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???displayArticle.abstract??? Pescadillo is a multifunctional, nuclear protein involved in rRNA precursor processing, ribosomal assembly, and transcriptional regulation. Pescadillo has been assigned important functions in embryonic development and tumor formation. We previously identified pescadillo as a potential downstream target of non-canonical Wnt-4 signaling. Here we have investigated for the first time the function of the Xenopus laevis homolog of pescadillo during early embryogenesis on a molecular level. Loss of function analysis indicates that pescadillo is required for eye development and neural crest migration. BrdU incorporation and TUNEL assays indicate that a loss of pescadillo function affects proliferation and triggers apoptosis through a p53-mediated mechanism. Furthermore, pescadillo affects the expression of early eye-specific marker genes, likely independent of its function in regulating proliferation and apoptosis, and in addition migration of cranial neural crest cells. Our data indicate that pescadillo has multiple important functions during X. laevis development and that its function is highly conserved among different species.
Fig. 1. Spatial expression of Xenopus laevis pescadillo. Spatial expression of pescadillo was analyzed by whole-mount in situ hybridization. (A) Specific expression could first be detected at stage 18 in neural tissue with strongest expression in the anterior neural plate (arrow). (B) At stage 23, pescadillo can be detected in migrating cranial neural crest cells (arrow) and the developing eye (arrow head). (C) Similarly, expression of pescadillo at stage 26 can be detected in the neural crest cells and the eye. Dashed line indicates level of cross-section shown in panel G. (D) Dorsal view of an embryo at stage 31. Specific expression can be found at the isthmus, the midbrain–hindbrain boundary (arrow). (E) Expression at stage 31 persists in derivatives of cranial neural crest cells, the eye and the pronephros (arrow). White dashed line indicates level of section as indicated in panel H, whereas the black dashed line indicates level of section as shown in panel I. (F) Magnification of an embryo at stage 31. Pescadillo expression is enriched in the region of the ciliary marginal zone of the eye (arrow). (G) Transverse section of an embryo at stage 26. Expression of pescadillo can be found in the isthmus (arrow head) and outer layer of the eye, the periocular mesenchyme (arrow). (H) Horizontal section at stage 31 indicates expression of pescadillo in the branchial arches. (I) Horizontal section at stage 31 indicates expression to be likely within the periocular mesenchyme (arrow).
Fig. 2. Expression of pescadillo depends on Wnt-4 and Fz-3. Xenopus laevis embryos were unilaterally injected into one dorsal-animal blastomere at 8-cell stage with either antisense morpholino oligonucleotides (MO) against Wnt-4 (A), Frizzled-3 (B) or a control MO (A, B). Expression of pescadillo was visualized by whole-mount in situ hybridization. Expression of pescadillo in the eye (white arrows) and the cranial neural crest cells (black arrows) depends on Wnt-4 as well as Frizzled-3 function.
Fig. 3. Characterization of an antisense morpholino oligonucleotide directed against Xenopus laevis pescadillo. (A) Binding site of the pescadillo MO within the pescadillo mRNA. The ATG start codon is highlighted. Sequence of the pescadillo_Mut RNA in which 9 nucleotides have been changed as indicated in red. (B) Coupled transcription and translation using radioactively labeled methionine. Plasmids encoding pescadillo or pescadillo_Mut were used as indicated. Morpholino oligonucleotides were added as indicated. Pescadillo MO but not a control MO interferes with translation of the pescadillo RNA. Pescadillo MO does not block translation of Pescadillo_Mut. (C) Efficiency of pescadillo MO was also tested in whole embryos. Oligonucleotides representing the sequences as given in panel A were cloned in frame to and in front of GFP. RNA coding for the corresponding constructs (pescadillo-GFP or pescadillo_Mut-GFP) was injected together with pescadillo MO or control MO as indicated. GFP expression was monitored at stage 25. Pescadillo MO but not control MO interfered with translation of Pescadillo-GFP. Pescadillo_Mut-GFP was not targeted by pescadillo MO.
Fig. 4. Loss of pescadillo function results in severe eye and craniofacial cartilage defects. (A–F) Unilateral injection of pescadillo MO but not control MO into one dorsal-animal blastomere of 8-cell stage embryos results in severe eye phenotypes. (G–I) Histological analysis of pescadillo MO injected embryos indicates strong reduction or severe morphological eye defects in comparison to control MO injected embryo. (J–O) Embryos were unilaterally injected into one dorsal-animal blastomere of 8-cell stage embryos and cultured until stage 48. Cartilage development was visualized using an Alcian blue staining. Loss of pescadillo results in cartilage defects of diverse severity that correlates with the eye phenotype. ba: Branchial arches, ta: tectum anterius, mc: Meckel's cartilage, lc: infrarostralcartilage. (P) The effect of pescadillo MO onto eye development is dose dependent. n: Number of independent experiments, N: number of embryos scored in total. Error bars indicate standard error of the mean.
