Fig.2. Using zebrafish model to investigate the role of Rpl22 in T cell development.

(A, B) Targeted knockdown of Rpl22 in zebrafish embryos. (A) For Rpl22, the MO is designed to specifically block the initiation of translation at the start codon in exon 1. The short black line and blue arrow indicate the positions of sequences targeted. (B) The effect of 1ng Rpl22-MO knockdown on expression of endogenous Rpl22 is assessed by immunoblotting on 5dpf embryos. (C) Rpl22-MO injected (1 ng), but not MM (5-base pair mismatch) control injected, Tg(lck:EGFP) fish exhibit a loss of EGFP-marked T cells at 5 dpf (lateral view, red circles; dorsal view, yellow rectangles). Numbers refer to fraction of morphants with the depicted phenotypes. (D) Injection of 1 ng Rpl22-MO causes apoptosis in the thymus at 3.5 dpf, while injection of the same quantity of 5-base pair mismatch control morpholino (Rpl22-MM) caused no change in the thymus (green dashed rectangles). The white dashed line delimits the eye. Numbers refer to fraction of morphants with the depicted phenotypes. (E-H) The use of a heat-shock inducible, bidirectional expression construct to restore T cell development in rpl22 morphants. (E) Upper panels depict schematics of the heat-inducible expression plasmid pSGH2-mRpl22. (F) The heat shock-inducible expression plasmid associated with I-sceI mediated transient overexpression is injected into 1-cell stage embryos. At 3dpf, the embryos are heat shocked at 37°C for 1hr and then effects on T cell development are analyzed at 5 dpf using WISH for lck, a marker of thymic progenitors. (G) After heat-shock, GFP+ embryos are selected for subsequent WISH analysis (pink arrowheads). (H) Heat shock induction of mouse Rpl22 expression rescues T cell development in rpl22 morphants (5dpf lck WISH staining, red rectangles). Numbers refer to fraction of morphants with the depicted phenotypes.