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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. |
