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First published online 21 January 2009
doi: 10.1242/dev.024703

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Identification of direct T-box target genes in the developing zebrafish mesoderm

¹ Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley CA 94720, USA.
² Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Road, Cambridge, CB2 1QN, UK.
³ Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK.
⁴ Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁵ California Institute for Quantitative Biosciences, Berkeley, CA 94158, USA.
⁶ Department of Physiology, Development and Neuroscience, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK.

View larger version (47K): [in this window] [in a new window]   Fig. 1. Microarray analysis identifies candidate spt and ntl target genes. (A) Gene expression levels in MO-injected embryos relative to wild-type embryos at 75% epiboly. Downregulation is shown in shades of yellow (2 to >8-fold) and upregulation (2 to 3-fold) is shown in light purple. Only genes with a two-fold or greater decrease in expression level are shown. (B) spadetail (spt) and no tail (ntl) gene expression profiles between 3 and 48 hpf are shown (top) with the expression profiles of putative targets below. These gene expression profiles were retrieved from another dataset (Mathavan et al., 2005). The expression level for each gene was normalized to its highest expression level over the time course. (C) mRNA expression of spt, ntl and four targets at midgastrula (75% epiboly, 8 hpf) and early segmentation (11-13 hpf). These patterns have been previously reported: ntl (Schulte-Merker et al., 1992), spt (Griffin et al., 1998), tbx6 (Hug et al., 1997), dld (Haddon et al., 1998; Hans and Campos-Ortega, 2002), cdx4 (Joly et al., 1992) and msgn1 (Yoo et al., 2003).

View larger version (43K): [in this window] [in a new window]   Fig. 2. Spt and Ntl bind similar sites in vitro. (A,B) Representative gel shifts using recombinant Spt-GST (A) and Ntl-GST (B). (C,D) WebLogo (Crooks et al., 2004) binding site models based on the sequences of >70 bound oligos from the in vitro selection assays are shown for Spt-GST (C) and Ntl-GST (D) with the count matrices for the sequenced binding sites below. Regions of binding sites contained in the non-random primer region of the oligonucleotide were not counted, therefore some columns have fewer total counts.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Ntl binds clusters of T-box sites near tbx6 and deltaD in vivo. (A,C) The genomic loci of tbx6 (A) and dld (C) are schematized with sequence identity between the orthologous zebrafish and Fugu sequences plotted above the gene schematics using VISTA (Mayor et al., 2000; Frazer et al., 2004) (the Fugu tbx6 ortholog has not yet been sequenced). Matches to the Spt and/or Ntl binding site models with a PATSER P-value 0.001 are depicted as hatch marks below the gene schematics, with all T-box sites shown first, followed by those that belong to a cluster of 5 and 7 sites within 500 bp of one another. Previously identified dld neural regulatory elements HI and HII (Hans and Campos-Ortega, 2002) are shown in grey in C. (B,D) Chromatin immunoprecipitation was carried out using an antibody against Ntl. Fold enrichment in the Ntl-immunoprecipitated samples is shown. Error bars are not assigned to the Ntl-ChIP data since it represents the median of ratios.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Spt binds clusters of T-box sites near tbx6 and deltaD in vivo. We used qPCR to measure the enrichment of the genomic sequences highlighted in blue in B-E in DNA precipitated using a Spt antibody. (A,B) Regions of the T-box motif cluster upstream of tbx6 (B) are 7.9±1.8 and 6.2±0.39-fold enriched in Spt-bound DNA (A). A region of the intronic T-box motif cluster in the dld second intron (C) is enriched 4.3±0.95 fold in Spt-bound DNA (A). As a negative control, we determined the enrichment level of parts of the rhodopsin (rho) (D) and v-maf musculoaponeurotic fibrossarcoma oncogne protein b (mafba) (E) upstream regions in Spt-bound DNA using qPCR and saw no enrichment in Spt-bound DNA (A).

