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Fig. S1. Microarray analysis. (A) Frontal view of embryos labeled by in situ hybridization for genes indicated. Arrows indicate the presumptive primary mouth. cg, cement gland. Scale bars: 200 µm. cDNAs used for making in situ probes are vgl-2 (GA# BC056001, image clone 4930090, Open BioSystems), CPN (GA# BC059995, image clone 4030455, Open Biosystems), pitx-3 GA# AF297713.1, provided by Hollemann (Pommereit et al., 2001) and six-1 GA# AF279254, provided by Moody (Pandur and Moody, 2000). (B) Expression levels of genes from Table S1, as determined by microarray (M) and qRT-PCR (R). Shading and colors match scheme shown in Fig. 1, in which the anterior dorsal region (AD) is shaded dark gray, the cement gland and ventral region (CG+V) light gray, and the presumptive primary mouth (PMo) is colored red. (C) Expression of Wnt pathway components in the primary mouth (red) relative to the anterior dorsal region (AD; dark gray), and a ventral region, including the ventral half of the cement gland (CG+V; light gray) as determined by microarray. The expression level of each gene was plotted relative to each other so that a single bar represents the total expression in all three regions, scaled to 100. In the primary mouth anlage expression of wnt-8, wnt-8b, wnt-3a and wnt-2 is significantly depleted (<6% relative to the flanking regions) and expression of wnt-4, β-catenin, axin and the Frizzled receptors frz-3 and frz-9 are also depleted (<20% relative to the flanking regions). Other Wnt inhibitors, dkk-1 and wif-1, are enriched (>57% relative to the flanking regions).
Fig. S2. In situ hybridizations to show expression patterns sfrps during neurula and early tailbud stages. sfrp1 (A-D), sfrp2 (E-H) and sfrp5 (I-L) are labeled purple/blue, whereas the cement gland marker, XCG, is labeled orange. A, C and E are frontal views; B, D and F are sagittal sections (anterior to the left) of the same stage; G-L are frontal views. Arrows indicate the presumptive primary mouth. cg, cement gland. Scale bars: 200 µm. cDNAs used for making in situ probes were obtained from Open Biosystems; sfrp-1 (GA# BC106360, image clone 7392081), sfrp-2 (GA# BC044687, image clone 5571003) and sfrp-5 (GA# BC082632, image clone 5512233).
Fig. S3. Characterizing the morpholino phenotype. (A, parts a,b) Whole embryo phenotype. (a) frzb-1/crescent morphants at stage 23-24 compared with control morpholino-injected embryo. Note the overall similarity in the embryos, suggesting that the positioning and specification of many of the anterior structures are normal. (b) frzb-1/crescent morphants at stage 40-41 compared with control morpholino-injected embryo. Note the curved axis and reduced head structures. Scale bars: 1000 µm (c,d) Alcian Blue staining of cartilage at stage 45 in (c) control embryos, and (d) frzb-1 and crescent morphants. Note the almost complete lack of Alcian Blue staining in the morphants. Scale bars: 340 µm. (e,f) Axon tracts of the CNS at stage 40 were visualized by acetylated tubulin immunohistochemistry in (e) controls and (f) frzb-1/crescent morphants. Note the unorganized axon tracts in frzb-1/crescent morphants. Scale bars: 370 µm. (g,h) Phalloidin labeling of somites at stage 45 in (g) controls and (h) frzb-1/crescent morphants, using F-actin labeling with Phalloidin conjugated to Alexa Fluor 488. Scale bar: 1200 µm. Insets show a 100× magnification of the somites, note the unorganized muscle fibers in frzb-1/crescent morphants. Scale bar: 1200 µm. (B, parts a,b) Embryos and tadpoles were processed for terminal deoxynucleotidyl transferase-mediated dTP nick-end labeling (TUNEL) as described (Dickinson and Sive, 2006), using the Apoptag kit (Chemicon). TUNEL labeling (green) in the primary mouth region of (a) control and (b) frzb-1/crescent morphants; embryos were counterstained with propidium iodide (red). Note that there are few TUNEL-positive cells in the presumptive primary mouth at stage 24-26, and no observable difference in frzb-1/crescent morphants (n=5; two experiments, not quantified). Scale bars: 100 µm. (c,d) Phosho-histone3 (pH3) immunohistochemistry (green) using a polyclonal anti-phosphohistone3 antibody diluted 1:1000 and a secondary goat anti-rabbit Alexa Fluor-conjugated antibody diluted 1:500, counterstained with propidium iodide (1:1000). (c) Control. (d) frzb-1/crescent morphants have similar amounts of pH3-positive cells to controls (6.33%, no significant difference) in the presumptive primary mouth regions (four stages examined, n=5 at each stage, one stage in one experiment quantified). Arrows indicate the presumptive primary mouth. cg, cement gland. Scale bars: 100 µm.
Fig. S4. Requirement of the frzb-1/crescent expression domain for primary mouth formation. Fate mapping has shown that the region expressing frzb-1 becomes the primary mouth (data not shown) (Dickinson and Sive, 2006). Removing the superficial layers of ectoderm does not affect primary mouth formation (Dickinson and Sive, 2006), probably because frzb-1/crescent expression is restricted to deeper layers. We tested whether the frzb-1/crescent expression domain was required for primary mouth formation by performing extirpations. Arrows indicate the presumptive primary mouth; cg, cement gland. (A-C) Schematic of dissections at stage 23-24 depicting anterior ectoderm (blue), foregut epithelium (yellow), and dissected regions (dotted red box). (D-F) frzb-1 in situ hybridization immediately after dissection. frzb-1, purple/blue; xcg, orange. Scale bars: 200 µm. (D) In un-operated embryos, the frzb-1 expression domain is normal 100% (n=10). (E) In 91% of embryos with superficial extirpations, the frzb-1 expression domain is normal (n=11). (F) Embryos receiving the deep extirpations rarely (8.3%) contain significant frzb-1 expression (n=12) (G-I) Frontal view of representative embryos at stage 40, showing the effect of the extirpations on the formation of the primary mouth; the opening is outline by a dotted black line. Scale bars: 250 µm. (G) 100% of un-operated embryos have a normal primary mouth (n=10). (H) 100% of embryos with the superficial ectoderm removed have a normal primary mouth (n=10). (I) 90% of embryos having the deep ectodermal extirpations do not form a primary mouth (n=10).
Fig. S5. Measurement of Wnt signaling in the primary mouth region using β-catenin protein and activity as a readout. Schematic depicts the experimental design. Embryos at the 1-cell stage were injected with (A) frzb-1 mRNA or GFP mRNA or (B) frzb-1 RNA or GFP RNA plus the TOPFLASH plasmid and the renilla reference plasmid, then at stage 20-22 the primary mouth and area just surrounding it was dissected from 10 embryos. Tissue was pooled and processed for (A) western blot or (B) the TOPFLASH assay. The western blot shows a 27% decrease in β-catenin protein in tissue collected from embryos injected with frzb-1 mRNA compared with controls (n=10, two experiments). The TOPFLASH assay shows an 88% decrease in luciferase activity in tissue collected from embryos injected with frzb-1 mRNA compared with controls injected with GFP (n=10, two experiments, s.d.=1.42).
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