Volume 11, Issue 4, Pages 282-287 (February 2001) TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish  Ferenc Müller, Lòrànt Lakatos, Jean-Christophe Dantonel, Uwe Strähle, Làszlò Tora  Current Biology  Volume 11, Issue 4, Pages 282-287 (February 2001) DOI: 10.1016/S0960-9822(01)00076-8

Figure 1 Inhibition of TLF by expression of a dominant negative mutant (TLFmutB) interferes with epiboly and transcription in the zebrafish embryo. (a–d) Expression of TLFmutB results in severe inhibition of the epibolic spread of the blastoderm over the yolk cell. (a) Noninjected control at 50% epiboly stage. Expression of wild-type TLF (b) did not cause significant inhibition of epiboly.(c) Expression of the dominant negative TLFmutB had similar blocking effect on epiboly as injection of embryos with α-amanitin (d). (e,f) Zygotic expression of green fluorescent protein (gfp) from the reporter construct CMV::gfp is inhibited by coinjection of dominant-negative TLFmutB (f) but not by wild-type TLF (e). (g–i): Expression of notail (dark ring in [g]) is inhibited by expression of TLFmutB (h) or injection with α-amanitin (i). (j–l) The correct phosphorylation of the CTD of Pol II on serine 2 is impaired in TLFmutB-injected embryos. Immunohistochemical staining of noninjected embryo (j), TLFmutB-injected embryo (k), or α-amanitin-injected embryo (l) at 50% epiboly stage using the H5 antibody. All embryos are shown at 50% epiboly stage in lateral views, animal pole up (a–d), or in dorsal views of the animal pole (e–l) Current Biology 2001 11, 282-287DOI: (10.1016/S0960-9822(01)00076-8)

Figure 3 TLF and TBP differentially regulate early zygotic transcription. Embryos were injected with either the dominant-negative TLFmutB or with the antisense TBP-MO. Embryos were split when controls had reached the 40%–50% epiboly stage and subjected to in situ hybridization with evenskipped (eve1) (a–c), notail (ntl) (d–f), forkhead4 (fkh4) (g–i), sonic hedgehog (shh) (j–l), spadetail (spt) (m–o), t-box6 (tbx6) (p–r), and goosecoid (gsc) (s–u) probes. Expression was abolished by inhibition of either TLF or TBP in case of shh (87.5%, n = 23; 95.0%, n = 20, respectively), spt (88.9%, n = 18; 95.2%, n = 21), tbx6 (85.0%, n = 20; 100.0%, n = 22), and gsc (90.0%, n = 20; 100.0%, n = 17). In contrast, ntl and fkh4 were downregulated only by inhibition of TLF ([e,h] 94.3%, n = 35, and 87.9%, n = 33, respectively) while blocking of TBP had no or little effect on these genes (ntl, 0.0%, n = 42; fkd4, 23.8%, n = 21). Expression of eve1 was abolished by inhibition of TBP ([c] 100.0%, n = 18); however, TLF-blocked embryos showed no effect or an expansion of eve1 mRNA expression into the animal pole area ([b] 72.8% normal or ectopic expression, n = 22). View onto animal pole with dorsal up Current Biology 2001 11, 282-287DOI: (10.1016/S0960-9822(01)00076-8)

Figure 2 Inhibition of TBP expression by antisense morpholino oligonucleotides (MOs) impairs epiboly and blocks correct phosphorylation of the CTD of Pol II. (a–d) A TBP-MO overlaping the first AUG codon of zTBP mRNA blocks epiboly in a dose-dependent manner. In contrast to embryos injected with control MOs (a), increasing doses of TBP-MO (0.3 mM, 1 mM, 5 mM in [b–d], respectively) inhibit epiboly. Embryos are oriented with the animal pole up. (e) TBP expression in extracts from MO-injected embryos. Injection of the TBP-MO but not of the control MO (c-MO) completely eliminated zebrafish TBP (zTBP) expression at 50% epiboly stage. Furthermore, expression of human TBP (hTBP) from a coinjected plasmid (CMV:hTBP) was clearly detectable at 75% epiboly in the embryos; however, the endogenous TBP expression at this stage is significantly lower than at 6 hr gastrula stage. As a loading control (Pol II), an antibody raised against the CTD of Pol II was used (10% SDS-PAGE). (f–h) Phosphorylation of the CTD of Pol II on serine 2 is abolished in TBP-MO-injected zebrafish embryos. Immunohistochemical staining of control-MO injected embryo (f) and zTBP-MO injected embryo (g) at 50% epiboly stage using the H5 antibody. (h) Western blot analysis of protein extracts from 50% epiboly stage embryos using the H5 or the 7G5 mAbs. The hyperphosphorylated (Pol IIO) and the hypophosphorylated (Pol IIA) forms of the large subunit of Pol II are labeled (8% SDS PAGE). ns: non-specific loading control Current Biology 2001 11, 282-287DOI: (10.1016/S0960-9822(01)00076-8)