View larger version (28K): [in this window] [in a new window]   Fig. 5. T-box proteins bind putative regulatory sequences in vitro in a T-box binding site-dependent manner. (A-F) To test whether Spt and Ntl bind putative regulatory sequences in vitro, we performed gel shifts using equal volumes of recombinant Spt-GST and Ntl-GST, and radiolabeled DNA corresponding to putative tbx6 and dld genomic regulatory sequences (shown as blue boxes in A and D and magnified in B and E to indicate Spt/Ntl binding motifs a-d and a-g, respectively, as grey rectangles). Spt and Ntl bind a 336 bp region containing four T-box sites just upstream of tbx6 (C; lane 1 versus lane 2). Addition of 10-250x excess unlabeled competitor competes well for binding and virtually eliminates DNA-protein complex formation (C; lanes 3-5); however, excess unlabeled competitor containing mutated T-box sites competes poorly (C; lanes 6-8). Similarly, a mixture of equal volumes of Spt-GST and Ntl-GST binds a 475 bp fragment from the dld second intron containing seven putative T-box sites (F, lane 1 versus 2). Unlabeled competitor containing four of the T-box sites competes for binding (F; lanes 3-5), but fails to compete when three of the T-box sites are mutated (F, lanes 6-8).

View larger version (48K): [in this window] [in a new window]   Fig. 6. The tbx6 regulatory element drives mesodermal gene expression and requires Spt and Ntl for activity. The 1.7 kb region upstream of tbx6 is shown with gray rectangles representing predicted Spt- and/or Ntl-binding site motifs (A). This region drives reporter expression at the margin at mid-gastrulation (B), but not if embryos are co-injected with spt and ntl MOs (C) (mCherry, red; GFP, green). (D-G) Endogenous tbx6 expression is decreased in spt- (F) or ntl-depleted (E) embryos when compared with expression in wild-type embryos (D). tbx6 expression is undetectable when both ntl and spt are depleted (G).

View larger version (68K): [in this window] [in a new window]   Fig. 7. T-box sites are required for activity of the tbx6 mesodermal regulatory region. (A) The 1.7kb region upstream of tbx6 is shown with gray rectangles representing predicted Spt- and/or Ntl-binding site motifs. (B) A detailed view of 300 bp upstream of the tbx6 transcription start site [gray rectangles indicate putative T-box sites (a-d) and diamonds indicate Tcf/Lef-binding sites important for regulatory element function (Szeto and Kimelman, 2004)]. (C,D) mCherry expression driven by the full-length construct is robust at mid-gastrulation (8 hpf) (C) and mid-segmentation (14 somites) (D). (E-P) During gastrulation, mCherry expression driven by constructs in which one T-box site has been mutated (E,H,K,N) is reduced (F,I,L,O), but is normal during segmentation (G,J,M,P). (Q-S) When all four sites are mutated (Q) expression is abolished at both stages (R,S). The percentages of embryos with robust, faint or no mCherry expression are given in Table 1.

View larger version (47K): [in this window] [in a new window]   Fig. 8. The second intron of deltaD contains a conserved cluster of T-box sites required for T-box-dependent mesodermal expression. (A) The second intron from fish dld orthologs (there is an unsequenced gap in the Medaka intron). Grey rectangles represent T-box binding motifs, diamonds indicate putative Tcf/Lef sites and grey ovals represent putative Suppressor of Hairless-binding sites. (B) An alignment of the second intron of fish dld orthologs reveals two particularly well-conserved Spt/Ntl-binding motifs (sites c and d). (C-F) A construct containing 1.2 kb upstream and 1.3 kb downstream sequences relative to the dld transcription start site, including the known neural regulatory element (HII) (Hans and Campos-Ortega, 2002) and the T-box site cluster (C,D) drives robust mCherry expression in the non-axial margin during gastrulation (E) and in the tail bud and somites during segmentation (F). (G-I) When two well-conserved T-box sites (c,d) and a T-box consensus sequence (e) are mutated (G), mCherry expression is greatly reduced (H,I). The percentages of embryos with robust, faint or no mCherry expression is given in Table 1.

View larger version (76K): [in this window] [in a new window]   Fig. 9. The mesodermal deltaD regulatory element requires spt and ntl function. (A-I) Stable transgenics carrying the dld reporter construct (A) express mCherry at the blastoderm margin during mid-gastrulation (G), and in the tail bud and hindbrain (C,D,H,I). Transgene expression begins by 30% epiboly (E,F; animal pole views). (J-M) Stable dld transgenics were injected with MOs targeting ntl, spt or both transcripts, and analyzed by whole-mount in situ hybridization for mCherry. Most uninjected control (J) and Ntl-depleted embryos (K) expressed mCherry [76% (38/50) and 72% (26/36), respectively]. Only 67% (22/33) of Spt-depleted embryos expressed mCherry, and only 9% (3/33) did so at a level comparable with that of uninjected controls (L). No spt+ntl MO-injected embryos (0/54) expressed mCherry (M). (N-Q) In situ hybridization revealed that endogenous dld responds to Spt and Ntl depletion in a similar manner to the transgene.

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