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Fig. 6. Abolishing localisation of h transcripts reduces Hairy protein activity. (A-C) h transcripts (red) in wild-type (A) or transgenic (B,C) blastoderm embryos carrying one copy of a h transgene either with (B, st2-h^wtD) or without (C, st2-h^nlocD) a functional localisation signal under the control of the eve stripe 2 enhancer. Numbers of endogenous h stripes are indicated. Red bar indicates approximate region of enhancer activity. (D) Incidence of defects in thoracic segment 2 (T2) induced by st2-h activity in different transgenic lines. Numbers above bars indicate number of first instar larvae scored. (E) Levels of st2-h transcripts (relative to actin 5C transcripts) in different lines, as determined by real-time PCR, normalised to that of wtA. Provided RNA levels are not very high (nlocD and wtD), lines with localising st2-h transcripts have stronger phenotypic effects than those with nonlocalising transcripts, even when the localising transcript is expressed at significantly lower levels [e.g. P<0.01 for different transcript levels for wtA versus nlocB or nlocC and P=0.05 for wtB versus nlocC (t-test)]. (F-H) Lateral views of st2-h first instar larvae showing examples of (F) a normal T2 denticle belt, (G) a deletion of a ventral region of the T2 denticle belt leading to a characteristic curvature of the thorax (arrowhead points to remaining dorsal cuticle) or (H) a complete deletion of the T2 denticle belt. st2-h has been previously shown to have stronger effects ventrally (Wu et al., 2001). (I) Lateral (dorsal upwards) and (I') ventral views of st2-h^nlocC/st2-h^nlocC blastoderm embryos stained for ftz mRNA. In ventral views, stripes 1, 2 and 3 are shown. Anterior is towards the left. (J,J') Same views as in I,I' of st2-h^wtB/st2-h^wtB embryos. Ectopic h expression partially deletes ftz stripe 2 with a stronger effect ventrally. See Table S2 for quantification of ftz defects in different lines. (K-M) Confocal images of Hairy protein distribution in wild-type (K, +/+), (L) st2-h^nlocC/st2-h^nl°^cC and (M) st2-h^wtB/st2-h^wtB blastoderm embryos. More Hairy protein accumulates in the nuclei between endogenous stripes 2 and 3 in st2-h^wtB/st2-h^wtB than it does in st2-h^nlocC/st2-h^nlocC, even though the former expresses significantly less st2-h mRNA. Consistent results were seen in several embryos for each genotype. Red bars in L-N show approximate regions of enhancer activity. (N) Mean fluorescent intensity/pixel within nuclei of corresponding images (data were collected with Kinetic Imaging AQM6 software). Scale bar: 50 µm in A-C; 180 µm in F-H; 130 µm in I-J', 20 µm in K-M.

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