Academic journal article Genetics

Genomewide Expression Profiling in the Zebrafish Embryo Identifies Target Genes Regulated by Hedgehog Signaling during Vertebrate Development

Academic journal article Genetics

Genomewide Expression Profiling in the Zebrafish Embryo Identifies Target Genes Regulated by Hedgehog Signaling during Vertebrate Development

Article excerpt

ABSTRACT

Hedgehog proteins play critical roles in organizing the embryonic development of animals, largely through modulation of target gene expression. Little is currently known, however, about the kinds and numbers of genes whose expression is controlled, directly or indirectly, by Hedgehog activity. Using techniques to globally repress or activate Hedgehog signaling in zebrafish embryos followed by microarray-based expression profiling, we have discovered a cohort of genes whose expression responds significantly to loss or gain of Hedgehog function. We have confirmed the Hedgehog responsiveness of a representative set of these genes with whole-mount in situ hybridization as well as real time PCR. In addition, we show that the consensus Gli-binding motif is enriched within the putative regulatory elements of a sizeable proportion of genes that showed positive regulation in our assay, indicating that their expression is directly induced by Hedgehog. Finally, we provide evidence that the Hedgehog-dependent spatially restricted transcription of one such gene, nkx2.9, is indeed mediated by Gli1 through a single Gli recognition site located within an evolutionarily conserved enhancer fragment. Taken together, this study represents the first comprehensive survey of target genes regulated by the Hedgehog pathway during vertebrate development. Our data also demonstrate for the first time the functionality of the Gli-binding motif in the control of Hedgehog signaling-induced gene expression in the zebrafish embryo.

DURING animal development, cells communicate with each other to coordinate their proliferation and differentiation and to ensure that the right kind of tissues are assembled spatially and temporally within the embryo. Surprisingly, only a handful of intercellular signaling molecules have so far been identified and shown to participate in this process, their reiterative use in different cellular situations governing the generation of cellular diversity. The Hedgehog (Hh) family of lipid-modified secreted proteins are one such group of intercellular signals that have profound effects on the regulation of embryonic development of all animals (INGHAM and MC MAHON 2001; HOOPER and SCOTT 2005). Although the signaling pathway was first discovered in Drosophila, where it primarily acts to pattern the cuticle of the larva and the appendages of the adult fly, in vertebrates, a multitude of developmental processes in the embryo and the adult organism have now been shown to be regulated by Hh activity. These include effects on cell proliferation and cell survival as well as cell fate determination. Consistent with all of these influences of Hh on normal development and physiology, loss of Hh signaling in humans has been linked to a number of congenital abnormalities like holoprosencephaly, whereas excessive activity of the pathway appears to be the etiological factor in the initiation and growth of some of the most common forms of cancers (ALTABA et al. 2002; MC MAHON et al. 2003).

Much of our understanding of the molecular details of the Hh pathway has come from genetic and biochemical experiments with components required for transduction of the signal in Drosophila. From all of these investigations it is evident that nuclear access of the transcriptional activator form of the zinc finger protein, Cubitus interruptus (Ci), and its activation of target gene transcription marks the culmination of events in the Hh transduction cascade (INGHAM and MC MAHON 2001; MC MAHON et al. 2003; HOOPER and SCOTT 2005). In vertebrates, three distinct Ci homologs, the Gli proteins, have subsumed the function of Ci in the regulation of target gene expression. Because Drosophila Ci and the Gli3, and possibly Gli2, proteins of vertebrates undergo phosphorylation and proteolytic processing in the absence of Hh to yield truncated transcriptional repressor forms, optimal induction of target gene expression is critically determined by the ratio of the activator (Gliact) vs. …

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