. The results were compared using the unpaired t-test; P<0.05 was considered statistically significant. *P<0.05; **P<0.01.