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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). |