Fig. 7. Activation of SpGsc expression depends on cell-cell interactions and nuclear ß-catenin. (A) SpGsc transcription requires cell-cell interactions. Cultures of single cells obtained from embryos dissociated in Ca2+- and Mg2+-free sea water at the two-cell stage were analyzed by RNase protection assays at very early blastula (12 hours), mesenchyme blastula (25 hours) and gastrula (36 hours) stages for the levels of SpGsc and SpHE (hatching enzyme) transcripts. RNA samples were checked for concentration and quality by electrophoresis through formaldehyde-containing gels (bottom panel). Negative controls were carried out with yeast tRNA (ytRNA). (B) Cell-cell interactions are required for SpGsc transcription until early blastula stages. RNase protection assays were carried out with a mixture of SpGsc and SpHE probes on samples isolated from cultures of embryos dissociated into single cells at either two-, 32-, 60-, 200- or 500-cell stages and assayed at the 600-cell gastrula stage. C refers to embryos that remained intact throughout the experiment. (C) SpGsc expression depends on the canonical Wnt signaling pathway because transcript levels are strongly reduced in 24-hour embryos injected with cadherin mRNA. RT-PCRs with primers specific for the Endo16 vegetal plate marker or for SpGsc were sampled at either cycle 22 or cycle 25 and signals were quantitated by phosphorimagery and normalized for embryo number using the mitochondrial (Mito) 12S rRNA signal as described in the legend to Fig. 4. A similar analysis was carried out for the accumulation of SpBMP2/4 message that is not inhibited by loss of ß-catenin function. –RT refers to samples that were not reverse transcribed and Mito refers to detection of 12S mitochondrial rRNA to normalize for embryo loads among samples.
