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. 2007 Sep;8(9):858-63.
doi: 10.1038/sj.embor.7401045. Epub 2007 Aug 3.

Paf1 complex homologues are required for Notch-regulated transcription during somite segmentation

Takashi Akanuma  1 , Sumito Koshida, Akinori Kawamura, Yasuyuki Kishimoto, Shinji Takada
Affiliations

Paf1 complex homologues are required for Notch-regulated transcription during somite segmentation

Takashi Akanuma et al. EMBO Rep. 2007 Sep.

Abstract

Members of the yeast polymerase-associated factor 1 (Paf1) complex, which is composed of at least five components (Paf1, Rtf1, Cdc73, Leo1 and Ctr9), are conserved from yeast to humans. Although these proteins have been implicated in RNA polymerase II-mediated transcription, their roles in vertebrate development have not been explained. Here, we show that a zebrafish mutant with a somite segmentation defect is deficient in rtf1. In addition, embryos deficient in rtf1 or ctr9 show abnormal development of the heart, ears and neural crest cells. rtf1 is required for correct RNA levels of the Notch-regulated genes her1, her7 and deltaC, and also for Notch-induced her1 expression in the presomitic mesoderm. Furthermore, the phenotype observed in rtf1-deficient mutants is enhanced by an additional deficiency in mind bomb, which encodes an effector of Notch signalling. Therefore, zebrafish homologues of the yeast Paf1 complex seem to preferentially affect a subset of genes, including Notch-regulated genes, during embryogenesis.

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Figures

Figure 1
Figure 1
Isolation and molecular characterization of a zebrafish rtf1kt641 mutant defective in somite formation. (A–F) Lateral views of wild-type (wt) (A,B,E) and kt641 homozygotes (C,D,F) at the 17-somite stage (A–D) and at 36 h post-fertilization (E,F). Panels (B) and (D) show views of panels (A) and (C) respectively, at higher magnification. Somite boundaries are disrupted in the posterior trunk of the kt641 mutant (square bracket in (D)). At later stages, the kt641 mutation causes reduced pigmentation, limited tail growth and abnormal heart (arrow) and ear (arrowhead) development (F). (G) Meiotic and physical mapping of the kt641 mutation. Horizontal grey bars represent contiguous sequences deposited in linkage group 13. (H) Schematic diagrams of zebrafish Rtf1 proteins encoded by wild-type and kt641 alleles.
Figure 2
Figure 2
The rtf1kt641 mutation reduces the expression of genes involved in somite segmentation. (A–E) A comparative representation of expression patterns of her1 (A–C), her7 (D) and deltaC (E) messenger RNAs between wild-type siblings (wt; left halves) and rtf1kt641 mutants (kt641; right halves) indicates a reduction of these genes in rtf1kt641 mutants. Each half panel represents the cyclic expression patterns of wild-type siblings and rtf1kt641 mutants at comparative oscillation phases. Three consecutive expression phases of her1 mRNA are represented (A–C). Arrowheads indicate the anterior-most stripes in rtf1kt641 mutants, which are shifted in the posterior direction. (F–I) Expression patterns of fgf8 (F,G) and her13.2 (H,I) mRNA in wild-type siblings (F,H) and rtf1kt641 mutants (G,I). (J,K) her1 nascent transcript hybridized with intron probe in wild-type siblings (J) and rtf1kt641 mutants (K). Expression of her1 is reduced at the transcriptional level in rtf1kt641 mutants. Embryos are at the 10- or 12-somite stage and the anterior is top in (A–K). (L) Reverse transcription–PCR analysis of transcript abundance for genes involved in somitogenesis. Data are presented as means+s.d. from triplicate assays.
Figure 3
Figure 3
Functional cooperation between Rtf1 and Notch signalling. (A,B) her1 expression in wild-type (wt; A) and kt641 mutant (B) embryos injected with synthetic messenger RNA encoding Notch1a intracellular domain (NICD). In kt641 mutant embryos, the elevated expression is reduced, although the pattern of her1 expression is still abnormal. (C–F) Lateral views of 12-somite-stage embryos from crossing of rtf1kt641/+;mibta52b/+ fish. Wild type (C), kt641 mutant (D), mib mutant (E) and kt641;mib compound mutant (com; F) were generated at a ratio (57.1:18.1:19.3:5.5%; n=590) close to the expected mendelian frequency (56.3:18.8:18.8:6.3%). Square brackets in (D–F) indicate abnormally segmented somites. The kt641;mib double mutation enhances the somite segmentation defect. (G) Number of fully segmented somites in kt641 (n=47), mib (n=59) and kt641;mib (com; n=15) at 18 h post-fertilization. Data are presented as means+s.d. P<0.001 for differences between single mutants (kt641 or mib) and compound mutants (kt641;mib) by Student's t-test are represented. (H,I) In addition to the somite defect, kt641;mib mutants (I) show more severe defects in the eye, pigmentation and tail elongation than kt641 (Fig 1F) and mib single mutants (H) at 36 h post-fertilization. (J–M) In contrast to wild-type siblings (J), kt641 mutants, which show cyclic but reduced her1 expression (K), and mib mutants, which lack the striped expression of her1 (L), kt641;mib compound mutants show markedly reduced her1 expression without stripes (M) at the 12-somite stage.
Figure 4
Figure 4
Rtf1 cooperates with the Paf1 complex during somitogenesis. (A,B) Knockdown of ctr9 results in defects that are similar to but weaker than those of kt641 mutants. Embryos injected with ctr9 MO1 (A), but not 5-mismatched MO1 (B), show reduced pigmentation, abnormal cardiogenesis (arrow), small ears (arrowhead), slightly shortened tails and disorganized somite boundaries in the tail (square bracket) at 36 h post-fertilization. Two independent ctr9 MOs cause similar morphological defects. (B) 5-mispaired ctr9 MO1 hardly impairs development. (C–E) At the 12-somite stage, no marked defects were observed in ctr9 MO-injected embryos (C). 5-mispaired control MO1 had no effect on the kt641 phenotype (D), whereas injection of ctr9 MO1 into kt641 mutant embryos resulted in an enhanced segmentation defect with few segmentation boundaries (E). The square brackets in (D) and (E) indicate the posterior somites with obscure boundaries. (F) Number of segmented somites in ctr9 MO1-injected wild-type embryos (n=82), ctr9 5-mismatched MO1-injected kt641 mutants (n=18) and ctr9 MO1-injected kt641 mutants (n=23) at 24 h post-fertilization. Error bars represent the s.d. P<0.001 between single mutants (kt641<5-mismatched MO1 or wt<ctr9 MO1) and double mutants (kt641<ctr9 MO1) are represented. (G–J) her1 expression in 5-mismatched control MO1-injected (G,I), ctr9 MO1-injected (H,J) wild-type (G,H) or kt641 mutant (I,J) embryos. In comparison with wild-type siblings injected with 5-mismatched control MO1 (G), those injected with the ctr9 MO1 show reduced her1 expression (H). This reduction is enhanced in kt641 mutant embryos injected with ctr9 MO1 (J). MO, morpholino oligonucleotides.
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