1. Volume 6, Issue 3, Pages 715-722 (September 2000)
TBP-like Factor Is Required for Embryonic RNA Polymerase II Transcription in C. elegans 
Jean-Christophe Dantonel, Sophie Quintin, Lòrànt Lakatos, Michel Labouesse, Làszlò Tora 
Molecular Cell 
Volume 6, Issue 3, Pages (September 2000)
DOI: /S (00)

2. Figure 1
CeTLF Protein Is Nuclear and Present in All Cells throughout Embryogenesis
(A) Schematic representation of the Caenorhabditis elegans (Ce) tlf-1 genomic structure. The cDNA (starting with the SL1 splice leader) was aligned to the genomic sequences. Open boxes indicate the exons, and gray regions indicate the exons that encode the two direct repeats of CeTLF (Dantonel et al. 1999).
(B) Homology of CeTLF amino acid sequence with the C. elegans TBP (CeTBP). The percentage of similarity between the core domains (shown in gray) is indicated. “n.s.” denotes nonsignificant.
Wild-type embryos (WT) were stained with polyclonal antibodies against CeTLF-1 (D, F, and H) and the DNA-specific dye DAPI (C, E, and G).
(C and D) Two-cell stage embryo.
(E and F) Mid-embryogenesis embryo (end of proliferation). All cells express CeTLF, and the signal is nuclear.
(G and H) Mid-embryogenesis embryos that were coincubated with polyclonal antibodies against CeTLF and an excess of competitor peptide used to generate the anti-CeTLF antibody. The nuclear signal is abolished.
(I and J) The nuclear signal is eliminated in tlf-1(RNAi) embryos. In this and subsequent figures, embryos are oriented with anterior to the left and dorsal up; the scale bar indicates 10 μm.
(K) Forty WT or tlf-1(RNAi) embryos were collected at different time points after injections (as indicated) and allowed to reach their terminal phenotypes. The four different time points correspond to the four time points shown in Table 1. Embryos were boiled directly in 10 μl of SDS–PAGE sample buffer, and proteins were analyzed by Western blot using antibodies raised against CeTLF, CeTBP, and the CTD of Pol II. The hyperphosphorylated (IIO) and the hypophosphorylated (IIA) forms of the large subunit of Pol II are labeled.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
tlf-1(RNAi) Embryos Arrest with Three Different Terminal Phenotypes
Tlf-1 dsRNA was injected into strains bearing integrated pha-4::gfp, hlh-1::gfp, or hlh-2::gfp markers (as indicated), which are expressed in the digestive system, the muscles, and the nervous system, respectively. The top line shows the phenotype seen by Nomarski optics of the embryos tested for pha-4::gfp. Wild-type embryos (WT) are shown either at less than 80 cell stage (A–D) or at the comma stage (I–L) when cell division stops in WT embryos. tlf-1(RNAi) embryos were found to display three main phenotypic classes (denoted S1–S3, see top line) depending on how long after injection they were collected. (E–H) S1-class embryos arrested with approximately 50 cells and were positive for all GFP markers tested. (M–P) S2-class embryos arrested at the approximately 300-cell stage and showed no GFP expression. (Q–T) S3 embryos arrested with more than 600 cells (without any apparent differentiation) and showed some hlh-1::gfp and hlh-2::gfp expression but did not express pha-4::gfp. (U) Poly(A)+ RNA isolated from either the three phenotypic classes of tlf-1(RNAi) embryos (S1, S2, and S3) or WT embryos (having the same cell numbers as the corresponding mutant embryos) was subjected to quantitative RT-PCR analysis to monitor tlf-1 expression (28–30 cycles). Transcription from the tbp gene (28–30 PCR cycles) was used for normalization. “C”, control RT-PCR using 0.5 μg of total RNA from adult worms. Results from two independent experiments were scanned, and the mRNA levels of the different tlf-1(RNAi) and WT embryos were calculated relative to the amount present in the WT embryos with less than 80 cells (which was taken as 100%). The tlf-1 levels are as follows: lane 1, 14% (S1); lane 2, 100% (WT 80 cells); lane 3, 8% (S2); lane 4, 56% (WT 300 cells); lane 5, 15% (S3); lane 6, 32% (WT 600 cells).
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Lack of CeTLF Can Lead to Either Derepression or to Absence of Endogenous Gene Expression
Embryos were stained with DAPI (top), the anti-LIN-26 antiserum (middle), and the monoclonal antibody 1CB4 (bottom). Endogenous LIN-26 is expressed in all nonneuronal ectodermal cells, and the 1CB4 antibody recognizes intestinal cells. Wild-type control (WT) embryos are shown either at less than 50-cell stage (A–C) or at the comma stage (G–I) when cell division stops in WT embryos. (D–F) S1-class tlf-1(RNAi) embryos. Despite the early arrest, the two differentiation markers were turned on in the same cells (shown by arrows). (J–L) S2-class tlf-1(RNAi) embryos. These embryos arrested with no apparent differentiation. (M–O) S3-class tlf-1(RNAi) embryos. These embryos showed no morphogenesis, but differentiation markers were turned on in variable cell numbers.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Tlf-1(RNAi) Embryos Divide Abnormally, Fail to Initiate Gastrulation, and Are Impaired in the Correct Phosphorylation of Pol II
Wild-type (A and C) and tlf-1(RNAi) (B and D) embryos were recorded by Nomarski time-lapse video microscopy. Graphs show the sequence of divisions observed in a wild-type embryo (A) and a S2-class tlf-1(RNAi) embryo (B). The time scale (10 min) is shown on the left of the panels. Timing is unaffected until the first division of E daughter cells (arrow). (C and D) One particular focal plane is shown to illustrate the plane of division of E daughter cells, which is left-right in the wild-type embryo (not visible in a single focal plane) and anterior-posterior in the mutant embryo (arrows). The asterisk shows where Ea and Ep ingress toward the interior of the embryo at the onset of gastrulation in WT embryos.
(E–H) tlf-1 (RNAi) prevents phosphorylation of the CTD of Pol II on serine 2. Embryos were stained with the monoclonal antibodies H5 (E and F) and H14 (G and H), which recognize the large subunit of RNA polymerase II isoforms phosphorylated on serine 2 of the CTD and serine 5 of the CTD, respectively. (E–G) Wild-type embryos. (F–H) tlf-1(RNAi) embryos. In 85% of tlf-1(RNAi) embryos (n = 250), the H5 staining was absent. In contrast, the nuclear H14 signal was unaffected in 77% of tlf-1(RNAi) embryos (n = 171).
Molecular Cell 2000 6, DOI: ( /S (00) )