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EMBOJ-2007-63895 Sander et al., 2007 Supplementary Figure legends Supplementary Figure 1 Gsc MO causes cell-autonomous ventralization of dorsal blastomere fates. Embryos were injected with either lacZ mRNA (2.4 ng) or a mix of lacZ mRNA and Gsc MO (68 ng) into a single B1 blastomere at 32-cell stage. The fate of the injected cell was visualized at tadpole stage. (A) In control embryos, the B1 blastomere gives rise to neural ectoderm in the head, as well as notochord in the trunk and tail. The magnification shows the characteristic coins-in-a-stack intercalation of notochord cells (arrow in A’; n=15). (B, C) In embryos injected with Gsc MO the labeled descendants populate the somites instead of the notochord. The β-Gal staining is seen in a large portion of the somites, as well as in the head and the dorsal fin (B and B’; n=24), or in the nuclei of medial somite cells (arrow in C’) and neural ectoderm (C and C’; n=25). Medial somites represent the more dorsal compartment of the somite (Niehrs et al., 1993). As expected from a repressor of Vent1/2, our results in the Gsc loss-of-function situation are similar to the fate changes observed in the Vent1 or Vent2 gain-of-function studies (Gawantka et al., 1995; Onichtchouk et al., 1996). df, dorsal fin; ne, neural ectoderm; no, notochord; so, somites. Material and methods For lacZ lineage tracing, embryos injected with nuclear lacZ mRNA were fixed for 20 min. in MEMFA (Sive et al., 2000), and washed twice in PBS for 10 min. β-Gal staining was performed in 0.5 M K3Fe(CN)6, 0.5 M K4Fe(CN)6, 0.5 M MgCl2 and 100 mg/ml 5Bromo-6-chloro-3-indolyl-β-D-galactopyranoside (in DMSO) in PBS at 4˚C over night. Stained embryos were washed twice in PBS and re-fixed in MEMFA for 2 hours. 1 Supplementary Figure 2 Removal of Gsc re-specifies dorsal mesodermal fates in a dose-dependent manner. Embryos were injected four times at 2- to 4-cell stage with increasing amounts of Gsc MO (as indicated on the abscissa). Dorsal marginal zone (DMZ) explants were excised at stage 10 and cultured until sibling embryos reached stage 13. For RNA extraction and cDNA synthesis, 8 DMZs were used per sample. To study the responses in gene expression upon increasing doses of Gsc MO, quantitative RT-PCRs for marker genes of prechordal plate (Frzb1) and anterior brain (Otx2), notochord (Collagen type IIA) and somites (MyoD), as well as of ventral mesoderm (Vent1, Wnt8) were performed. The starting level of gene expression (0 ng Gsc MO) was set to 1, and the changes in gene expression after MO injection (68 ng, 136 ng and 272 ng Gsc MO) were calculated as fold change. (A) Anterior neural and prechordal plate genes are highly expressed in the presence of Gsc, and are downregulated upon MO injection, indicating that head and prechordal plate tissue formation requires maximum amount of Gsc. (B) Notochord and somite markers are expressed at low levels in uninjected DMZs, but their expression is upregulated at low and intermediate doses of Gsc MO, while injection of the highest amount of Gsc MO resulted in a decrease of Col2A and MyoD expression back to control levels and below. These curves suggest that notochord and somites are formed in regions of intermediate Gsc. We observed very similar expression profiles for notochord and somites - rather than more separate peaks, with notochord being formed at lower Gsc MO doses than somites, as expected from previous histological work of Niehrs et al. (1994). This might be due to Col2A being expressed not only in the notochord, but also in peri-notochordal somite tissue at neurula stages (Su et al., 1991; Larrain et al., 2000). (C) The expression of the ventral mesodermal marker genes Vent1 and Wnt8 (as well as Vent2 and Scl, data not shown) is barely detectable in control DMZs, but significantly increases after Gsc knockdown, indicating that Gsc inhibits ventral mesoderm on the dorsal side of the embryo. Primer sequences: Frzb1, fwd: GACCACTGAATGTAGCCAGGA, rev: GGAGATGCAGACTCCTCTGTCA; Col2A, fwd: AGGCTTGGCTGGTCCTCAAGG, rev: TGTAACGCATAGGGTCGGGTC. References Larrain J, Bachiller D, Lu B, Agius E, Piccolo S, De Robertis EM (2000) BMP-binding modules in chordin: a model for signalling regulation in the extracellular space. Development 127: 821-830 Su M-W, Suzuki HR, Bieker JJ, Solursh M, Ramirez F (1991) Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts. J Cell Biol 115: 565-575 2 Supplementary Figure 3 Depletion of Wnt8 rescues the Gsc MO phenotype. (B) Injection of Gsc MO ventralizes the embryo (n=29). (C) Wnt8 MO, in contrast, leads to