Fig. 1. (A) Expression patterns of region C constructs. (a-e) CHZ. (f,g) Hoxb4 promoter-region C construct. (h-l) CR1 deletions. (A) Lateral and dorsal views of a 10 somite (So) stage embryo, showing anterior boundaries of expression: neural tube adjacent to So 4 (yellow arrow); somitic and flank mesoderm at So 6/7 (black arrow). Typical of Hox gene expression domains, staining is weaker in So 7 (light blue triangle) than in So 8 (dark blue). (b) 9.5 dpc: somitic boundary regresses to So 13/14 (triangles); neural boundary regresses to So 6/7 (yellow arrow). (c) 10.5dpc: staining in flank mesoderm (fm). (d) 12 dpc: strong staining in spinal ganglia up to the first cervical nerve (cn1) and ventral neural tube, extending anteriorly (open triangle). (e) Transverse section (TS), forelimb level of a similar embryo (drg, dorsal root ganglia; sg, sympathetic ganglia; f, floorplate; v, ventral root of spinal nerve). (f) 11 dpc embryo (Hoxb4 promoter, construct 5) (Whiting et al., 1991): note strong anterior domains of expression in the neural tube up to the spinal cord/hindbrain boundary (yellow arrow) and in the somitic mesoderm between the anterior So 6/7 boundary and So14 (white arrows). (g) TS forelimb level of the same embryo: note strong staining in sclerotomal derivatives (open triangles) and the dorsal aorta (d). (h,i) Lateral and dorsal views of a 12 dpc embryo (CHZ559-599): boundaries of expression in the neural tube (yellow arrow) and in the somitic mesoderm (blue triangle) are indicated. (j,k) Lateral and dorsal views of a 12dpc embryo (CHZ515-607): consistent expression is restricted to a domain in the ventral neural tube (vnt). (l) TS thoracic level of the same embryo. Scale bars: 100µm. (B) Sequence alignment showing a comparison between CR1 of the mouse Hoxb4 intron and those of other paralogous group 4 Hox genes, identical bases are highlighted in black. Numbering is with respect to that of region C (bp 1 is the first base of the SalI site in exon 1, +321 of Hoxb4). The number of base pairs (bp) in each aligned sequence is shown on the right. The extents of the two deletions in constructs CHZ559-599 and CHZ515-607 are marked with blue lines and the boundaries of a possible cis-positive regulatory element (bp 515-558), identified by these deletions, are marked by red triangles. The margins of the 28bp HB-1 element (bp 574-601) are shown (black triangles). The locations of four conserved motifs are marked below (I-IV). (C) Schematic diagram of the transgenes in A. The hsp68 promoter-lacZ reporter, which is common to each construct, is not shown to scale. Exons are shaded grey. CR1 is represented by a black rectangle within the intron (white rectangle) flanked by MunI and SfiI restriction sites. CHZ515-607 carries a deletion of the entire CR1 region (Aparicio et al., 1995). An example is shown for comparison. Exp. # denotes the total number of independent transgenic F₀ embryos and lines generated with each construct giving a consistent pattern of expression.

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