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Figure 1. Conservative evolution of vertebrate zfp36 genes.
(A) Conserved syntenic regions between human (Hsa), mouse (Mmu) and Xenopus tropicalis (Xtr) chromosome regions containing zfp36, zfp36l1 and zfp36l2. Gene names symbols are according to HUGO. Boxes with the same colour correspond to the same gene; white boxes correspond to genes without annotation or without orthologues in the species shown here. The drawing is not to scale to avoid complexity and dashes represent long chromosome regions. (B) Conserved structural organization of vertebrates zfp36 genes between evolutionary distant animals. Exons (1, 2) are figured in open boxes and intron as a solid line respectively. Shaded box, untranslated region. TZF, Tandem Zing Finger domain.
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Figure 2. mRNA expression of Xenopus zfp36 genes during development.
(A) RT-PCR analyses showed that all zfp36 genes are maternally expressed. zfp36, zfp36l1 and zfp36l2 mRNAs are expressed at a constant level throughout development from stage 2 to stage 33 while zfp36l4 mRNA level decreases after the mid-blastula transition (MBT, arrow). (B) In situ hybridization showed that all four zfp36 mRNA are localized at the animal pole in 4-cell stage (a) and morula stage (i) embyos. e correspond to histological sections from embryos shown in a. (C) RT-PCR analysis showed that zfp36 mRNAs are preferentially expressed in the animal pole region of blastula embryos. (D) RT-PCR analysis showed that zfp36 mRNAs are expressed throughout the embryo at the gastrula stage. An, animal pole; DM, dorsal marginal zone; Emb, whole embryo; Ve, vegetal pole; VM, ventral marginal zone. A control embryo (Emb) assayed by RT-PCR for the expression of control genes chordin and wnt8. odc was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-).
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Figure 3. Zfp36 has a distinct spatial expression from zfp36l1 and zfp36l2 in the embryo.
The localization of zfp36 mRNAs was detected by in situ hybridization in embryos from stage 24 (a, d, g, j), stage 28 (b, e, h, k) and stage 35/36 (c, f, i, l). A close up view of a transverse section at the level of pronephros is shown in c. Zfp36l4 expression was never detected in the embryos at any stage. ba, branchial arches; br, brain; cg, cement gland; du, pronephric duct; mb, midbrain; no, notochord, ov; otic vesicle; pn, pronephros anlage, pronephric tubule; so, somites.
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Figure 4. The different zfp36 genes respond differently to growth factors treatment in animal cap explants.
(A) RT-PCR analysis of zfp36 gene expression in animal cap explants treated with 12.5 or 50 ng/ml of FGF2 or with the FGF inhibitor in the absence (SU) or in the presence of 50 ng/ml FGF2 (SU+F). (B) RT-PCR analysis of zfp36 gene expression in animal cap explants treated with 1, 5 or 25 ng/ml of activin or with the activin inhibitor in the absence (SB) or presence of 25 ng/ml activin (SB+A). (C) RT-PCR analysis of zfp36 genes in on animal cap explants from embryos injected with 0.5 ng or 1 ng of BMP2 mRNA. Stage 20 embryo (Emb) or uninjected embryo or untreated animal caps (-) were assayed by RT-PCR for the expression of control genes msr, myl1 and globin. Odc was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-RT).
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Figure 5. Zfp36 mRNA overexpression induces somites segmentation defects.
250 pg of mouse zfp36 mRNA (a, b) or Xenopus zfp36 (d, e), zfp36l1 (I, j), zfp36l2 (k, l) or zfp36l4 (m, n) mRNA were injected into one blastomere of two-cell stage embryos and developing embryos were fixed at stage 28 before immunhistochemistry analysis with the somite specific marker 12/101. Embryos were embedded in paraffin then sectioned longitudinally (c, f) or treated for scanning electronic microscopy (g, h). The arrows mark the alteration of segmentation on the injected side (Inj) by comparison with the uninjected side (Uninj). no, notochord; so, somite.
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Figure 6. Zfp36 mRNA overexpression does not prevent mesoderm induction nor myogenic factor expression. (A) Two-cell stage embryos were injected with 250 pg of the different Xenopus zfp36 mRNAs or mouse zfp36 mRNA (mzfp36) and animal caps were explanted at stage 8.5 then treated with 10 ng/ml of activin before analysis by RT-PCR for xbra expression when control embryos reached stage 12. Stage 12 embryo (Emb) or untreated animal caps (-) were assayed by RT-PCR in parallel. Odc was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-RT). (B) 250 pg of Xenopus zfp36 (a, b) and zfp36l1 (c, d) or mouse Zfp36 (mZfp36, e, f) mRNAs were injected in one blastomere of two-cell stage embryos and developing embryos were fixed at stage 28 and analyzed by in situ hybridization for myod expression.
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Figure 7. Zfp36 mRNA overexpression alters the expression of notch signalling pathway members esr5 and hairy2a. 250 pg of Xenopus zfp36 (a, b, i, j), zfp36l1 (c, d), zfp36l2 (e, f) or mouse zfp36 (mzfp36, g, h, k, l) mRNAs were injected into one blastomere of two-cell stage embryos and developing embryos were fixed at stage 25 (a and k, l) or stage 28 (i, j) and analyzed by in situ hybridization for esr5 and hairy2a expression. Arrows in a, c, e and g mark the presomitic mesoderm (psm) and the pronephros region (pn) in i respectively. Stars in a, c, e and g mark the first two somitomeres.
