Fig. 2.

cNkd1 and cLef1 are Notch dependent and reduced Wnt signalling increases the periodicity of cLfng oscillations in the chick PSM. Chick explant pairs cultured for 3–4 h on a Millipore filter floating on 100 μM DAPT, 200 μM CKI-7 or 4–10 μM sFRP2 or control media supplemented with DMSO, PBS 0.1% BSA or ethanol respectively. Panels A′, F′ are schematic representations of panel A and panel F respectively showing cLfng expression in the PSM only. (A, A`) DAPT treatment led to loss of cLfng in the PSM. Neural tube expression was unaffected. (B) Expression of intronic cLef1 was also lost. (C) Expression of cNkd1 was also severely down regulated. (D) Expression of exonic cAxin2 was also lost. (E) Exonic cTBX6 expression was not affected. (F, F`) CKI-7 slowed the period of cLfng. Note the treated explant is phase 1 and the control is early phase 3 of the same cycle and has formed a new somite boundary. (G) CKI-7 led to loss of intronic cLef1 expression. (H) Exonic cNkd1 and (I) exonic cAxin2 expression are severely down regulated on exposure to CKI-7. (J) Exonic cSprouty2 expression is lost on exposure to CKI-7. (K) Exposure to CKI-7 led to the formation of fewer somites and stripes of cUncx4.1 expression. (L) Explants showing cycling cLfng in 200 μM CKI-7 with the fixed explant showing phase 3 while the cultured explant has progressed to late phase 1. (M) Exonic cTBX6 expression was not affected on exposure to CKI-7. (N) sFRP2 treatment down regulates cLef1 expression. (O) The explant treated with sFRP2 is in early phase 2 and the control explant in early phase 3 of the cLfng cycle. (P) Schematic representation of clock gene expression during one oscillation in the PSM. (Q) Box plot showing spread of TUNEL staining measurements or phosphoH3 staining in explants treated with CKI-7 compared to untreated controls. Arrow heads show last somite boundary formed in culture. “+” indicates treated half and dashed line indicates control half.