Transgene Expression In Embryos Research Articles

Characterization of the transvection mediating region of the abdominal-B locus in Drosophila.

Genetic studies have identified an unusual transvection process in the Abdominal-B (Abd-B) locus of Drosophila. In some cases distal infraabdominal (iab) regulatory domains continue to activate the Abd-B promoter even when translocated onto different chromosomes. Transvection depends on an approx. 10 kb genomic DNA sequence, termed the transvection mediating region (tmr), located immediately downstream of the Abd-B transcription unit. Here we report a detailed analysis of this region. Different DNA fragments from the tmr were inserted into a variety of P-transformation vectors. Analyses of reporter gene expression in transgenic embryos and adults identify at least three cis-regulatory elements, including two enhancers (IAB7 and IAB8) and a new insulator DNA (Frontabdominal-8, Fab-8). Evidence is also presented for a Polycomb Response Element (PRE) linked to the IAB8 enhancer, and an internal promoter in the iab-8 domain, which transcribes the iab-7 and iab-8 cis-regulatory DNA, including the Fab-8 insulator. We discuss the significance of these findings with regard to Abd-B transvection and long-range enhancer-promoter interactions in mammalian globin loci.

An Enhancer Element in the EphA2 (Eck) Gene Sufficient for Rhombomere-specific Expression Is Activated by HOXA1 and HOXB1 Homeobox Proteins

In the hindbrain of the mouse embryo, there is often coincident rhombomere-restricted expression of Eph receptor tyrosine kinases and Hox homeobox genes, raising the possibility of regulatory interactions. In this paper, we have identified cis-acting regulatory sequences of the EphA2 (Eck) gene, which direct node and hindbrain-specific expression in transgenic embryos. An 8-kilobase region of mouse genomic DNA element was sufficient to drive rhombomere 4 (r4)-specific expression while conferring patchy expression in the node. Further analysis localized the rhombomere-specific enhancer to a 0.9-kilobase sequence. This element contains multiple Hox-Pbx consensus binding sites that bind to both HOXA1/Pbx1 and HOXB1/Pbx1 proteins in vitro. Co-expression of either HOXA1 or HOXB1 with Pbx1 transactivated EphA2 enhancer-dependent reporter gene expression. These results, together with observations of reduced EphA2 expression in hoxa1 and hoxb1 double mutant mice, suggest that expression of EphA2 gene in rhombomere 4 is directly regulated by Hoxa1 and Hoxb1 homeobox transcription factors.

A binding site for multiple transcriptional activators in the fushi tarazu proximal enhancer is essential for gene expression in vivo.

The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. We previously identified nine binding sites for multiple Drosophila nuclear proteins in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. We showed previously that the nuclear receptor Ftz-F1 interacts with this repeated module. Here we purified additional proteins interacting with this module from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, we have shown that both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1-Adf-1-Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks.

Lineage-Specific Regulation of the Neural Differentiation GeneMASH1

Mash1 is a transcription factor required during embryogenesis for the development of multiple neural lineages. It is expressed in restricted domains at specific stages in the developing central and peripheral nervous systems and in the developing olfactory epithelium. We have investigated the regulation ofMash1expression during embryogenesis using transgenic mice containingMash1/lacZreporter constructs.Cis-acting regulatory elements controllingMash1expression in the central nervous system are located within an 8-kb sequence upstream of theMash1coding region. This 8-kb sequence does not contain elements directing expression to the peripheral nervous system, olfactory epithelium, or retina. Sequences outside this 8 kb but within 36 kb of theMash1locus contain elements responsible for expression in the autonomic division of the peripheral nervous system. However, transgene expression in embryos containing the 36-kb sequence was never detected in the olfactory epithelium and retina. Thus, regulatory elements driving expression in these lineages may be at even greater distances from theMash1coding region. These data provide evidence for complex regulation ofMash1expression in which multiple lineage-specificcis-acting regulatory regions span greater than 36 kb of theMash1locus. Further characterization of these regions will facilitate the study of factors that regulate the temporal and spatial expression ofMash1during development. In addition, the regulatory sequences identified here can direct expression of heterologous genes to developing neural lineages that normally expressMash1,thus providing an important tool for examining the function of candidate regulatory genes in mammalian nervous system development.

Dacapo, a Cyclin-Dependent Kinase Inhibitor, Stops Cell Proliferation during Drosophila Development

Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. We show that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest.

