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Fig. 4. EFHC1b plays a role in Wnt signaling. In situ hybridization shows defects (red arrows) in central nervous system (En2) patterning (A, B) and neural crest (Sox9) migration (D, E) in EFHC1b morphants at stage 18 and stage 25, respectively. These phenotypes were rescued by co-injection of EFHC1b-GFP-rescue RNA (C, F). Embryos were injected with either control MO, EFHC1b MO, or EFHC1b MO together with EFHC1b-GFP RNA. All embryos (A–C) were injected with RNA encoding β-galactosidase (red) as a lineage tracer. The loss of neural patterning markers (En2, Krox20, Tubb2b) as well as neural crest markers (Sox9, Twist1) was also rescued by δD3-C-GFP (G). The percentage of embryos that were “abnormal”, that is displayed reduced marker gene expression, was calculated from two independent experiments (N=55 to 60 embryos per condition). H: RNA levels in control and EFHC1b morpholino explants were analyzed at stage 18 using RT-PCR; EFHC1b morphant explants displayed decreased levels of Tubb2 RNA and increased levels of Wnt8a RNA. Levels of BMP4, Noggin and FGF8 RNA were unchanged. I: qPCR analyses (N=3) of control and EFHC1b morphant explants co-injected with EFHC1b-GFP-rescue, Dkk1, or SRFP2 RNAs. Both EFHC1b-GFP-rescue and the two Wnt signaling inhibitors returned all RNAs to control levels. J, K: Embryos were injected with TOPFLASH and FOPFLASH (control) plasmid DNAs (100 pgs/embryo) together with δG-β-catenin RNA (100 pgs/embryo) either alone or together with GFP or EFHC1b-GFP (100 pgs/embryo) RNAs or control, EFHC1b MO (10 ngs/embryo) and EFHC1b MO with SFRP2 RNA. The Y-axis indicates the fold-increase relative to the control TOPFLASH/FOPFLASH value (set equal to 1, N=3). L: qPCR analyses (N=3) of control and EFHC1 morphant explants co-injected with EFHC1b-GFP-rescue, SRFP2, or EFHC1b mutant RNAs on Wnt8 RNA. Besides the EFHC1b-GFP and the Wnt inhibitor SFRP2, injection of δD3-C-GFP RNA (100 pgs/embryo) also returned the Wnt8 RNA to control levels. Significant differences between conditions are marked by horizontal bars; in each case, data are represented as mean±SD, * for p<0.05 and ** for p<0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) |
