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The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development. To address these questions, we measured RNA synthesis and decay rates genome wide via metabolic labeling of whole cultured Drosophilalarval brains using 4-thiouridine. Our analysis revealed that decay rates span a range of more than 100-fold, and that RNA stability is linked to gene function, with mRNAs encoding transcription factors being much less stable than mRNAs involved in core metabolic functions. Surprisingly, among transcription factor mRNAs there was a clear demarcation between more widely used transcription factors and those that are expressed only transiently during development. mRNAs encoding transient transcription factors are among the least stable in the brain. These mRNAs are characterized by epigenetic silencing in most cell types, as shown by their enrichment with the histone modification H3K27me3. Our data suggest the presence of an mRNA destabilizing mechanism targeted to these transiently expressed transcription factors to allow their levels to be regulated rapidly with high precision. Our study also demonstrates a general method for measuring mRNA transcription and decay rates in intact organs or tissues, offering insights into the role of mRNA stability in the regulation of complex developmental programs.

One of the many advantages of Drosophila melanogasteras a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.5–1 kb, thereby rapidly identifying deficiencies that affect only the gene of interest. In addition, we used polymorphism profiling to map existing deficiencies. The method can also be applied to map the extent of deletions generated by x-rays and to identify targeted mutations generated by engineered zinc-finger nucleases in Drosophilaand other polymorphic model organisms (e.g., zebrafish, mouse, Caenorhabditis elegans).

Objective. To investigate the effects of practice on performance and retention of a balance task in persons with Parkinson disease (PD).Methods. Ten persons with PD and 10 age and gender-matched healthy control subjects were tested on an anticipatory, static base of support, limits of stability (LOS) balance task on a force plate. The motor learning paradigm utilized for all subjects included an acquisition phase and retention tests at 24 h and 1 week after acquisition. A force plate was used for testing and to collect outcome measures including movement velocity (MVL), endpoint excursion (EPE), and directional control. Data were analyzed for differences between groups and change over time.Results.Persons with PD demonstrated performance deficits relative to controls for MVL at all testing periods (P< 0.05), and initially for EPE (P< 0.05), but were able to maintain significant improvements through retention testing relative to baseline (P< 0.05).Conclusions. Persons with PD demonstrated unimpaired capacity for motor learning in a LOS balance task for MVL and EPE, although performance deficits remained for MVL. The results concur with previous motor learning research of upper extremity tasks by suggesting that individuals with mild to moderate PD exhibit a preserved ability to benefit from practice as a means of improving balance task performance.

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.

We present a technique for single-beam measurement of the optical nonlinearity in GaAs for photon energies above the bandgap. We measured the real and the imaginary parts of the nonlinear refractive index of a bulk crystal by using the change in reflection of dye laser pulses (10 ns, 538 nm). The values obtained, n_2 = (7.8 ± 0.6) × 10^-8 cm^2/W and κ_2 = (-2.8 ± 0.7) × 10^-8 cm^2/W, are discussed.

We measured the refractive-index change on a liquid sample, using the reflection of a polarized Gaussian laser beam close to the angle of total reflection. We applied this technique to a solution of nickel (ii) phthalocyanine tetrasulfonated (NiPTS) in water–ethanol (1/1 v/v), in which the nonlinearity of the refractive index is due to optically induced thermal effects. We show that close to the angle of total reflection the sensitivity of this technique is four times bigger than at normal incidence.

Somitic segmentation provides the framework on which the segmental pattern of the vertebrae, some muscles and the peripheral nervous system is established. Recent evidence indicates that a molecular oscillator, the 'segmentation clock', operates in the presomitic mesoderm (PSM) to direct periodic expression of c-hairy1 and lunatic fringe (l-fng). Here, we report the identification and characterisation of a second avian hairy-related gene, c-hairy2, which also cycles in the PSM and whose sequence is closely related to the mammalian HES1 gene, a downstream target of Notch signalling in vertebrates. We show that HES1 mRNA is also expressed in a cyclic fashion in the mouse PSM, similar to that observed for c-hairy1 and c-hairy2 in the chick. In HES1 mutant mouse embryos, the periodic expression of l-fng is maintained, suggesting that HES1 is not a critical component of the oscillator mechanism. In contrast, dynamic HES1 expression is lost in mice mutant for Delta1, which are defective for Notch signalling. These results suggest that Notch signalling is required for hairy-like genes cyclic expression in the PSM.

Signals derived from antigen-presenting cells (APC) influence the functional differentiation of CD4⁺ T cells. We report here that Serrate1 (Jagged1), a ligand for the Notch1 receptor, may contribute to the differentiation of peripheral CD4⁺ T cells into either helper or regulatory cells. Our findings demonstrate that antigen presented by murine APC overexpressing human Serrate1 induces naive peripheral CD4⁺ T cells to become regulatory cells. These cells can inhibit primary and secondary immune responses, and transfer antigen-specific tolerance to recipient mice. Our results show that Notch signalling may help explain `linked' suppression in peripheral tolerance, whereby tolerance induced to one epitope encompasses all epitopes on that antigen during the course of an immune response.

Signals derived from antigen-presenting cells (APC) influence the functional differentiation of CD4(+) T cells. We report here that Serrate1 (Jagged1), a ligand for the Notch1 receptor, may contribute to the differentiation of peripheral CD4(+) T cells into either helper or regulatory cells. Our findings demonstrate that antigen presented by murine APC overexpressing human Serrate1 induces naive peripheral CD4(+) T cells to become regulatory cells. These cells can inhibit primary and secondary immune responses, and transfer antigen-specific tolerance to recipient mice. Our results show that Notch signalling may help explain 'linked' suppression in peripheral tolerance, whereby tolerance induced to one epitope encompasses all epitopes on that antigen during the course of an immune response.

