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grhl1 transcription is controlled in an autoregulatory, timeand cell type-dependent matter. (A-M′) Lateral views (anterior/ventral to the left) of whole mount in situ hybridisations with probes indicated in the lower right corner. (H,J,K-M′) Cells at the anterior end of the trunk (H,K-M′) or at the head (J). (H) In situ hybridisation was followed by an immunostaining for p63. Stages are indicated in the upper right corner and genotypes in the lower left corner and apply to all panels left. MO indicates grhl1 morphants. Orange arrows in (M,M′) indicate cells coexpressing grhl1 and pvalb8. (N) Coexpression of skin cell markers and grhl1. It shows a graphical illustration of the average total numbers of grhl1 expressing cells (dark blue), NaR cells (yellow), HR cells (grey) and pvalb8 cells (orange), quantified for the left side of the trunk of 10 WT and 10 grhl1 morphant embryos at 24 hpf. The proportion of co-expressing cells is indicated, grhl1 positive NaR cells (green), grhl1 positive HR cells (light blue) and grhl1 positive pvalb8 cells (dark red). It can be seen that total numbers of ionocytes and pvalb8 cells do not change between WT and morphants, while the proportion of co-expressing cells increases. Please note that in the grhl1-splicemorphants shown in this figure, the grhl1 mRNA was located primarily in the nuclei. This is in contrast to the cytoplasmic localisation of other mRNAs, see (K-M′), and in contrast to grhl1 mRNA in wild-type (see Fig. 2A) or in grhl1-ATG-MO-injected embryos (compare Supplementary Fig. S5C with S5D). Since splicing of hnRNA occurs in the nucleus, incorrectly or not fully spliced grhl1 transcripts appear to be incapable of nuclear export. Apart from this different RNA localisation, identical results with progressively increasing numbers of grhl1-positive cells were obtained for both grhl1-ATGMO and grhl1-splice-MO.
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