A Genomic Regulatory Network for Development Eric H. Davidson, Jonathan P. Rast, Paola Oliveri, Andrew Ransick, Cristina Calestani, Chiou-Hwa Yuh, Takuya Minokawa, Gabriele Amore, Veronica Hinman, César Arenas-Mena, Ochan Otim, C. Titus Brown, Carolina B. Livi, Pei Yun Lee, Roger Revilla, Alistair G. Rust, Zheng jun Pan, Maria J. Schilstra, Peter J. C. Clarke, Maria I. Arnone, Lee Rowen, R. Andrew Cameron, David R. McClay, Leroy Hood, and Hamid Bolouri

Supplementary Material

Supplemental Figure 1. The expression pattern of PKS as visualized by whole mount in situ hybridization (39). A digoxigenin (DIG)-labeled antisense RNA probe of about 500 bp in length was used at a final concentration of 0.02 ng/l as in Ransick et al., 1993 (3). The 20 hour embryo is viewed from the vegetal pole. At this stage the mesodermal precursor cells express the gene. The 38 hour and 72 hour embryos are viewed from the side. The pigment cells express the gene at these stages. Evidence for gcm autoregulation comes from QPCR data posted at the Science Web site.

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Supplemental Figure 2. [See (43).] (Top panel) In situ hybridization of embryos with g a 0.1 ng/l mixture of two 500 bp probes corresponding to the coding region of Gata-e (excluding the zinc fingers) The procedure is modified from Ransick et al. (1993) (3), by substituting a heat treatment at 95°C for 5 min for the proteinase K digestion.The left column shows side views and the right column vegetal views of the embryos. Gata-e appears to be expressed in cells of the veg2 lineage at the hatching blastula stage (18 hour). At initiation of gastrulation, Gata-e appears to be expressed in some cells of the veg1 lineage as well. (Bottom panel) Embryos at mesenchyme blastula (24 hour) and gastrula (48 hour) stages from eggs that were injected with a 30 M solution of morpholino antisense oligonucleotide specific for Gata-eGata-e treated embryos do not have a vegetal plate at the mesenchyme blastula stage. Primary mesenchyme ingression appears normal. Gastrulation does not occur in these embryos. The presence of pigment cells in 48 hour embryos show that at least some mesoderm specification has taken place in the treated embryos.

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Supplemental Figure 3. The effect of a FoxA MASO on brachyury spatial expression in S. purpuratus embryos (46). Combined fluorescent and brightfield images of embryos expressing a GFP reporter gene controlled by the intron cis-regulatory region of the brachyury gene. In the embryo co-injected with a morpholino antisense oligonucleotide for FoxA (labeled anti-FoxA) the region of expression was extended into the archenteron. Zygotes were injected with 2500 copies of the 6 kb reporter gene , alone or in combination with 5 pg of FoxA antisense morpholino oligonucleotide. Injected embryos were cultured for 48 hours to the gastrula stage and observed on an upright compound microscope with epifluorescence attachment. Images were collected by digital camera and processed in Photoshop.

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Supplemental Figure 4. Whole mount in situ hybridization (WMISH) of AmBra in (A) normal and (B) AmFoxA antisense morpholino-oligonucleotide injected midgastrula stage Asterina miniata embryos (47). The expression ofAmBra has moved into the invaginating archenteron in B (white arrow) compared with the normal embryo (A). AmBra is also expressed in the oral ectoderm in both (arrowheads). The AmFoxA antisense morpholino oligonucleotide was injected into 1-cell embryos at a final concentration of 5 micromolar, which results in a mild phenotype in which gastrulation can still proceed. WMISH was performed with a 1110 nt antisense DIG -labeled RNA probe which included regions of the 3'ORF and 3'UTR of the AmBra mRNA\ and followed the procedure of Hinman and Degnan [Dev. Genes Evol. 210, 129 (2000)].

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Supplemental Figure 5. Views of 72 hour S. purpuratus embryos that were injected just after fertilization with 200 micromolar solution of either anti-GCM morpholino(left) or control morpholino (right) (49). The anti-GCM morpholino targeted the translation start region of the GCM mRNA. The development of both types of injected embryo was normal until the pluteus lava stage, with the exception that those embryos injected with anti-GCM morpholino did not differentiate any pigment cells. The two examples shown here were viewed as live specimens using DIC optics and slightly compressed under a cover slip.

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