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Figure 2. Sfrp5 is required for foregut organogenesis. (A,D,G,J,M,P,S) Control embryos. (B,E,H,K,N,Q,T) Foregut development was impaired by injection of the sfrp5-MO (50 ng). (C,F,I,L,O,R,U) Foregut development was expanded by injection of ectopic sfrp5 RNA (500 pg) into the hindgut. Anterior is to the left and dorsal is up. (A–C) External morphology of stage 30 tailbud embryos. (D–F) Mid-sagittal H&E-stained sections of stage 30 embryos showing the collapsed foregut cavity in sfrp5-MO embryos (shown in E). (G–L) In situ hybridization to isolated gut tubes at stage 42 showing that the hhex-expressing liver bud (G–I) and pdx1-expressing ventral pancreatic bud (J–L) were hypoplastic in Sfrp5-depleted embryos and expanded by Sfrp5 overexpression. (M–O) The liver specification marker for1 was repressed by sfrp5 depletion and expanded by sfrp5 overexpression at stage 35. (P–R) At stage 20, the foregut marker hhex was repressed by sfrp5 depletion and expanded by sfrp5 overexpression. (S–U) At stage 20, the Wnt/β-catenin posterior target gene vent2 was ectopically expressed in the foregut of sfrp5-MO embryos (arrow) and repressed by sfrp5 overexpression. (V) Injection of the mismatch-MO (50 ng) had no effect, while coinjection of a synthetic sfrp5 RNA lacking the sequence targeted by the sfrp5-MO rescued hhex and for1 expression in sfrp5-depleted embryos. MOs were injected at the eight-cell stage followed by a targeted injection of the sfrp5 RNA (lacking the 5′-UTR; 100 pg) into the anterior endoderm D1 cells of the 32-cell stage. The histogram shows the percent of embryos with normal expression levels. The number of embryos for each condition is in parentheses, and representative examples are shown in Supplemental Figure S2. (W) Sfrp5-depleted embryos show increased cell death in the foregut at later stages of development, but not at stage 20. The percentage of TUNEL-positive foregut cells is presented ±standard deviation. The number of embryos counted is in parentheses, with ∼75 foregut cells per embryo at stage 20 and ∼150 foregut cells per embryo at stage 35. (*) P < 0.05 compared with age-matched controls in a Student t-test. |
