Paganelli AR , Ocaña OH , Prat MI , Franco PG , López SL , Morelli L , Adamo AM , Riccomagno MM , Matsubara E , Shoji M , Affranchino JL , Castaño EM , Carrasco AE .

	Fig. 1. Effects of Ps1 mRNA injection on neural plate development. WMISH was performed at stage 14–15. All are dorsal views. Anterior is up. The injected side (IS) is demarcated by the pale blue X-gal staining. NIS, non-injected side. (A) Suppression of primary neurogenesis, revealed by N-tubulin (N-tub) (85%, n=40). The trigeminal ganglion (arrowhead) was absent from the IS. m, i, and s, primary motorneurons, interneurons, and sensory neurons, respectively. (B) Expansion of the neural plate, revealed by nrp-1 (100%, n=15). (C) Reduction of Gli3 expression (39%, n=41). (D) Expansion of Zic2 domains (73%, n=40). (E) Reduction of X-Delta-1 expression (80%, n=10). (F) Rescue of primary neurogenesis by RA treatment in Ps1-injected embryos (100%, n=5).
	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 ï°… 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 ï°… 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 Ì„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.
	Fig. 3. Effects of presenilin and X-shh injection on proliferation and programmed cell death. (A) Western blot of PCNA at stage 12 (st. 12, gastrula) and stage 14 (st. 14, neurula). C, controls; Ps1, embryos bilaterally injected with Ps1 mRNA. One embryo equivalent was loaded per lane. Identical results were obtained from two independent experiments. On the left, molecular mass markers, in kDa. (B, C) Whole-mount inmunohistochemistry for PCNA after unilateral Ps1 (B) or X-shh (C) injection. Dorsal views of stage 14 embryos are shown; anterior is up. IS, injected side. NIS, non-injected side. (D) Ps1 and X-shh mRNA injection increased apoptosis. (D) Whole-mount TUNEL determination. Control embryos (D, G), embryos unilaterally injected with Ps1 (E, H) or X-shh mRNA (F, I). All are dorsal views. Anterior is up. The injected side (IS) is demarcated by the pale blue X-gal staining. NIS, non-injected side. (D) Stage 12 embryos. (G) Stage 15 embryos. (J) Caspase-3 activity determination. Embryos bilaterally injected with Ps1 or X-shh mRNA show an increased caspase-3 activity at stage 10.5 and at stage 12 (69% for Ps1, n ï°… 3; 97% for X-shh, n ï°… 3) when compared to controls (Ctrl, n ï°… 4). Caspase-3 activity declines at neurula stages (stage 15). Values are expressed as percentage of the activity from control extracts; SEMs are indicated by error bars; n indicates the number of independent caspase-3 determinations, each consisting of extracts from 30 embryos. The observation that at neurulation TUNEL staining revealed strong signal while caspase-3 activity declined is consistent with the fact that caspase-3 activation precedes DNA fragmentation during apoptosis (Liu et al., 1997). (K) Cross-section of the embryo shown in (B). (L) Cross-section of the embryo shown in (H). n, notochord.