Xenbase Image ID: 81352

Fig. 4. Wholemount in situ hybridization (WISH) indicates that ductin-dependent Hflux is upstream of RNAs in the CNC and placode signaling pathways. Embryos injected with xduct-noTM4 were probed for developmentally regulated mRNAs. In all images, red arrows point to abnormal and green to normal staining patterns. A: Dorsal/anterior views of WISH in stage-14 embryos showing the wildtype pattern (left), an injected embryo with lower than normal expression (center), and an injected embryo with higher than normal expression (right). A.i: xfz3, a marker of neural crest; no embryos were found that had loss of xfz3 staining on one side but were otherwise normal. A.ii: In many xduct-noTM4-injected specimens, cells staining positively for xfz3 appear larger than normal at early stages (stage 10 shown). B: slug, a marker of CNC cells. C: sox9, at this stage a marker for otic capsule formation. D: Embryos at stage 20. D: slug, a marker of CNC cells. E: sox9, a marker for otic capsule formation and CNC. F.i: otic placodeF.ii: Control using the pax8 sense strand lacks non-specific staining; all other sense strand controls were also negative (not shown). G: Expression of pax6, an eye-field marker. Middle column: b-gal, used to label the injected side of the embryo, shows that the disruption to pax6 expression is on the injected side. Right column: A red filter was placed on the camera to give a clear image of the WISH signal. H: Expression of otx2, a marker for olfactory placode and lens. Middle and right columns as in G. Like pax6, otx2 is posterior to its normal domain on the injected side. Ectopic staining is also apparent. I: At stage 22, expression of mitf, a marker for the neural crest melanocyte lineage, is visible in a region appearing to be ectopic neural fold. J: At stage 22, normal sized, one-sided expression domain of xnr-1 in a ductin-disrupted embryo shows that ductin inhibition does not affect other patterning genes. K: Stage-25 embryos stained for slug. The top embryo is an untreated control (cut to lie flat for imaging) showing the normal staining of slug in the pharyngeal arches. The lower embryo was injected with xduct-noTM4 into one blastomere at the 2-cell stage. The pharyngeal arches have been populated normally, but there is ectopic staining of slug; both of these observations are consistent with neural crest cell motility being unaffected by ductin disruption. Image published in: Vandenberg LN et al. (2011) Copyright © 2011. Image reproduced with permission of the Publisher, John Wiley & Sons. Experiment + Assay Source Phenotypes and Disease Xla Wt + atp6v0cdel + NF20 (in situ hybridization) Fig 4 G Expression Phenotype decreased amount otx2.S expression in rhombomere decreased amount pax6.L expression in eye primordium Xla Wt + atp6v0cdel + NF20 (in situ hybridization) Fig 4 H Expression Phenotype decreased amount otx2.S expression in rhombomere Xla Wt + atp6v0cdel + NF25 (in situ hybridization) Fig 4 K Expression Phenotype mislocalised snai2.L expression in dorsal trunk Xla Wt + atp6v0cdel + NF14 (in situ hybridization) Fig 4Aii Anatomical Phenotype increased size of the cell in cranial neural crest increased size of the cranial neural crest Expression Phenotype increased amount fzd3.L expression in neural crest Xla Wt + atp6v0cdel + NF14 (in situ hybridization) Fig 4B Expression Phenotype mislocalised snai2.L expression in neural crest Xla Wt + atp6v0cdel + NF14 (in situ hybridization) Fig 4C Expression Phenotype mislocalised sox9.L expression in otic placode Xla Wt + atp6v0cdel + NF20 (in situ hybridization) Fig 4D Expression Phenotype mislocalised snai2.L expression in migratory neural crest cell Xla Wt + atp6v0cdel + NF20 (in situ hybridization) Fig 4E Expression Phenotype mislocalised sox9.L expression in migratory neural crest cell Xla Wt + atp6v0cdel + NF20 (in situ hybridization) Fig 4Fii Expression Phenotype mislocalised pax8.L expression in migratory neural crest cell mislocalised pax8.L expression in neural plate mislocalised pax8.L expression in otic placode Larger Image Printer Friendly View

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