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frzb (frizzled related protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, (A) anterior view, dorsal up., and (A') in midsaggital section, anterior left, dorsal up.
Co-staining: muc2 (mucin 2, oligomeric mucus/gel-forming) marks cement gland (cg), and nrp1( neuropilin 1) marks brain in light orange; arrows indicate the position of the presumptive primary mouth. |
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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 . (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 . (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). |
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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 . 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). |
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frzb (frizzled related protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, (B) anterior view, dorsal up, and (B') in section.
Co-staining: muc2 (mucin 2, oligomeric mucus/gel-forming) marks cement gland (cg), and nrp1( neuropilin 1) marks brain and eyes in light orange.; arrows indicate the position of the presumptive primary mouth. |
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frzb (frizzled related protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, (C) anterior view, dorsal up, and (C') in midsaggital section, anterior left, dorsal up..
Co-staining: muc2 (mucin 2, oligomeric mucus/gel-forming) marks cement gland (cg), and nrp1( neuropilin 1) marks brain and eyes in light orange.; arrows indicate the position of the presumptive primary mouth. |
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frzb (frizzled related protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, (D) anterior view, dorsal up, and (D') in midsaggital section, anterior left, dorsal up.
Co-staining: muc2 (mucin 2, oligomeric mucus/gel-forming) marks cement gland (cg), and nrp1( neuropilin 1) marks brain and eyes in light orange.; arrows indicate the position of the presumptive primary mouth. |