Fig. 5. Analysis of domain requirements for pescadillo function. (A) Schematic drawing of pescadillo deletion mutants. (B) RNAs coding for pescadillo_Mut, pescadilloΔBRCT, or pescadilloΔSUMO are able to revert the pescadillo MO phenotype. PescadilloΔNLS is unable to revert the pescadillo MO phenotype. The eye was used as a read out. Representative embryos are shown. (C) Statistical evaluation of described experiments. Embryos were scored for strong reduction and morphological eye defects as shown in Fig. 4 panels A–D. Mild effects on eye size were not considered to be a phenotype. n: Number of independent experiments, N: number of embryos scored in total. Error bars indicate standard error of the mean.
Fig. 6. Pescadillo regulates apoptosis and proliferation through p53. Embryos were injected at 8-cell stage into one dorsal-animal blastomere with pescadillo MO (6 ng), control MO or p53MO (10 ng) as indicated. (A) Embryos were analyzed for apoptosis using TUNEL staining at stage 26. A statistical evaluation of embryos with an increased apoptosis is given. Coinjection of p53 MO together with pescadillo MO can reduce the number of embryos with an increased apoptosis. (B) Embryos were analyzed for changes in proliferation by BrdU assays at stage 25. A statistical evaluation of embryos with decrease of proliferation is given. Coinjection of p53 MO together with pescadillo MO can reduce the number of embryos with decreased proliferation. (C) Coinjection of pescadillo MO (6 ng) and p53MO (10–20 ng) does not rescue the pescadillo loss of function eye phenotype. Both doses tested (10 ng, n = 2; 20 ng, n = 2) had no rescuing activity. (D) Pescadillo MO was injected unilaterally into one dorsal-animal blastomere of 8-cell stage embryos and proliferation was monitored using BrdU assay at stage 25. Embryos were scored for unilateral reduction in BrdU incorporation as shown in Fig. 5B. Coinjection of RNAs coding for pescadillo_Mut or pescadilloΔSUMO but not GFP, pescadilloΔBRCT or pescadilloΔNLS were able to rescue the pescadillo MO phenotype on proliferation. n = Number of independent experiments. N = number of embryos examined in total. Error bars indicate standard error of the mean.
Fig. 7. Pescadillo affects differentiation within the early eye field. Pescadillo MO or control MO was injected unilaterally into one dorsal-animal blastomere of 8-cell stage embryos. (A) Expression of marker genes Pax-6, Rx, Otx-2, or MyoD was analyzed by whole-mount in situ hybridization at stage 23, as indicated. Expression of Pax-6, Rx, or Otx-2 in the eye field was reduced whereas expression of MyoD was not affected. (B) Statistical evaluation of reduced Rx expression in pescadillo MO injected embryos. Coinjection of pescadillo_Mut, pescadilloΔBRCT, or pescadilloΔSUMO but not pescadilloΔNLS was able to reduce the number or embryos with an unilateral reduction in Rx expression. n = Number of independent experiments. N = number of embryos examined in total. Error bars indicate standard error of the mean.
Fig. 8. Pescadillo affects Sox-3 expression in branchial arches. (A) Pescadillo is expressed in the neural crest cell derived part of the branchial arches, whereas Sox-3 is expressed in the pharyngeal pouches within the endodermal layer that touches the outer ectodermal layer. More dorsally, Sox-3 is expressed in the lateral line placode and the otic placode (Schlosser and Ahrens, 2004). (B) Unilateral injection of Wnt-4 MO or pescadillo MO but not control MO results in a reduction of Sox-3 expression in the branchial arches. Neither pescadillo MO nor control MO affects expression of MyoD or Emx1 at stage 32. (C) Statistical evaluation of embryos with reduced Sox-3 expression at stage 32 after unilateral injection of pescadillo or Wnt-4 MO. Pescadillo _Mut RNA can revert the pescadillo MO effect on Sox-3 expression. n = Number of independent experiments. N = number of embryos examined in total. Error bars indicate standard error of the mean.
Fig. 9. Pescadillo interferes with cranial neural crest migration but not specification. (A) Whole-mount in situ hybridization indicates a partial coexpression of Slug and Wnt-4 at stage 19. Frizzled-3 is expressed throughout the whole anterior neural plate with different intensities. (B) Frizzled-3 MO but not Wnt-4 MO, pescadillo MO or control MO interferes with slug expression at stage 17. Pescadillo MO does not interfere with FoxD3 expression at stage 17. (C) Unilateral injection of Wnt-4 MO and pescadillo MO results in a neural crest cell migration defect as indicated by staining for slug (stage 20) and Krox20 (stage 23/24). In case of pescadillo MO, FoxD3 was used as an additional marker (stage 20). Staining for FoxD3 indicates that neural crest cells stay in the dorsal part of the embryo and do not migrate towards the ventral side. A statistical analysis of these experiments is given at the bottom of the panel C. n = Number of independent experiments. N = number of embryos examined in total. Error bars indicate standard error of the mean.
