Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

Enhancer Traps in the Drosophila Bithorax Complex Mark Parasegmental Domains

Eight P elements carrying a β-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure.     

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs.     

Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis-acting elements

The Hoxc8 early enhancer that controls the initiation and establishment of Hoxc8 expression in the developing mouse embryo is found in different vertebrate lineages including mammals, birds and fish. Mouse and Fugu Hoxc8 early enhancers (200 bp) have diverged in the composition of elements located towards the 3' region. However, they share cis-acting elements A-E located in the 5' region. Mutations at these elements in the context of the mouse Hoxc8 early enhancer affect reporter gene expression in the posterior neural tube, somites and lateral plate mesoderm of day 9.5 mouse embryos. Here, we demonstrate that mutations introduced at the same elements but in the context of the Fugu Hoxc8 early enhancer had different consequences on the reporter gene expression in transgenic mouse embryos. Furthermore, in contrast to the mouse enhancer the Fugu enhancer does not utilize elements D and E in achieving posterior neural tube and somite expression. These results suggest that the diverged sequences prevent regulatory interactions at conserved cis-acting elements. We propose that divergent sequences modify regulatory interactions at conserved elements by providing a "contextual change". Our finding that the enhancer elements do not act in a unitary fashion but function in the context of the surrounding sequence brings a new dimension to the study of cis-regulatory evolution.