Fig S1. spt and ntl translation-blocking morpholinos phenocopy the appropriate single and double mutant phenotypes. (A) Sequences of MOs used to deplete embryos of Spt/Ntl activity. The ntla MO was used in an earlier study (Nasevicius and Ekker, 2000). MOs were injected at a total concentration of 5 ng/nl. To deplete both factors, all four MOs were combined at a concentration of 1.25 ng/nl each and for each single depletion the two appropriate MOs were combined at a concentration of 2.5 ng/nl each. Each embryo was injected with a total of 15 ng to 25 ng of MO. (B-E) Live pictures of 24 hpf embryos following MO injection into one- to four-cell stage embryos. The spt single MO and spt;ntl double MO phenotypes are more necrotic than the corresponding genetic mutants at this stage (Amacher et al., 2002). (F-I) shh expression in the ventral midline is completely abolished posteriorly in 24 hpf embryos depleted of spt and ntl function, and single and double morphant phenotypes mimic those previously demonstrated for genetic mutants (Amacher et al., 2002). Expression of the krox20 hindbrain marker, which labels hindbrain rhombomeres 3 and 5, is included for reference and is not affected in any of the MO-injected embryos. (J-M) Muscle tropomyosin expression is completely abolished in 24 hpf embryos depleted of spt and ntl function, and both single and double morphant phenotypes mimic those previously demonstrated for genetic mutants (Amacher et al., 2002).

References

Amacher S. L., Draper B. W., Summers B. R. and Kimmel C. B. (2002). The zebrafish T-box genes no tail and spadetail are required for development of trunk and tail mesoderm and medial floor plate. Development 129, 3311-3323.

Nasevicius A. and Ekker S. C. (2000). Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216-220.

Fig S2. Expression of putative T-box gene targets overlaps with ntl and spt expression during gastrulation. Shown are wild-type in situ hybridization patterns during gastrula stages (6-9 hpf) for genes downregulated at least 2.5-fold in embryos depleted of spt and ntl function. Expression patterns obtained from the ZFIN database (Thisse et al., 2001) are indicated. (A) ntl and spt expression patterns at mid-gastrulation. (B) Expression of 18 potential targets, arranged (from top left to bottom right) to indicate the magnitude to which they were affected (fold decrease indicated at the bottom right of each panel). Two additional targets are expressed in patterns that overlap with spt and/or ntl, but are not shown: during gastrulation, knypek (3.8×) is expressed in the axial mesoderm (Machingo et al., 2006) and six4.2 (2.8×) is expressed in the non-axial mesoderm (Kobayashi et al., 2000). An additional nine potential targets that were down-regulated at 2.5-fold or greater are not shown for the following reasons: the gene they identify is not known four genes: AI942866 (8×); AI584322 (6×); BM036916 (4.5×); and BI886648 (4×), their gastrula expression patterns have not been well characterized or described two genes: cxcl12a (2.8×) and itm2c (2.5×), their gastrula expression levels have been reported as low or not detectable two genes: ywhae1 (3.6×) and efemp2 (3×), or they have been described to be ubiquitously expressed during gastrula stages one gene: tuba812 (2.8×).

References

Kobayashi M., Osanai H., Kawakami K. and Yamamoto M. (2000). Expression of three zebrafish Six4 genes in the cranial sensory placodes and the developing somites. Mech. Dev. 98, 151-155.

Machingo Q. J., Fritz A. and Shur B. D. (2006). A β1,4-galactosyltransferase is required for convergent extension movements in zebrafish. Dev. Biol. 297, 471-482.

Fig S3. Spt-GST and Ntl-GST have differential in vitro binding activities. To extend the observation from the SELEX assays that Spt bound a C at position 1 in 25% of the sequenced oligos where as Ntl bound a C at this position in only 2% of the sequenced oligos (Fig. 2), we directly compared the ability of Spt and Ntl to bind TCACACCT versus CCACACCT. Shown are gel mobility shift assays using radiolabeled probes and increasing amounts of bacterially produced Spt-GST and Ntl-GST fusion proteins. Complexes were fractionated on native polyacrylamide gels. Spt-GST bound both probes, but bound TCACACCT probe (A) with higher affinity than the CCACACCT probe (B). As predicted, Ntl-GST also bound the TCACACCT probe (C), but we were unable to detect any Ntl-GST binding to the CCACACCT probe (D). This motif difference has been previously noted between Brachury and Tbx6 in mouse (White and Chapman, 2005), and our data are consistent with previous SELEX experiments with T-box factors, which show that Ntl orthologs do not select sequences with a C at position 1 (Kispert and Herrmann, 1993; Conlon et al., 2001) where as all other tested T-box factors are able to bind oligomers with a C at this position (Conlon et al., 2001; Ghosh et al., 2001; Yagi, 2005; White and Chapman, 2005).

