Self-regulation by Gsc and Vent1/2 V Sander et al This situation, in which the removal of a very important developmental gene results in normal development in certain mutant backgrounds, is reminiscent of the case of Nanos in the early embryonic patterning of Drosophila. The posterior morphogen Nanos clears ubiquitously distributed maternal hunchback transcripts from the posterior half of the embryo. Lack of the maternal determinant Nanos leads to severe abdomen defects. However, ﬂies double mutant for maternal nanos and hunchback develop completely normally (Struhl, 1989). Thus, the compensation mediated by the simultaneous removal of counteracting factors might help provide an understanding of the molecular mechanisms of genetic redundancy that goes beyond gene duplication hypotheses. Goosecoid and cancer Gsc is an old, intensely studied gene that might still yield more surprises, such as the one found in the present study for the mutual requirement of Gsc and Vent to self-regulate pattern. One particularly interesting recent development is the discovery by Weinberg and co-workers that Gsc is a major mediator of epithelial–mesenchymal transition in mammary tumor metastases (Hartwell et al, 2006). Thus, a gene that promotes cell migration in the prechordal plate in the Xenopus embryo (Niehrs et al, 1993), can be co-opted by cancer cells to promote invasiveness. This leaves us with the question of whether in these metastatic tumors the opposing interactions between Gsc and Vents, so important during Xenopus embryogenesis, might also come into play. Materials and methods Morpholino oligos Antisense MOs for X. laevis Gsc, Vent1, and Vent2 were obtained from Gene Tools LLC and consisted of the following sequences: Gsc MO, 50-GCTGAACATGCCAGAAGGCATCACC-30; Vent1 MO 50-GTCA ATAGAGAATCCCTGTTGAACC-30; and Vent2 MO 50-GTCATCTTG TCTGTATTAGTCCT-30. X. tropicalis Vent1 and Vent2 MOs had been reported previously (Polli and Amaya, 2002), but were not useful for the pseudotetraploid species X. laevis. The Chordin MO was as described (Oelgeschla¨ger et al, 2003). MOs were resuspended in sterile water to a concentration of 1 mM. Prior to microinjections, the MOs were heated at 951C for 30 s, placed on ice, and, unless indicated otherwise, injected four times vegetally (136 ng total). For the double Vent1/2 depletions, the two MOs were mixed at a ratio of 1:1:1 with water, the triple Vent1/2/Gsc MO mix was prepared at a ratio of 1:1:1, and a total dose of 45 ng per MO was injected in each case. References Ault KT, Dirksen M-L, Jamrich M (1996) A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos. Proc Natl Acad Sci USA 93: 6415–6420 Belo JA, Leyns L, Yamada G, De Robertis EM (1998) The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants. Mech Dev 72: 15–25 Blum M, Gaunt SJ, Cho KWY, Steinbeisser H, Blumberg B, Bittner D, De Robertis EM (1992) Gastrulation in the mouse: the role of the homeobox gene goosecoid. Cell 69: 1097–1106 Cho KWY, Blumberg B, Steinbeisser H, De Robertis EM (1991) Molecular nature of Spemann’s organizer: the role of the Xenopus homeobox gene goosecoid. Cell 67: 1111–1120 Danilov V, Blum M, Schweickert A, Campione M, Steinbeisser H (1998) Negative autoregulation of the organizer-speciﬁc homeo- box gene goosecoid. J Biol Chem 273: 627–635 De Robertis EM (2004) Goosecoid. In: Gastrulation, Stern CD (ed). pp 581–589. New York: Cold Spring Harbor Laboratory Press Embryological methods mRNA for mGsc was transcribed from a pBluescriptII KS( ) construct (Blum et al, 1992). Procedures for mRNA synthesis and whole-mount in situ hybridization are available at www.hhmi.u- cla.edu/derobertis/index.html. For animal cap explant studies, 2- to 4-cell embryos were injected four times into the animal pole with either Gsc MO, Vent1/2 MO, or all three MOs. Animal explants were isolated at stage 8, treated with 2 ng/ml recombinant human/ mouse/rat Activin A (R&D Systems), and cultured until stage 22 (Sive et al, 2000). UV irradiation was performed as described (Steinbeisser et al, 1995). DV bisections were prepared from embryos with strong DV polarity (Klein, 1987) at stage 9 in 0.3 Barth’s solution as described (Reversade and De Robertis, 2005). Quantitative RT–PCR Total RNA of either three whole embryos or 10 animal caps per sample was extracted using the Absolutely RNA Microprep kit (Stratagene), and cDNA synthesis was carried out using random hexamer priming and the StrataScript Reverse Transcriptase. Quantitative RT–PCR was performed on the Mx3000P (Stratagene) using the Brilliant SYBR Green QPCR Master Mix (Stratagene). Measurements were performed in quadruplicates and normalized to the expression levels of ODC (Ornithine decarboxylase). Fold change values (x) were calculated using the following formula: x ¼ 2 DDCt. Bars indicate standard deviations. The primer sequences were: a-Actin, fwd: TCCCTGTACGCTTCTGGTCGTA, rev: TCTCAAA GTCCAAAGCCACATA; Admp, fwd: GATGATGGAAGGAGAGGA, rev: TCATGTTCTGACCCAAAG; Chd, fwd: GTTGTACATTTGGTGGGAA, rev: ACTCAGATAAGAGCGATCA; Gsc, fwd: GCTGAT-TCCACCAGT GCCTCACCAG, rev: GGTCCTGTGCCTCCTCTTCCTCCTG; MyoD, fwd: AGGTCCAACTGCTCCGACGGCATGAA, rev: AGGAGAGAATCC AGTTGATGGAAACA; ODC, fwd: CAGCTAGCTGTGGTGTGG, rev: CAACATGGAAACTCACACC; Otx2, fwd: GGATGGATTTGTTACAT CCGTC, rev: CACTCTCCGAGCTCACTTCCC; Rx2a, fwd: AGACTGGT GGCTATGGAG, rev: ATACCTGCACCCTGACTT; Szl, fwd: GTCTTCC TGC-TCCTCTGC, rev: AACAGGGAGCACAGGAAG; Vent1, fwd: TTCCCTTCAGCATGGT-TCAAC, rev: GCATCTCCTTGGCATATTTGG; Wnt8, fwd: TATCTGGAAGTTGCAGCA-TACA, rev: GCAGGCACTCT CGTCCCTCTGT. The PCR cycling conditions for 40 cycles were: denaturation at 951C for 30 s, annealing at 551C for 60 s, and extension at 721C for 30 s. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). 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