cis–regulation and mis–regulation – Insights into genomic regulatory mechanisms underlying the control of fgf8a in zebrafish Danio rerio

Fibroblast growth factor 8 (Fgf8) is a potent vertebrate morphogen that plays decisive roles in multiple developmental processes, such as cellular proliferation, survival, differentiation, growth and migration. Fgf8 regionalizes the forebrain and has organizing activity in the midbrain–hindbrain boundary (MHB). Fgf8 induces limb formation and maintains the apical ectodermal ridge (AER), it controls development of the branchial arches and craniofacial skeleton, and its expression in the presomitic mesoderm (PSM) regulates axis elongation, and defines somite boundaries. It follws that the spatiotemporal activity and expression levels of Fgf8 should be tightly controlled, and this control likely occurs at multiple levels – from transcription to regulation of signaling pathways triggered by Fgf8. This thesis constitutes an analysis of mechanisms controlling the activity of Fgf8, and shows that slight modifications in its expression can alter multiple phenotypic traits. Comparative analysis of mammal:teleost conserved chromosomal regions combined with enhancer detection revealed functional chromosomal units called genomic regulatory blocks (GRBs). GRBs usually contain a developmental control gene, or target gene, whose expression is under the control of numerous highly conserved noncoding elements (HCNEs) distributed over a large area that can stretch into and beyond adjacent phylogenetically and functionally unrelated genes, called bystander genes, expressed in patterns unrelated to the target gene. Rearrangements in evolutionarily conserved GRB may have serious consequences during development and thereby contribute to human developmental disease. In all vertebrates, as well as the ascidian Ciona intestinalis, the Fgf8 gene is located in a block of conserved synteny, which also contains the bystander gene Fbxw4. The teleost genomes contain an additional gene upstream of fgf8, slc2A5, which was lost from the mammalian lineage, while the fish have lost BTRC, which is found in all tetrapod genomes. In addition, a whole genome duplication in the teleost lineage resulted in two fgf8 paralogs, fgf8a and –b. Along the fgf8a synteny block a number of HCNEs was detected, located in an area of about 200kb, including inside and around adjacent genes. Functional analysis of these HCNEs in transgenic zebrafish lines suggested that all of them acting as enhancers directed expression of the reporter GFP protein into domains characteristic for fgf8a expression, but not for fbxw4 or slc2a5. Even though in teleosts the fgf8a locus is inverted with respect to tetrapods, and HCNEs are located on the other site of the gene as a result, they still exhibit specific fgf8a activity. Therefore, fgf8a and the bystander genes fbxw4 and slc2a5 genes and cis–regulatory elements of fgf8a constitute a genomic regulatory block (GRB), conserved in all teleost genomes. Insertion of tumor viruses in the proximity of developmental control genes can cause misregulation of the gene and lead to cancer in the mouse. Long terminal repeats (LTRs) of murine tumor viruses contain steroid response elements, which can act as a long–range enhancers. Analysis of four enhancer detection lines in the zebrafish, where integrations of engineered murine proviral vectors had occurred in a 100kb region around the fgf8a gene, caused multiple developmental defects, which were linked to LTR activity. While the expression pattern of YFP reporter protein in all fgf8aCLGY lines mimicked, at least partially, the endogenous expression domains characteristic of fgf8a, fish from the transgenic lines showed extensive phenotypic disorders, including pigmentation anomalies, craniofacial, axial and dermal skeleton defects, and abnormal accumulation of subcutaneous and visceral fat. These phenotypic changes were connected to elevated expression of fgf8a and became manifest during metamorphosis. In addition, transgenic fish had an 80% higher risk of malignant lesions in the brain. The strength of the observed phenotype appeared to be dependent on the distance of the integration site from fgf8a. Progression of tumorigenesis tended to be more severe in males of one line, suggesting androgens as mediators of altered fgf8a activity. Morpholino knock–down experiments targeted to androgen receptor transcript in this transgenic line could reduce the observed elevated fgf8a level. Fgf8 signaling is also regulated at the protein level by specific antagonists, the sprouty proteins. A new member of this family was identified in zebrafish by enhancer detection. A transgenic line was isolated with an expression pattern overlapping fgf8a in forebrain, dorsal diencephalon, MHB, branchial arches, pectoral fin and PSM. Mapping by inverse PCR identified this locus as sprouty1. Sprouty1 is a downstream modulator of Fgf signaling and its expression is attenuated by the small molecular inhibitor of FGF receptor 1, SU5402. Together these findings highlight complex regulatory mechanisms underlying the control, in all vertebrates, of Fgf8, a potent morphogen, which controls multiple aspects of animal form and function