dorsalization (n=13). (D) Combined knockdown of Wnt8 and Gsc rescued the ventralized phenotype of the Gsc knockdown (n=35), suggesting an inhibitory regulation between Gsc and Wnt8. For each experiment, 68 ng of each MO were injected radially into 2- to 4-cell stage embryos. This result is in agreement with previous findings of Yao and Kessler (2001). Supplementary Figure 4 Partial redundancy of Vent1 and Vent2 at gastrula. (B, F) Injection of Vent1 MO did not affect the expression of Chd and Gsc in the organizer (Chd, n=17; Gsc, n=9). (C, G) Vent2 morphants showed slightly increased expression of Chd and Gsc (Chd, n=20; Gsc, n=10). (D, H) Vent1/2 double knockdown caused a strong expansion of Chd and Gsc expression (Chd, n=23; Gsc, n=17). 68 ng of each MO were injected radially into 2- to 4-cell stage embryos. These results correlate with our observation of mildly dorsalized Vent2 morphants and extreme dorsalization in Vent1/2depleted embryos at tadpole stage (Figure 4C and D). Thus, it seems that Vent1 and Vent2 are partially redundant, with Vent2 being more active in repressing dorsal genes – as previously concluded by Onichtchouk et al. (1998). Supplementary Figure 5 Vent1/2 knockdown is rescued by zebrafish Vega mRNA. (A) Control embryo stained for XAG1 (cement gland) and Scl (blood). (B) Dorsalized embryo after Vent1/2 MO knockdown (n=16). (C) Injection of mRNAs encoding zebrafish Vega1 and Vega2 strongly ventralized the embryo (n=15) (D) Co-injection of Vega1 and Vega2 mRNAs restored normal development (n=15). 2 pg of each mRNA, and 34 ng of each MO were injected radially into 2- to 4-cell stage embryos. Since Vega1/2 mRNA injection leads to a stronger ventralization than Gsc MO injection (compare panel C to Figure 1D), this indicates that Vent/Vega are also involved in Gsc-independent processes in the embryo. This is supported by the observation that blood formation (marked by Scl expression) cannot be restored in the Vent1/2/Gsc triple knockdowns (see Figure 5I). Supplementary Figure 6 Complete depletion of Gsc and Vent1/2 in triple Vent1/2/Gsc morphant embryos. (B, E, H and C, F, I) Individual injections of Gsc MO and Vent1/2 MOs in the same concentrations as used in the triple knockdowns (45 ng each) resulted in identical phenotypes to those of embryos injected with 2- or 3-fold higher concentrations of the same MOs. (D, G, J) The rescued phenotype of the triple Vent1/2/Gsc knockdown did not change upon injection of higher doses of all three MOs. However, the overall survival rates decreased with increasing doses of MOs. These results show that Gsc and Vent1/2 are completely inhibited, even at the lower doses of MOs used for the triple 3 knockdown. Therefore, the phenotypic rescue seen in Vent1/2/Gsc morphants is not caused by partial depletion of those transcription factors. Supplementary Figure 7 Chd is epistatic to Vent1/2. (A) Uninjected control embryo (B) Double knockdown of Vent1/2 showing dorsalization of the embryo (C) Chd depletion leads to ventralization, as shown by decreased size of eyes (Rx2a), cement gland (XAG1), hindbrain and notochord (Xbcan). (D) Co-injection of Chd MO in Vent1/2-depleted embryos (45 ng of each MO, radial injections) not only rescued the dorsalization caused by the double Vent1/2 knockdown, but caused mild ventralization in 80% of the tripledepleted embryos (n=41), as does the Chd MO single knockdown phenotype. Thus, the Vent1/2 depletion phenotype requires Chd. However, Chd is epistatic to Vent1/2, whereas Gsc MO only rescues the Vent1/2 knockdown back to a wild-type pattern. This suggests that Chd has Gsc-independent functions, in addition to mediating the downstream effects of overexpressed Gsc. Supplementary Figure 8 The secreted growth factors ADMP and BMP4 compensate for the loss of Gsc and Vent1/2. (A-C) Knockdown of Gsc leads to ventralization, while Vent1/2 depletion strongly dorsalize the embryo. Rhombomere 5 of the hindbrain is radialized in these embryos. The radial Krox20 expression is seen as a solid line at neurula stages, at later stages – as shown here – the line breaks into shorter segments. (D) The Vent1/2/Gsc triple knockdown rescued normal patterning. (E, F) Additional depletion of ADMP or BMP4 resulted in strongly dorsalized phenotypes (n=28 for Vent1/2/Gsc/ADMP MOs; n=34 for Vent1/2/Gsc/BMP4 MOs), showing that these two extracellular proteins are part of the molecular safety net mechanism that compensates for the loss of the transcription factors Gsc and Vent1/2. For the experiments in panel C to F, 34 ng of each MO were injected radially into 2- to 4-cell stage embryos. 4