ABSTRACTSurprisingly 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 Drosophilablastoderm embryos and that repression by GscRrequires Gro function. GscRand 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.

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 Drosophilablastoderm embryos and that repression by GscRrequires Gro function. GscRand 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.

Mutations of the segmentation gene Krüppel (Kr)cause deletions of contiguous sets of body segments from the middle region of the Drosophilaembryo. We have monitored expression in situof three other genes implicated in the establishment of the body plan, namely hairy (h), fushi tarazu (ftz)and engrailed (en), in mutant Krembryos. Our results show that the pattern of expression of all three genes depends upon Kr+activity and are consistent with a hierarchical model of segmentation gene activity. In addition, we find that the initial expression of the homoeotic selector gene Ultrabithorax(Ubx)follows a novel pattern in Kr‐embroys indicating a close integration of the spatial control of homoeotic and segmantation gene expression.

Drosophila pair‐rule transcripts accumulate exclusively apical of the layer of peripheral nuclei in syncytial blastoderm stage embryos. Here, we use aneuploid embryos to test zygotic gene requirements for pair‐rule transcript localization. As apical localization is maintained in all genotypes tested, the required components must be maternally encoded. In aneuploid embryos with multiple layers or cortical nuclei, pair‐rule transcripts lie apical of both superficial and internalized nuclei. In the latter case, the transcripts are ‘pseudo‐apical’, i.e. apical of the nuclei from which they derive, but basal of superficial nuclei. We show that internalized nuclei maintain their apico‐basal nuclear orientation, and that they lack the apical cytoskeletal assemblies which lie adjacent to superficial nuclei. These results support a mechanism of localizing pair‐rule transcripts by directional (vectorial) nuclear export.

The Hairy and Runt pair‐rule proteins regulate Drosophila segmentation by repressing transcription. To explore the ability of these proteins to function as promoter‐bound regulators in vivo, we examined the effects of Hairy and Runt derivatives containing heterologous transcriptional activation domains (HairyAct and RunAct). Using this approach, we find that Hairy and Runt efficiently target such activation domains to specific segmentation gene promoters, leading to rapid induction of transcription. Our results strongly suggest that Hairy normally acts as a promoter‐bound repressor of fushi tarazu, runt and odd‐skipped, and that Runt directly represses even‐skipped. We also show that expressing HairyAct in early blastoderm embryos causes ectopic Sex‐lethal expression and male‐specific lethality, implying that the Hairy‐related denominator element Deadpan represses Sex‐lethal during sex determination by directly recognizing the early Sex‐lethal promoter.

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 introduction of copia‐based vectors in Drosophila hydeicells 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 copiaessential for expression. We find that the essential sequences reside within the long terminal repeat, but 3′ to the site of initiation of copiaRNA. Deletion of the consensus enhancer‐like sequences from copiaappears to have no effect on vector expression.

We have developed an assay for transient gene expression using a dominant‐selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid‐precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene. This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine. Deletion analysis confirms that the LTRs are essential for gpt gene expression. Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol. wt. heat‐shock (hsp 70) promoter show regulated guanine incorporation when heat shocked. The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not. The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells.

In theDrosophilanervous system, lateral inhibition regulates commitment to a neural fate by preventing neighbouring cells from developing alike. This signalling process is mediated by two transmembrane proteins—Notch as receptor and Delta as its ligand. The Delta-related protein Serrate also acts as a Notch ligand inDrosophila,but in a different developmental process that organises patterning of the wing. We have previously shown that lateral inhibition operates at early stages of neurogenesis in vertebrates, via genes homologous toDrosophila DeltaandNotch.We report here the cloning of a chickSerratehomologue,C-Serrate-1.This gene is expressed in the central nervous system, as well as in the cranial placodes, nephric epithelium, vascular system, and distal limb-bud mesenchyme. In most of these sites, its expression is associated with expression ofC-Notch-1andC-Delta-1.All three genes are expressed in the ventricular zone of the hindbrain and spinal cord, throughout the period when neurons are being born. Within this zone,C-Delta-1andC-Serrate-1are expressed in complementary subsets of nondividing cells that appear to be nascent neurons:C-Serrate-1expression is restricted to specific locations along the dorsoventral axis, forming narrow bands extending from the anterior hindbrain to the tail. Our observations strongly suggest that Delta–Notch signalling delivers lateral inhibition not only early but throughout vertebrate neurogenesis to regulate neuronal commitment, and that Serrate–Notch signalling may act similarly in this process. By analogy with its role inDrosophilawing patterning,C-Serrate-1may also have a role in organising the dorso-ventral pattern of the neural tube. We argue that signalling via Notch maintains neurogenesis, both in vertebrates and in flies, by keeping a proportion of the neuroepithelial cells in an uncommitted stem-cell-like state.

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, Dl1dn, which we show renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1dn, 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.

1. The single gap voltage clamp technique (Kovács & Schneider, 1978) was used to monitor membrane charge movement in tendon‐terminated short segments of cut frog skeletal muscle fibres.

1. The current IQdue to membrane charge movement and the threshold pulse duration tthrequired to produce microscopically just‐detectable contraction were determined for pulses to a variety of membrane potentials in tendon‐terminated short segments of cut frog skeletal muscle fibres voltage‐clamped using a single gap technique.