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The multifunctional Eya1 protein plays important roles during the development of cranial sensory organs and ganglia, kidneys, hypaxial muscles and several other organs in vertebrates. Eya1 is encoded by a complex locus with candidate cis-regulatory elements distributed over a 329 kbp wide genomic region in Xenopus. Consequently, very little is currently known about how expression of Eya1 is controlled by upstream regulators. Here we use a library of Xenopus tropicalis genomic sequences in bacterial artificial chromosomes (BAC) to analyze the genomic region surrounding the Eya1 locus for enhancer activity. We used BAC recombineering to first create GFP reporter constructs, which were analysed for enhancer activity by injection into Xenopus laevis embryos. We then used a second round of BAC recombineering to create deletion constructs of these BAC reporters to localize enhancer activity more precisely. This double recombineering approach allowed us to probe a large genomic region for enhancer activity without assumptions on sequence conservation. Using this approach we were able to identify two novel cis-regulatory regions, which direct Eya1 expression to the somites, pharyngeal pouches, the preplacodal ectoderm (the common precursor region of many cranial sensory organs and ganglia), and other ectodermal domains.
Figure 1. Genomic region of Eya1 in Xenopus tropicalis. (A) Position of BACS 15 and 16 on chromosome 6 of X.tropicalis. Note that the precise position of the plus end (right side) for BAC16 has not been reported (see Suppl. Figure 1 and Table 1). (B,C) Detail of BAC15 (B) and BAC16 (C) with the positions of the GFP reporter cassette (insert 1, blue) and the regions deleted in various deletion constructs (orange) indicated. Deletion constructs depicted by faint orange color were not tested. Scale shows position on chromosome 6 of the Xenopus tropicalis genome (version 9.0; http://www.xenbase.org/). Blue double arrows show position of the two genomic regions with enhancer activity identified in the present study, BAC15P2D2-P2D3 and BAC16P2D3-P2D4.
Figure 2. Overview of procedure for generating BAC15 and BAC16 reporter constructs. BACs were obtained from a BAC library maintained in the DH10B strain of E. coli (1). BACs were then purified and transformed into strain SW102 (or EL250; not shown) competent for recombineering (2). Primers with overhangs complementary to the targeted BAC region (homology arms H1 and H2) were used to amplify a cassette containing the GFP reporter and an ampicillin resistance gene (Amp) from plasmid pIS-GATA2-GFP using PCR. The insert amplified with primers F1 and R1 was used for BAC15 and did not contain a promoter, whereas the insert amplified with primers F2, R2 was used for BAC16 and contained the GATA2 minimal promoter (3).The insert was transformed into SW102 bacteria containing the appropriate BAC to allow for recombineering (4).
Figure 3. Double recombineering strategy to localize enhancers in large genomic regions. (A) Overview of recombineering procedure. A target region for insertion is selected in the BAC, for example the first coding exon of the gene of interest containing the start codon (ATG) (1). An insert (containing an antibiotic resistance gene and possibly other elements such as a reporter gene) with appropriate homology arms (H1, H2) complementary to BAC target sequences is transformed into bacteria containing the BAC (2). The insert is inserted into the BAC by homologous recombination (recombineering) replacing the original BAC sequence between H1 and H2 (3). Proper insertion is verified by PCR using forward (FB) and reverse primers (RB) for BAC sequences flanking the insertion, while proper orientation is verified by PCR combining FB with a reverse primer for an insert specific sequence (RI) or a forward primer for an insert specific sequence with RB (4). (B) Creation of reporter BACs in the first recombineering step. Insert 1 (here the insert for BAC15) contains an ampicillin resistance gene and a GFP reporter gene (1). Homology arms are designed to target the insert to a position immediately after the start codon in the first coding exon of Eya1 during recombineering (2). BAC reporter construct after recombineering (3). (C) Creation of deletion constructs of BAC reporters in the second recombineering step. Sequences flanking large noncoding regions in the BAC are selected as new homology arms (HD1-HD5). Chosing different combinations of these for the insert 2 (containing a kanamycin resistance gene) will result in deletions of different extent (1). During recombineering the sequence D4 on the BAC between HD1 and HD2 (or between other homology arms chosen) will be deleted and replaced by the kanamycin resistance cassette (2). BAC reporter deletion construct after second step of recombineering containing insert 1 and 2 (3).
Figure 4. GFP reporter expression driven by BAC15. Embryos of Xenopus laevis at neural plate (A–D: dorsal view, anterior to the left) and tailbud stages (A’–D’: lateral view, anterior to the left) after injection with BAC15 reporter constructs at the one-cell stage. A,A’,B,B’: GFP expression after injection of full length BAC15 reporter (BAC15-FL) in living Xenopus embryos (A,A’) or as revealed by GFP in situ hybridization (B,B’). C,C’: Absence of GFP expression domains after injection of BAC15-P2D2 deletion construct. D,D’: GFP expression domains after injection of BAC15-P2D4 deletion construct recapitulate those observed after BAC15-FL injection. Insert in D’ shows somitic expression in trunk region of a different embryo (tail end to the right). Abbreviations: epi: epidermis; np: neural plate; pp: pharyngeal pouches; PPR: preplacodal region; som: somites.
Figure 5. GFP reporter expression driven by BAC16. Embryos of Xenopus laevis at neural plate (A–D: dorsal view, anterior to the left) and tailbud stages (A’–D’, Aâ€,Bâ€: lateral view, anterior to the left) after injection with BAC16 reporter constructs at the one-cell stage. A–Aâ€, B–Bâ€: GFP expression after injection of full length BAC16 reporter (BAC16-FL) in living Xenopus embryos (A–Aâ€) or as revealed by GFP in situ hybridization (B–Bâ€). C,C’: Absence of GFP expression domains after injection of BAC16-P2D3 deletion construct. D,D’: GFP expression domains after injection of BAC16-P2D4 deletion construct recapitulate those observed after BAC16-FL injection. Insert in D’ shows somitic expression in trunk region of a different embryo (tail end to the right). Abbreviations: epi: epidermis; le: lens placode; ll: lateral line placodes; np: neural plate; olf: olfactory placode; ot: otic placode/vesicle; pp: pharyngeal pouches; PPR: preplacodal region; som: somites.
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