Functional analysis of the transcriptional control regions of the copia transposable element.

The introduction of copia-based vectors in Drosophila hydei cells results in their high-level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3' to the site of initiation of copia RNA. Deletion of the consensus enhancer-like sequences from copia appears to have no effect on vector expression.

Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina.

BACKGROUND: Neurons of the vertebrate central nervous system (CNS) are generated sequentially over a prolonged period from dividing neuroepithelial progenitor cells. Some cells in the progenitor cell population continue to proliferate while others stop dividing and differentiate as neurons. The mechanism that maintains the balance between these two behaviours is not known, although previous work has implicated Delta-Notch signalling in the process. RESULTS: In normal development, the proliferative layer of the neuroepithelium includes both nascent neurons that transiently express Delta-1 (Dl1), and progenitor cells that do not. Using retrovirus-mediated gene misexpression in the embryonic chick retina, we show that where progenitor cells are exposed to Dl1 signalling, they are prevented from embarking on neuronal differentiation. A converse effect is seen in cells expressing a dominant-negative form of Dl1, Dl1(dn), which we show renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1(dn), essentially all progenitors stop dividing and differentiate prematurely as neurons, which can be of diverse types. Thus, Delta-Notch signalling controls a cell's choice between remaining as a progenitor and differentiating as a neuron. CONCLUSIONS: Nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. We propose that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues.

Point mutations in the Drosophila hairy gene demonstrate in vivo requirements for basic, helix-loop-helix, and WRPW domains.

The Drosophila pair-rule gene, hairy (h), encodes a nuclear basic helix-loop-helix (bHLH) protein that regulates embryonic segmentation and adult bristle patterning. In both cases, the h protein behaves as a transcriptional repressor. In this study, we determined the molecular nature of 12 h alleles. One mutation maps within the HLH domain, consistent with h function requiring homodimerization or heterodimerization with other HLH proteins. A second mutation lies in the basic domain, suggesting that DNA binding is required for h activity. Several mutations show that the h C terminus, in particular the WRPW domain, is also required for h activity, perhaps by interacting with other proteins to mediate transcriptional repression. We show that the h protein in Drosophila virilis closely resembles that in D. melanogaster and includes completely conserved bHLH and WRPW domains.

A chimeric enhancer-of-split transcriptional activator drives neural development and achaete-scute expression.

Drosophila melanogaster neurogenesis requires the opposing activities of two sets of basic helix-loop-helix (bHLH) proteins: proneural proteins, which confer on cells the ability to become neural precursors, and the Enhancer-of-split [E(spl)] proteins, which restrict such potential as part of the lateral inhibition process. Here, we test if E(spl) proteins function as promoter-bound repressors by examining the effects on neurogenesis of an E(spl) derivative containing a heterologous transcriptional activation domain [E(spl) m7Act (m7Act)]. In contrast to the wild-type E(spl) proteins, m7Act efficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Act activity, suggesting that its effects are mediated through direct DNA binding. m7Act causes ectopic transcription of the proneural achaete and scute genes. Our results support a model in which E(spl) proteins normally regulate neurogenesis by direct repression of genes at the top of the neural determination pathway.

A conserved motif in goosecoid mediates groucho-dependent repression in Drosophila embryos.

Surprisingly small peptide motifs can confer critical biological functions. One example is the WRPW tetrapeptide present in the Hairy family of transcriptional repressors, which mediates recruitment of the Groucho (Gro) corepressor to target promoters. We recently showed that Engrailed (En) is another repressor that requires association with Gro for its function. En lacks a WRPW motif; instead, it contains another short conserved sequence, the En homology region 1 (eh1)/GEH motif, that is likely to play a role in tethering Gro to the promoter. Here, we characterize a repressor domain from the Goosecoid (Gsc) developmental regulator that includes an eh1/GEH-like motif. We demonstrate that this domain (GscR) mediates efficient repression in Drosophila blastoderm embryos and that repression by GscR requires Gro function. GscR and Gro interact in vitro, and the eh1/GEH motif is necessary and sufficient for the interaction and for in vivo repression. Because WRPW- and eh1/GEH-like motifs are present in different proteins and in many organisms, the results suggest that interactions between short peptides and Gro represent a widespread mechanism of repression. Finally, we investigate whether Gro is part of a stable multiprotein complex in the nucleus. Our results indicate that Gro does not form stable associations with other proteins but that it may be able to assemble into homomultimeric complexes.

Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.

We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos. Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site. This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction. Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation. In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes. We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S. cerevisiae.

Genetic Characterization of the 87c Region of the Third Chromosome of DROSOPHILA MELANOGASTER.

Ethyl methanesulphonate (EMS) was used to induce 39 lethal and 13 karmoisin mutations within Df(3R)kar(3J), a nine-band deficiency extending from 87C1 to 87C9 (inclusive). Five complementation groups (four lethal and one visible) were identified and cytologically mapped between 87C4-5 and 87C9, one complementation group per band, with the exception of complementation group A, which is localized to 87C4-5. These positions were determined using a set of overlapping deficiencies, each having at least one breakpoint in the 87C1-9 region. Mutations within a single complementation group have similar lethal phases or subvital phenotypes, consistent with the notion that each complementation group represents a single functional locus. No mutations localized to 87C1-C3. The inability to induce mutations in the 87C1 heat-shock puff locus is consistent with the current interpretation of a duplication of coding sequences at the 87A7 and 87C1 heat-shock puffs.

Genetic characterization of the region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster.

The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.

Genetic Analysis of the Hairy Locus in DROSOPHILA MELANOGASTER.