References

Conlon F. L., Fairclough L., Price B. M., Casey E. S. and Smith J. C. (2001). Determinants of T box protein specificity. Development 128, 3749-3758.

Ghosh T. K., Packham E. A., Bonser A. J., Robinson T. E., Cross S. J. and Brook J. D. (2001). Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome. Hum. Mol. Genet. 10, 1983-1994.

Kispert A. and Hermann B. G. (1993). The Brachyury gene encodes a novel DNA binding protein. EMBO J. 12, 3211-3220.

White P. H. and Chapman D. L. (2005). Dll1 is a downstream target of Tbx6 in the paraxial mesoderm. Genesis 42, 193-202.

Yagi K., Takatori N., Satou Y. and Satoh N. (2005). Ci-Tbx6b and Ci-Tbx6c are key mediators of the maternal effect gene Ci-macho1 in muscle cell differentiation in Ciona intestinalis embryos. Dev. Biol. 282, 535-549.

Fig S4. Method for cross-species binding site cluster comparison. The method used for detecting conservation of binding site clusters is shown using the deltaD locus as an example. The zebrafish genome sequence surrounding genes downregulated in Spt/Ntl-depleted embryos was searched for 500 bp clusters of Spt/Ntl-binding sites. The location of a cluster of eight binding sites in the second intron of deltaD is shown in magenta on the zebrafish sequence. (A) Genomic sequences around fish orthologs for putative spt/ntl target genes, including 5 kb upstream and downstream were aligned. The broken lines indicate the bases in each fish species that align to every thousandth base pair in the zebrafish sequence. The magenta boxes in the Medaka, Fugu and stickleback sequences correspond to the regions whose ends align with the ends of the zebrafish T-box cluster. If the region from another fish species had less than 15% sequence identity in the cluster region it was classified as unalignable. (B) The positions of T-box binding motifs in the dld T-box cluster in the various fish species. The number of sequences matching the Spt and/or Ntl position-specific score matrices with a PATSER P-value≤0.001 in the aligned region in each gene examined from each species are shown in Table S1. T-box motifs occurring in the coding sequence were not included in the binding site count.

Fig S5. Transgene expression is temporally and spatially regulated like the endogenous gene and is dependent on Spt and Ntl function. (A) Embryos were injected with the tbx6 transgene with or without spt and ntl MOs and the percentage of embryos expressing mCherry at the margin was recorded at various stages of development. (A) The tbx6 transgene is initially activated at shield stage, as has been reported for endogenous tbx6 (Hug et al., 1997) (unbroken line). Injecting spt and ntl MOs eliminated mCherry expression (broken line). Similar experiments examining transgene expression in dld:mCherry stable lines are shown in Fig. 9. (B-E) Double in situ hybridizations for mCherry (in red) and tbx6 (B,C) and deltaD (D,E) (in blue) were performed at 75% epiboly. C and E are higher magnification pictures of the embryos in B and D. In all cases, the majority of mCherry expression occurred in regions that also expressed the endogenous gene, or where the endogenous gene was recently expressed.

Fig. S6. Tcf/Lef and Suppressor of hairless binding sites used to construct position specific score matrices. (A) Tcf/Lef-binding sites identified in zebrafish regulatory elements. The name of the gene regulated by the element containing each group of binding sites is given with the paper in which they were identified. (B) Binding sites for Suppressor of Hairless identified in regulatory elements for Enhancer of Split genes in Drosophila.

References

Bailey, A. M. and Posakony, J. W. (1995). Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9, 2609-2622.

Dorsky, R. I., Raible, D. W. and Moon, R. T. (2000). Direct regulation of nacre, a zebrafish MITF homolog required for pigment cell formation, by the Wnt pathway. Genes Dev. 14, 158-162.

Ryu, S. L., Fujii, R., Yamanaka, Y., Shimizu, T., Yabe, T., Hirata, T., Hibi, M. and Hirano, T. (2001). Regulation of dharma/bozozok by the Wnt pathway. Dev. Biol. 231, 397-409.