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Figure 8. Zfp36 mRNA overexpression alters the formation of pronephros and affects pronephric marker genes expression. (A) 250 pg of Xenopus zfp36 mRNA were injected into one ventral blastomere of 8-cell stage embryos and developing embryos were fixed at stage 39 before immunhistochemistry analysis with the pronephros specific markers 3G8 and 4A6. Arrows in b and d mark the pronephros (pn) alteration on the injected side. (B) 250 pg of Xenopus zfp36 (a) or zfp36l1 (e) mRNA was injected into one ventral blastomere of 8-cell stage embryos and developing embryos were fixed at stage 22 (a, b and e) or stage 26 (c, d) before in situ hybridization analysis for pax8 or lim1 expression. Arrows in b, d, f and h mark the pronephros (pn) alteration on the injected side. (C) Two-cell stage embryos were injected or not (NI) with 250 pg of the different Xenopus zfp36 mRNAs. Animal caps were explanted at stage 8.5 and treated with activin plus retinoic acid (RA) before analysis by RT-PCR for smp30 expression when control embryos reached stage 35. Stage 35 embryo (Emb) or untreated animal caps (-) were assayed by RT-PCR in parallel. Odc was used as control of loading and a reaction was performed in the absence of reverse transcriptase to check for genomic DNA contamination (-RT).
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Figure 9. Zfp36 and zfp36l1 morpholino knock down induces pronephros alterations. 20 ng of morpholinos directed against zfp36 (a, b) or zfp36l1 (c, d) mRNAs or control morpholinos (e, f) were injected into one ventral blastomere of 8-cell stage embryos with 250 pg of lacZ mRNA. In rescue experiments, 10000 pg of mouse zfp36 mRNA were co-injected with 20 ng of MO zfp36 (g, h). Developing embryos were fixed at stage 40 before lacZ staining and immunohistochemistry analysis to reveal the expression of pronephros specific markers, 3G8 and 4A6. Arrows and arrowheads in b, d, f and h, mark the pronephros proximal tubule (tu) and duct (du) respectively on injected sides of the embryos. I, Close up views of anterior region showing uninjected or injected sides of representative phenotypes for zfp36 morphants (i) and zfp36l1 morphants (m).
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Figure 10. Zfp36 depletion does not affect somitogenesis nor early pronephros specification. (A) 20 ng of zfp36 morpholinos (MO) were injected into one dorsal blastomere of 4-cell stage embryos with 250 pg of lacZ mRNA. Embryos were fixed at stage 15 for the detection of myod by in situ hybridization (a) or at stage 28 for immunohistochemistry with 12/101 antibody (d). a, b and c are representative phenotypes and f and g are close up views of d and e respectively. Arrowheads indicate regularly segmented somites on the injected side. (B) 20 ng of zfp36 morpholinos (MO) were injected into one ventral blastomere of 8-cell stage embryos with 250 pg of lacZ mRNA. Embryos were fixed at stage 29/30 (a, b, e, f), 33/34 (c, d) or 27 (g, h) and analysed for the expression of lim1, pax8, wnt4 and wt1 by in situ hybridization.
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Figure S1. Phylogenetic tree showing the evolutionary relationship between zfp36, zfp36l1 and zfp36l2 genes. The tree was made from the amino acids sequence of the tandem zinc finger domain using mega4 program. Ae, Aedes aegypti; Aq, Amphimedon queenslandica; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila Melanogaster; Hm, Hydra magnipapillata; Hs, Homo sapiens; Io, Ilyanassa obsoleta; Mm, Mus musculus; Nv, Nematostella vectensis; Sc, Saccharomyces cerevisiae; Sp, Strongylocentrotus purpuratus; Sk; Socoglossus kowalevskii; Tc, Tribolium castaneum; Xt, Xenopus tropicalis.
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Figure S2. Efficacy of zfp36 mRNA translation inhibition by morpholinos. (A) 500 pg of zfp36 mRNA were in vitro translated in reticulocyte lysate and translation products were analyzed by SDS PAGE followed by autoradiography. Lane 0, mock translation without mRNA; lane 1, no zfp36 MO; lane 2, 50 ng of zfp36 MO; lane 3, 100 ng of zfp36 MO; lane 4, 100 ng of Control (Co) MO. (B) 250 pg of zfp36 mRNA were injected in embryo alone (lane 1) or with 80 ng of zfp36 MO (lane 2) or 80 ng of control MO (lane 3). Embryos were fixed at stage 12 and protein extracts were analyzed by western blot with an anti flag antibody. The migration of zfp36 protein is indicated by an arrow. Lane 0, uninjected embryo. Non specific signal (ns).
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Figure S3. mpaired pronephros morphogenesis caused by zfp36 depletion is largely independent of Notch pathway, proliferation or apoptosis. 8-cell stage embryos were injected unilaterally with 20 ng of zfp36 morpholinos together with 250 pg of lacZ mRNA tracer and analysed at stage 33/34 for Hairy2a expression by whole mount in situ hybridization (a, b), at stage 32 by TUNEL assay (c, d) or at stage 28 by immunohistochemistry with anti-phospho-Histone H3 antibody (Phospho-H3) (e, f).
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