The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Background: The application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene. Results: A single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes. Conclusions: Our results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation

Additional file 4: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

MO-induced gro/tle loss of function phenotypes. Embryos at the 1-cell stage were co-injected with morpholino oligonucleotides and 1 μg/ml FITC-dextran. Injections were performed using either a single morpholino directed against tle1 (C), tle2b (D,F), and tle3b (E), or combinations directed against tle1 + 2b (H), tle1 + 3b (I), tle2b + 3b (J), and tle1 + 2b + 3b (K). Single morpholino oligonucleotides were injected at a concentration of 600 μM (C-F) and combinatorial injections (H-K) were performed using 300 μM of each MO. Phenotypes of FITC-dextran positive embryos were observed after the beginning of eye pigmentation at stage 32 (B-F) and stage 28 (G-K). (B,G) Wild type control embryos were injected with 1× Yamamoto’s and FITC-dextran. All embryos are shown in dorsal view with anterior at the top. Compared to the wild type controls (B,G), morpholino injected embryos developed smaller eyes that were shifted towards the midline (D,E,J,K) or cyclopic eyes (C,H,I). In rare cases the eyes were lost entirely (E). (A) Phenotypes of FITC-positive embryos were categorized at stage 28-32 into weak and strong phenotypes. Weak phenotypes developed smaller eyes that were shifted towards the midline, whereas strong phenotypes showed cyclopic eyes. Eye-less phenotypes were included into the group of strong phenotypes. Abbreviations: MO, morpholino oligonucleotide; WT, wild type. Scale bar 100 μM. (PDF 10028 kb

Additional file 3: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Mammalian two-hybrid analysis of Aes-mediated Gro/Tle repression. 20 ng of the expression constructs (pMCamVP16, pMCTle4VP16 or pMCTle1VP16) were co-transfected with 70 ng of firefly luciferase reporter construct pLucF24ZF, 20 ng of the expression construct for the six-domain of human Six3 (pMChSix3(85-203)mZFb6) and full length Aes (pKCAes at the indicated concentrations) into HeLa cells. The interaction of the artificial leucine zipper domain “acid” and “base” [63] was used as a control. All three interactions were set 100% in the absence of Aes (acid/base corresponds to a more than 100 fold activation of the luc reporter construct compared to a control without bait, similar induction rates were seen for Tle4/Six3 and Tle1/Six3). Addition of the Aes expression construct at the indicated concentrations did not affect the acid/base interaction (blue bars), but strongly reduced the Tle4/Six3- (red bars) and the Tle1/Six3 interaction (green bars). (PDF 10028 kb

Additional file 2: of The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

Gro/Tle dependence of 1 tcf3 in gain-of-function experiments. Embryos at the 1-2 cell stage were co-injected with 40 ng/μl of the indicated gfp:HSE:Tcf3 constructs. Heat treatment (10 min, 43.5 °C) was applied at stage 14. (A) Statistical overview of the phenotype distribution. Whole mount in situ hybridization experiments for rx2 were performed on embryos at stage 21. (B) dorsal view of a stage 31 embryo with anterior at the top, (C) lateral view with anterior at the left. Arrowheads indicate ectopic otic vesicles, the arrow points to the endogenous otic vesicle. Scale bar 100 μm; B and C. (PDF 10028 kb