Christine Rushlow

Characterization and localization of the even-skipped protein of Drosophila.

On the basis of homeo box cross‐homology we have isolated the pair‐rule gene even‐skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta‐galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila

The maternal morphogen dorsal (dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.

The Drosophila Gene brinker Reveals a Novel Mechanism of Dpp Target Gene Regulation

decapentaplegic (dpp), a Drosophila member of the TGFbeta family of secreted molecules, functions as a long-range morphogen in patterning of the embryo and the adult appendages. Dpp signals via the SMAD proteins Mad and Medea. Here we show that in the absence of brinker (brk), Mad is not required for the activation of Dpp target genes that depend on low levels of Dpp. brk encodes a novel protein with features of a transcriptional repressor. brk itself is negatively regulated by Dpp. Dpp signaling might relieve brks repression of low-level target genes either by transcriptional repression of brk or by antagonizing a repressor function of brk at the target gene promoters.

The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila

In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic “hotspot” regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo.

The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc.

The Drosophila segmentation gene, hairy (h), acts to regulate embryonic segmentation and bristle pattern. We present the DNA sequence of the h gene and of h cDNAs, thereby deducing the organization of the h transcripts. The h gene encodes a 337 amino acid protein that acts in both embryonic segmentation and adult bristle patterning. The h protein includes a domain that shows extensive similarity to a domain of the proto‐oncogene N‐myc that may be involved in DNA binding and/or protein dimerization. We discuss mechanisms of h action as a transcriptional regulator.

The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in drosophila

A gradient of the maternal morphogen dorsal (dl) establishes dorsal-ventral (D-V) polarity in the early Drosophila embryo. The dl concentration gradient is initiated by regulated nuclear transport, and only protein that enters nuclei is active in the D-V patterning process. Here we show that dl is a DNA-binding protein that specifically interacts with distal sequences of the zerknüllt (zen) promoter, one of the genetic targets of the morphogen. These zen sequences have the properties of a silencer element and can act over long distances to repress the expression of a heterologous promoter. The dl protein recognizes a sequence motif similar to that of the mammalian transcriptional activator NF-kappa B, which was shown to contain extensive homology with dl and the oncoprotein rel. We present evidence that the DNA-binding activity of the dl protein is mediated by the region of homology (the rel domain) conserved in the rel and NF-kappa B proteins.

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo.

Individual dorsal morphogen binding sites mediate activation and repression in the Drosophila embryo.

The dorsal (dl) morphogen gradient is responsible for initiating the differentiation of the mesoderm, neuroectoderm and dorsal ectoderm in the Drosophila embryo. dl encodes a sequence‐specific DNA binding protein that belongs to the Rel family of transcription factors. Previous studies have shown that dl activates the mesoderm determinant twist (twi); here we use a combination of site‐directed mutagenesis and P‐transformation assays to demonstrate that it also functions as a direct transcriptional repressor of a second target gene, zerknüllt (zen). By exchanging dl binding sites between the promoters we show that activator sites from twi can mediate repression when placed in the context of the zen promoter, and that repressor sites from zen can mediate activation in the context of the twi promoter. This represents the first demonstration that common binding sites for any DNA binding protein can mediate both activation and repression in a developing embryo. Evidence is also presented that the affinities of dl binding sites are important for the efficiency of repression, but are not the sole determinants of the threshold response to the dl gradient.

Conversion of a silencer into an enhancer: evidence for a co-repressor in dorsal-mediated repression in Drosophila.

The dorsal (dl) protein gradient determines patterns of gene expression along the dorsal‐ventral axis of the Drosophila embryo. dl protein is at peak levels in ventral nuclei of the embryo where it activates some genes (twist and snail) and represses others [zerknullt (zen), decapentaplegic and tolloid]. It is a member of the rel family of transcription factors and interacts with specific DNA sequences in the regulatory regions of its target genes. These sequences (dl binding sites), when taken from the context of either an activated or repressed promoter, mediate transcriptional activation of a heterologous promoter, but not repression. We found that T‐rich sequences close to the dl binding sites in the silencer region of the zen promoter are conserved between three Drosophila species. Using this sequence information we defined a minimal element that can mediate repression of a heterologous promoter. This element interacts with at least two factors present in embryonic extracts, one of which is dl protein. The other factor binds to the T‐rich site. Point mutations in either site abolish ventral repression in vivo. In addition, mutations in the T‐rich site cause ectopic expression in ventral regions indicating that the minimal silencer was converted into an enhancer.