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Fig. 2. Presenilin modulates X-shh expression. (A, B) Quantitative RT-PCR analysis of X-shh transcripts at stage 145. After Ps1 mRNA bilateral injection, Ps1 increased 122% the X-shh/Ef1a ratio (P D 0X001, n ﰅ 3) (A). Bilateral delivery of 2 ng of the mutant D385A Ps1 mRNA did not signiantly increase the X-shh/Ef1a ratio. However, delivery of 4 ng of the mutant Ps1 mRNA increased 85% the X-shh/Ef1a ratio (P D 0X01, n ﰅ 2) (B). The constitutively expressed Ef1a transcript was used for normalizing results. Expression levels were compared using the t-test; SEMs are indicated by error bars; n, number of independent experiments, each consisting of RNA samples from ten control or injected embryos that were reverse transcribed and then subjected to quantitative PCR analysis by quadruplicate. On the top of each panel is shown the corresponding ethidium bromide-stained agarose gel of one representative determination. C, control embryos. Minus RT (PRT), PCR ampliation without reverse transcriptase. (C) WMISH for X-shh showing that presenilin is able to modulate X- shh expression. The pale blue staining indicates the location of the nucb -gal tracer. IS, injected side. NIS, non-injected side. Dorsal views of control (C), X-Ps- a - (D), standard control morpholino oligo- (control Mo) (G), X-Ps-a Mo- (H), and X-Ps-a Mo I X-Ps-a - (I) injected embryos, stage 134. Cross-sections of control (E) and Ps1-injected (F) embryos, stage 14. Compare arrowheads between (E, F), showing an expanded distribution of X-shh transcripts in the ̄oor plate domain. The notochord (n) was unaffected. (J) Western blots of neural plate extracts with anti-PSCT. An 180-kDa immunoreactive band consistent with X-PS CTF endoproteolytic product is reduced in X-Ps-a Mo-injected embryos. |