Sequence and functional analysis of an upstream regulatory region of human HOXA7 gene

The Hox genes have been known to be involved in pattern formation during vertebrate development through differential expression along the anteroposterior body axis. Human homologue of position-specific regulatory region of murine Hoxa-7 was cloned from human genomic library. The restriction map of the 18-kb insert was determined, of which a 3.9-kb region was sequenced. Homology plot between the murine and the corresponding human sequence showed high sequence conservation over 70% in several regions. The homologous region has been reduced to about 1.1 kb ( HCR: human control region), which contained several putative factor binding sites. The function of HCR was analyzed in transgenic mice and turned out to be a position-specific regulatory element of human, setting the precise anterior boundary of expression in transgenic embryos; at day 12.5 post-coitum a distinct anterior limit of expression was noted at the level of C5 in neural tube and spinal ganglia in transgenic embryos. These results indicate that the regulatory sequences as well as the molecular mechanism for Hox gene expression are highly conserved among vertebrates.

Functional dissection of the mouse Hox-a5 gene.

The Hox genes are clustered in evolutionarily conserved complexes and encode DNA binding proteins that determine positional identity. Ubiquitous expression of fly or mammalian Hox proteins in Drosophila embryos provides an assay for gene function, since different Hox genes induce characteristic homeotic transformations. Drosophila Sex combs reduced (Scr) and its murine cognate Hox-a5 produce identical transformations in transgenic flies. To study the contributions of domains conserved between the two proteins, truncated versions of mouse Hox-a5 were assayed for their ability to activate transcription in cultured cells and to induce homeotic transformation and activate target gene expression in transgenic embryos. The homeodomain is essential for protein function and/or nuclear targeting; the N-terminal region contributes to transcription activity and transformation potential in the embryo, but plays no role in determining functional specificity. The YPWM motif is essential for biological specificity, although it does not contribute to transcriptional activation potential. It was recently shown that the Hox-a5 YPWM motif is necessary for in vitro interactions with the co-factor Pbx1. Our results suggest that this type of protein-protein interaction may be essential for the biological activities of Hox-a5 and Scr.

Multiple modes of dorsal-bHLH transcriptional synergy in the Drosophila embryo.

Synergistic interactions between the maternal regulatory factor dorsal (dl) and basic helix-loop-helix (bHLH) activators are essential for initiating differentiation of the mesoderm and neuroectoderm in the early Drosophila embryo. Here we present evidence that dl-bHLH interactions mediating gene expression in the neuroectoderm and mesoderm are fundamentally distinct. Close proximity of dl and bHLH binding sites is essential for the synergistic activation of gene expression in the lateral neuroectoderm, where there are diminishing levels of the dl regulatory gradient. In contrast, sharp on/off patterns of gene expression in the presumptive mesoderm do not require linkage of these sites. Analysis of minimal and synthetic promoter elements suggests that dl and bHLH activators, such as twist, might interact with different rate-limiting components of the transcription complex. These results are consistent with two distinct modes of dl-bHLH synergy: cooperative binding to DNA (requiring linkage of sites) and synergistic contact of basal transcription factors (not requiring linkage). Finally, the characterization of a 57 bp synthetic minimal stripe unit (MSU) provides evidence for a third tier of dl-bHLH synergy. Tandem copies of the MSU function as a bona fide enhancer and can mediate neuroectoderm expression in transgenic embryos even when placed 4.5 kb downstream of a test promoter. Multiple copies of the MSU function synergistically only when linked, but not when separated. We propose that this linkage requirement provides the basis for the evolution of modular promoters composed of discrete, non-overlapping enhancers.

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.

Importance of the CArG box in regulation of β-actin-encoding genes

The β-actin-encoding gene ( Act) in carp is regulated by several cis-acting regulatory elements including the evolutionarily conserved CC(A/T) 6GG ( CArG box or serum-response element) sequences positioned in the promoter region between the CAAT and TATA boxes and in the first intron. To address the roles of the two CArG boxes on gene expression, we replaced them with linker sequences. The CArG box in the proximal promoter was not required for promoter activity in tissue-cultured cells, but was required in conjunction with a second CArG box in the first intron to give full expression in transgenic embryos. Likewise, the geometry of cis-acting transcriptional elements in the proximal promoter was more important for expression of transgenic constructs in developing embryos than in tissue-cultured fibroblasts. Mobility-shift and exonuclease mapping experiments indicated that the same or similar protein factors bind around the two CArG boxes, suggesting that interactions between the promoter and the first intron are involved in Act regulation.