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???displayArticle.abstract??? POSH (Plenty of SH3s) has distinct roles as a scaffold for specific c-Jun N-terminal kinase (JNK) signaling modules and as an E3 ubiquitin ligase. The physiological function of POSH remains unclear, however, and its possible involvement in developmental processes motivated the present study wherein the Xenopus orthologue of POSH (xPOSH) was examined for its potential role during Xenopus early embryogenesis. Loss-of-function analysis using morpholino oligonucleotides demonstrated that POSH was essential for Xenopus anterior neural development, although not Spemann organizer formation and early neurogenesis, through the formation of an active JNK signaling complex. Moreover, POSH-mediated JNK pathway was essential for apoptosis in anterior neural tissues. Finally, the present findings demonstrate that RING (Really Interesting New Gene) domain-mediated E3 ubiquitin ligase activity of POSH was not involved in POSH-mediated JNK pathway in vivo. Together, these data suggest that the active JNK signaling complex formed of POSH and the JNK module is essential for the expression of anterior neural genes and apoptosis in Xenopus anterior development.
Fig. 1. Expression pattern and antisense morpholino of Xenopus POSH (xPOSH). (A) RT-PCR analysis of temporal expression of POSH. Developmental stages are shown above each lane. Ornithine decarboxylase (ODC) was used as a loading control. (B) In situ hybridization analysis of spatial expression of POSH during early Xenopus development. (C) Scheme of the myc-tagged 5′-UTR POSH construct, the myc-tagged ORF POSH construct, and the morpholino sequence targeting the POSH 5′-UTR sequences. (D) xPOSH MO specifically inhibits the translation of its cognate mRNA. 5′-UTR (500 pg) and ORF (500 pg) xPOSH-Myc mRNA were injected with xPOSH MO (20 ng) or control MO (20 ng) into the animal region of embryos at the four-cell stage. Animal cap explants isolated at early gastrula stages were then subjected to immunoblot (IB) analysis with anti-Myc antibodies. Actin served as a specificity control.
Fig. 3. POSH is required for Xenopus anterior neural development, but not organizer formation and early neurogenesis. (A–B) xPOSH depletion reduced several anterior neural markers. (A) Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng) and Co MO (20 ng), allowed to develop to stage 22, and analyzed by RT-PCR for expression of the indicated markers. ODC was used as a loading control. (−) RT denotes the no reverse transcription control sample. (B) Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), and analyzed by whole-mount in situ hybridization for of the regional neural markers Otx2, En2, or Krox20. Nuclear β-gal mRNA (200 pg) was coinjected to allow identification of the xPOSH MO-injected side as a lineage tracer. No; not injected, Inj; injected. (C) RT-PCR analysis showed xPOSH also did not affect the organizer markers. Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with xPOSH MO (20 ng), Co MO (20 ng), and xPOSH mRNA (2 ng). Dorsal marginal zone (DMZ) explants were isolated at stage 10.5 and analyzed by RT-PCR, using primers specific for Goosecoid (Gsc), Chordin (Chd), and Cerberus (Cer). ODC was used as a loading control. (−) RT denotes the no reverse transcription control sample. (D) xPOSH MO did not alter expression of the organizer markers Cer or Goosecoid, and early neural marker Sox2. Four-cell stage embryos were injected at their single dorsal blastomeres with xPOSH MO (20 ng), cultured until stage 10.5 (left and middle) or stage 13 (right). Nuclear β-gal mRNA (200 pg) was used as a lineage tracer. No; not injected, Inj; injected.
Fig. 4. POSH regulates Xenopus anterior neural development through JNK signaling pathway. (A–D) Two blastomeres of four-cell stage embryos were injected at their dorsal region with the indicated mRNAs. (A) Coinjection of xPOSH MO (20 ng) with XeJNK1 rescued the phenotypic defects of xPOSH MO in a dose-dependent manner. In addition, coinjection of JNK1 and CO MO did not exhibit the anterior neural defects. These phenotypes were classified into four classes: Index I, normal head and eye; Index II, weak head defect with small size eye; Index III, strong head defect with pin-shaped eye; Index IV, complete defect with no eye. (B–E) Whole-mount in situ hybridization with Otx2, En2, and Krox20 as probes. The reduction of anterior neural markers caused by xPOSH MO (20 ng; C) was reversed by XeJNK1 mRNA (2 ng; D). Coinjection of JNK1 and CO MO did not alter the expression of anterior neural genes (E).
