Fig. 2. Structure of the her5 genomic locus and reporter constructs and corresponding GFP expression. (A) Construction of her5PAC:egfp by ET-cloning-mediated recombination of the egfp cDNA within exon 2 of her5. The her5 locus comprises 3 exons (blue), of which exon 2 encodes the basic and first helix domain of the Her5 protein (bHLH domain labeled in red as b, H1, L and H2). Recombination arms (a',b') matching exon 2 were amplified in frame with the egfp sequence and a floxed zeocine resistance cassette (zeo) (top construct). The resulting product was inserted in vitro within a her5-containing PAC by ET-mediated homologous recombination (Muyrers et al., 2000; Muyrers et al., 1999). The zeo cassette was subsequently deleted by Cre excision in vitro, generating the herPAC:egfp construct (bottom line). (B) Reporter constructs used to localise her5 regulatory elements in transient (black lines) or transgenic (red lines) assays. Most constructs were generated from her5PAC:egfp (bottom construct) by PCR amplification and contain egfp in frame within her5 exon 2. Numbering to the left of each fragment refers to the length of upstream sequence from the transcriptional start site, in bp. The expression profile driven by each construct is written to the right. Note that the enhancer element(s) driving endodermal expression are located within 240 bp of upstream sequence and/or intron 1, and that sequences driving specific MH expression are recovered with 2.9 kb of upstream sequence. (C) Endogenous her5 transcription at 70% epiboly (onset of neural her5 expression) revealed by whole-mount in situ hybridisation (blue staining). her5 is expressed in a V-shaped domain at the AP level of the MH anlage (MH) and in a subset of anterior endodermal precursors (e) (see also Bally-Cuif et al., 2000). (D-H) Selected examples of GFP protein expression driven by representative reporter constructs [bright field (top) and fluorescent (bottom) views of transgenic embryos, constructs as indicated below each panel]. All constructs illustrated drive expression to the anterior endoderm. Constructs comprising more than 2.9 kb of upstream sequence (D,E) drive selective neural expression to the MH. Intermediate constructs (F,G) drive unrestricted anterior neural expression.

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