Fig. 6. SRp38 is required for regulated neurogenesis. (A) Dorsal views, in situ hybridization for neuronal specific ß-tubulin. Top, left to right: control embryos show expression of ß-tubulin in neurons and trigeminal ganglia; SRp38-injected embryos show a loss of ß-tubulin-expressing cells; injection of antisense morpholino oligonucleotides (AMOs) does not perturb expression of ß-tubulin. Bottom, left to right: inhibitory Delta (Delta^stu) injection results in increased and disorganized expression of ß-tubulin; co-expression of Delta^stu and SRp38 results in almost normal embryos (compare with +SRp38 above); co-injection of Delta^stu and AMOs results in increased expression of ß-tubulin (compare with +AMO embryo above and to controls). (B) In situ hybridization for nrp1, dorsoanterior view, stage 17. Left: control embryos show nrp1 staining in the neural plate and eye primordia. Middle: SRp38 overexpression inhibits expression of nrp1, red arrows. Right: co-injection of SRp38 with the function-blocking sequence C3 rescues expression of nrp1, red arrows. (C) RT-PCR analysis on animal cap ectodermal explants. Expression of 100 pg of neurogenin in the animal cap results in expression of neuronal ß-tubulin and a mild decrease in Id3 (lane 3). Co-expression of neurogenin and 5ng of C3 results in a greater increase of neuronal ß-tubulin and concomitant decrease in Id3 (compare control lane 5 with lane 3). C3 alone (lane 6, 5 ng) results in complete loss of Delta and no effect on Id3 expression. CyclinD1 and p27^xic1 expression are unchanged in neuralized explants upon addition of C3 (compare lane 5 to lane 4). (D) TUNEL staining indicates apoptotic cells. Dorsal views of stage 15 embryos. Control embryos show very few TUNEL-positive cells (black arrowhead), while embryos injected with 250 pg of SRp38 RNA in one cell at the two-cell stage show a variable, though clear increase, in the number of TUNEL-positive cells (red arrowheads). (E) Primary neurogenesis is increased in the absence of SRp38 and Delta function.