Mutations of the hairy locus in Drosophila may affect both adult chaeta differentiation and embryonic segmentation. In an effort to understand this phenotypic complexity, we have analyzed 30 mutant alleles of the locus. We find that the alleles fall into four groups according to their complementation properties, suggesting a structurally complex locus in which two distinct functions share a common coding region.

Cloning, mapping and expression of UBL3, a novel ubiquitin-like gene.

A novel Drosophila melanogaster gene UBL3 was characterized and shown to be highly conserved in man and Caenorhabditis elegans (C. elegans). The human and mouse homologues were cloned and sequenced. UBL3 is a ubiquitin-like protein of unknown function with no conserved homologues in yeast. Mapping of the human and mouse UBL3 genes places them within a region of shared gene order between human and mouse chromosomes on human chromosome 13q12-13 and telomeric mouse chromosome 5 (MMU5).

Apical localization of pair-rule transcripts requires 3' sequences and limits protein diffusion in the Drosophila blastoderm embryo.

The peripheral cytoplasm (periplasm) of the Drosophila blastoderm embryo is subdivided into apical and basal compartments by a layer of nuclei. We have demonstrated three classes of periplasmic transcript localization: apical, basal, and unlocalized (apical and basal), each of which depends on 3' sequences. We define 3' apical localization signals within the even-skipped, fushi tarazu, and hairy pair-rule segmentation genes and the alpha 1-tubulin and bicoid genes. 3' human alpha-globin sequences direct transcripts basally. Transcript destination depends on transcript structure, not on transcript stability or chromosomal location. Apical transcripts direct apical compartmentalization of cytoplasmic protein. We propose that apical localization of pair-rule transcripts restricts lateral protein diffusion, thereby allowing pair-rule proteins to define sharp boundaries and precise spatial domains.

wimp, a dominant maternal-effect mutation, reduces transcription of a specific subset of segmentation genes in Drosophila.

wimp is a dominant maternal-effect mutation that interacts with a specific subset of early-acting maternal and zygotic Drosophila genes. We show that wimp is a change-of-function mutation, allelic to mutations of the 140-kD subunit of RNA polymerase, which causes reduced transcription of interacting genes. Loci that do not interact with wimp are expressed at normal levels. We discuss these results in terms of specific interactions between transcription factors and RNA polymerase. Embryos from wimp mothers show unaltered fate maps and develop normally, despite the reduction of transcript levels at least twofold. We suggest that spatial cues are determined by a balance of segmentation gene products rather than their absolute concentrations. We also demonstrate powerful genetic screens for otherwise undetected loci required for segmentation, sex determination, and other early functions.

Individual stripe regulatory elements in the Drosophila hairy promoter respond to maternal, gap, and pair-rule genes.

Striped expression of the pair-rule gene hairy (h) plays a central role in regulating segmentation in Drosophila. We have used h-lacZ reporter gene fusions to delineate h sequences that drive individual stripe expression. We show that 14 kb of 5'-flanking DNA directs expression of seven lacZ stripes in the blastoderm embryo. Within this region, we identify discrete sequences required for expression of individual stripes 1, 5, 6, and 7, and dispersed elements active in the stripe 2 domain. Only the stripe 1 element directs lacZ expression in an accurate h stripe; stripes 5, 6, and 7 are displaced by one to two cells relative to their h counterparts. These results indicate that regulatory sequences are dispersed within the h promoter. We have determined the sensitivity of the lacZ stripes to maternal, gap, and pair-rule gene mutations. Our results suggest that different but overlapping subsets of gap genes regulate each stripe and that activation and repression are both important in generating the stripe pattern.

Mis-regulating segmentation gene expression in Drosophila.

We have used the hunchback (hb) gap-gene promoter to drive ectopic expression of the pair-rule genes fushi tarazu (ftz), even-skipped (eve) and hairy (h). Unexpectedly, flies transformed with such constructs are viable, despite spatial and temporal mis-regulation of pair-rule expression caused by the fusion genes. We show that fusion gene expression is transcriptionally regulated, such that ectopic expression is suppressed when pattern is established, and present evidence indicating that interstripe hb-ftz expression is repressed by eve. These results are considered in terms of redundant control of pair-rule gene striping. We also discuss the potential dangers of using mis-regulated gene expression to analyse normal function.

Pattern abnormalities induced by ectopic expression of the Drosophila gene hairy are associated with repression of ftz transcription.

The pair-rule segmentation gene hairy (h) is expressed and required in alternate metameres in the early Drosophila embryo. We show that h expression outside these domains, driven by an hsp70 promoter, suppresses the expression of fushi tarazu (ftz). The kinetics of action favor h acting directly as a transcriptional repressor. The resulting pattern defects, and the patterns of en and Ubx activity, can be explained if h acts via ftz and other pair-rule genes in the establishment of stable en domains. We present evidence that ftz is required for the initiation of Ubx transcription, but not for its maintenance.

Drosophila hairy pair-rule gene regulates embryonic patterning outside its apparent stripe domains.

The hairy (h) segmentation gene of Drosophila regulates segmental patterning of the early embryo, and is expressed in a set of anteroposterior stripes during the blastoderm stage. We have used a set of h gene deletions to study the h promoter and the developmental requirements for individual h stripes. The results confirm upstream regulation of h striping but indicate that expression in the anterodorsal head domain depends on sequences downstream of the two transcription initiation sites. Surprisingly, the two anterior-most h domains appear to be dispensable for head development and embryonic viability. One partial promoter deletion expresses ectopic h, leading to misexpression of other segmentation genes and embryonic pattern defects. We demonstrate that h affects patterning outside its apparent stripe domains, supporting a model in which primary pair-rule genes act as concentration-dependent transcriptional regulators, i.e. as local morphogens.