Fig. 4. Expression of ttrap and its role during Kupffer's vesicle formation. (A) Transcripts are detectable by RT-PCR throughout the first 24 hpf (MBT, mid-blastula transition; epi, epiboly; ss, somite stage). β-actin sequences are shown as loading control (NC, negative control; -RT, no reverse transcriptase). (B-E) Whole-mount ISH (WISH) analysis of ttrap. (B) Maternal ttrap mRNA contribution during early cleavage stages. (C) At 30% epiboly, transcripts are distributed throughout the blastula. Lateral views, animal pole at top. (D) At 60% epiboly, expression in DFCs becomes detectable (arrowheads). Dorsal view, animal pole at top. (E) In addition to the uniform expression throughout the embryo, stronger expression within tailbud and surrounding Kupffer's vesicle (KV, arrow) from 5 ss was observed. (F-I) Absence of KV in Ttrap^DFCMO embryos. (F) KV is present in control^DFCMO embryo and not detectable in (G) Ttrap^DFCMO embryo (16 ng MO; arrowheads, KV). Embryos scored live (5-9ss). Posterior views; nc, notochord. (H,I) WISH for cxcr4a (5-9 ss) to confirm KV in (H) control^DFCMO embryo and (I) absence in Ttrap^DFCMO (arrowheads). Posterior views.