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Fig. 1. Temporal and spatial pattern of kermit2 expression. (A) RT-PCR analysis of kermit2 temporal expression. Total RNA was isolated from embryos at different stages and analyzed by RT-PCR. -RT, negative control without reverse transcriptase. ODC was used as a loading control. (B) RT-PCR analysis of kermit2 spatial expression. Stage 16 embryos were dissected into dorsal, ventral, anterior and posterior pieces, as shown in the diagram. Stage 20 embryos were dissected into anterior and posterior pieces. EF1α was used as a loading control. kermit2 is equally expressed in anterior, posterior, dorsal and ventral regions at mid-neurula stage (stage16), but is localized to the anterior region by stage 20. Anterior marker Otx2, posterior dorsal marker HoxD1, and posterior ventral marker Vent-1 were used as positive controls. (C-F) In situ hybridization of neurula and tadpole stage embryos. (C,D) Stage 20, anterior view, dorsal towards the top. (C) Sense control; (D) antisense probe. At stage 20, kermit2 is expressed in the cement gland and neural plate border, which is adjacent to and/or overlapping the presumptive eye region. (E,F) Antisense probe. By tailbud stages, kermit2 expression becomes further restricted, strongly expressed in the cement gland and otic vesicles (ov: arrow) at stage 26 (E), and is also expressed in the pronephros and branchial arches in stage 33 tadpoles (F). |
