The maternal Dorsal regulatory gradient initiates the differentiation of several tissues in the early Drosophila embryo. Whole-genome microarray assays identified as many as 40 new Dorsal target genes, which encode a broad spectrum of cell signaling proteins and transcription factors. Evidence is presented that a tissue-specific form of the NF-Y transcription complex is essential for the activation of gene expression in the mesoderm. Tissue-specific enhancers were identified for new Dorsal target genes, and bioinformatics methods identified conserved cis-regulatory elements for coordinately regulated genes that respond to similar thresholds of the Dorsal gradient. The new Dorsal target genes and enhancers represent one of the most extensive gene networks known for any developmental process.
The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential CiBra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation. Received March 22, 1999; revised version accepted May 3, 1999. Brachyury encodes a sequence-specific activator that contains a T-box DNA-binding domain (Herrmann et al. 1990;Kispert et al. 1995;Conlon et al. 1996). In vertebrates, Brachyury is initially expressed throughout the presumptive mesoderm, and during later stages the expression pattern is gradually restricted to the developing notochord and tailbud. Brachyury notochord differentiation is essential in all vertebrates that have been studied, including mice, frogs, and zebrafish (for review, see Herrmann and Kispert 1994;Smith 1997;Papaioannou and Silver 1998).Brachyury is expressed exclusively in the notochord precursor cells of two divergent ascidians, Halocynthia roretzi (Yasuo and Satoh 1993) and Ciona intestinalis (Corbo et al. 1997a). The spatial and temporal patterns of the gene expression coincide with the clonal restriction of the notochord lineages. In H. roretzi, notochord formation is induced at the 32-cell stage by signals emanating from the adjacent endoderm (Nakatani and Nishida 1994). Overexpression of the Halocynthia Brachyury gene (As-T) via RNA injection results in notochord formation without a requirement for the inductive event at the 32-cell stage (Yasuo and Satoh 1998). In addition, misexpression of As-T also causes transformation of endoderm and neuronal lineages into notochord (Yasuo and Satoh 1998). These results indicate that the ascidian Brachyury gene is a critical determinant of the notochord. Here we report that the misexpression of the Brachyury gene (Ci-Bra) of C. intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra overexpression. We isolated and characterized 38 different notochord-specific genes that may include potential targets of the ascidian Brachyury. Results and DiscussionThe fork head/HNF-3 gene of C. intestinalis (Ci-fkh) is expressed in the endoderm, endodermal strand, notochord, and ventral ependymal cells of the neural tube (Corbo et al. 1997b). A 2.6-kb genomic DNA fragment from the 5Ј-flanking region of Ci-fkh is sufficient to direct the expression of a lacZ reporter gene in these tissues after electroporation into one-cell embryos (Fig. 1A). The Ci-Bra gene was misexpr...
Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.
The evolution of animal diversity depends on changes in the regulation of a relatively fixed set of protein-coding genes. To understand how these changes might arise, we examined the organization of shared sequence motifs in four coordinately regulated neurogenic enhancers that direct similar patterns of gene expression in the early Drosophila embryo. All four enhancers possess similar arrangements of a subset of putative regulatory elements. These shared features were used to identify a neurogenic enhancer in the distantly related Anopheles genome. We suggest that the constrained organization of metazoan enhancers may be essential for their ability to produce precise patterns of gene expression during development. Organized binding sites should facilitate the identification of regulatory codes that link primary DNA sequence information with predicted patterns of gene activity.
The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.
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