Fig. 4. Morpholino knockdown of SpGsc blocks oral endoderm and vegetal differentiation. (A) Confocal images of 3-day-old embryos injected either with 30% glycerol (top panels) or SpGsc morpholino in 30% glycerol (bottom panels). Embryos were stained either with antibodies against EctoV (red), which stains late oral ectoderm and foregut, and Spec1 (green), which stains aboral ectoderm at this stage (left side), or with antibody 6e10 that recognizes ingressed primary mesenchyme cells. (B) RT-PCR analysis of Endo16 mRNA levels in 24-hour embryos injected either with glycerol, or morpholinos against SpGsc (SpGsc-M) or SpKrl (SpKrl-M); RT, reverse transcriptase. Samples were analyzed at cycles 22 and 25 to verify that signals were compared during the linear phase of PCR amplification. Endo16 signals were normalized with respect to mitochondrial 12S rRNA values and these were set to a value of 1 for the positive control, which was RNA from glycerol-injected embryos. As SpKrl is required for Endo16 expression, SpKrl-M (morpholino) provides a negative control (Howard et al., 2001). (C) 2-day embryos that had been injected with glycerol (top) or the SpGsc morpholino (bottom) were stained with antibodies specific for the Sp1 epitope that is expressed on pigment cells (green) or for 6e10 (PMCs; red). Weakly Sp1-positive cells in SpGsc morpholino-injected embryos are indicated by arrowheads. Bars: 20 µm in A,C.