Search

Relatively little is known about the molecular mechanisms involved in transcriptional repression, despite its importance in development and differentiation. Recent evidence suggests that some transcriptional repressors act by way of adaptor molecules known as corepressors. Here, we use in vivo functional assays to test whether different repressor activities are mediated by the Groucho (Gro) corepressor in the Drosophila embryo. Previously, Gro was proposed to mediate repression by the Hairy-related family of basic helix-loop-helix proteins. Our results indicate not only that repression by Hairy requires Gro, but that a repressor domain from the Engrailed (En) homeodomain protein is also Gro dependent. The latter result correlates with an ability of this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Krüppel, and Knirps transcription factors are effective in the absence of Gro. These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En.

Drosophila melanogasterneurogenesis 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, m7Actefficiently induces neural development, indicating that it binds to and activates target genes normally repressed by E(spl). Mutations in the basic domain disrupt m7Actactivity, suggesting that its effects are mediated through direct DNA binding. m7Actcauses ectopic transcription of the proneural achaeteand scutegenes. 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.

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.

We have used a polyclonal antiserum specific for the Drosophila segmentation gene, hairy (h), to analyse its expression during embryogenesis. The pattern of wild-type expression resembles that of h transcription, being expressed in stripes at the blastoderm stage. h is also expressed later in the stomodaeum, proctodaeum, tracheal pits and mesoderm. We demonstrate that h protein stripes show consistent phase relationships to those of the even-skipped (eve) pair-rule gene. We examine h protein patterns in embryos mutant for other segmentation genes, including h itself. We show that lack of h activity appears not to affect h striping, arguing that h expression is not under autoregulatory control. We also show that h activity is not needed for tracheal invagination. Mutations that are rearranged upstream of the h gene cause the loss of specific stripes, indicating that the h promoter includes activating elements that respond to specific spatial cues. Our observations suggest that pair-rule striping may be under redundant control, and we discuss possible implications for hierarchical models of pair-rule gene action.

Patterning of the non-segmental termini of the Drosophila embryo depends on signalling via the Torso receptor tyrosine kinase (RTK). Activation of Torso at the poles of the embryo triggers restricted expression of the zygotic gap genes tailless (tll) and huckebein (hkb). In this paper, we show that the Groucho (Gro) corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; the former leads, in turn, to lack of abdominal expression of the Krüppel and knirps gap genes. We show that torso signalling permits terminal gap gene expression by antagonising Gro-mediated repression. Thus, the corepressor Gro is employed in diverse developmental contexts and, probably, by a variety of DNA-binding repressors.

Chick embryonic feather buds arise in a distinct spatial and temporal pattern. Although many genes are implicated in the growth and differentiation of the feather buds, little is known about how the discrete pattern of the feather array is formed and which gene products may be involved. Possible candidates include Notch and its ligands, Delta and Serrate, as they play a role in numerous cell fate decisions in many organisms. Here we show that Notch-1 and Notch-2 mRNAs are expressed in the skin in a localized pattern prior to feather bud initiation. In the early stages of feather bud development, Delta-1 and Notch-1 are localized to the forming buds while Notch-2 expression is excluded from the bud. Thus, Notch and Delta-1 are expressed at the correct time and place to be players in the formation of the feather pattern. Once the initial buds form, expression of Notch and its ligands is observed within each bud. Notch-1 and -2 and Serrate-1 and -2 are expressed throughout the growth and differentiation of the feathers whereas Delta-1 transcripts are downregulated. We have also misexpressed chick Delta-1 using a replication competent retrovirus. This results in induction of Notch-1 and-2 and a loss of feather buds from the embryo in either large or small patches. In large regions of Delta-1 misexpression, feathers are lost throughout the infected area. In contrast, in small regions of misexpression, Delta-1 expressing cells differentiate into feather buds more quickly than normal and inhibit their neighbors from accepting a feather fate. We propose a dual role for Delta-1 in promoting feather bud development and in lateral inhibition. These results implicate the Notch/Delta receptor ligand pair in the formation of the feather array.

Little is known about the tissue interactions and the molecular signals implicated in the sequence of events leading to the subdivision of the somite into its rostral and caudal compartments. It has been demonstrated that rostrocaudal identity of the sclerotome is acquired at the presomitic (PSM) level. However, it is not known whether this compartment specification is fully determined in the PSM or whether it is dependent upon maintenance cues from the surrounding environment, as is the case for somite epithelialization. In this report, we address this issue by examining the expression profiles of C-Delta-1 and C-Notch-1, the avian homologues of mouse Delta-like1 (Delta1) and Notch1 which have been implicated in the specification of the somite rostrocaudal polarity in mouse. In chick, these genes are expressed in distinct but partially overlapping domains in the PSM and subsequently in the caudal regions of the somites. We have used an in vitro assay that consists of culturing PSM explants to examine the regulation of these genes in this tissue. We find that PSM explants cultured without overlying ectoderm continue to lay down stripes of C-Delta-1 expression, although epithelialization is blocked. These results suggest that somite rostrocaudal patterning is an autonomous property of the PSM. In addition, they demonstrate that segmentation is not necessarily coupled with the formation of somites. Dev. Genet. 23:77–85, 1998. © 1998 Wiley-Liss, Inc.

Circular copies of the transposable element copia have been isolated from cultured Drosophila cells. These molecules, which are present at approximately 1 copy per 10–50 cells, comprise a heterogeneous family of related species. Most are composed of a complete copy of the internal section of copia combined with either one or two copies of the flanking direct repeat sequences. Such structures are strikingly analogous to the circular proviral forms of retroviruses.

Several species related to Drosophila melanogaster have two loci containing sequences which code for the major heat-shock protein of molecular weight 70,000 (hsp70). These sequences are arranged as two pairs of inverted repeats. The structure of the 87C locus in D. melanogaster seems to have arisen through DNA insertion. Hsp70-coding sequences at the two loci are not evolving independently. Gene conversion between hsp70 genes has apparently occurred both within and between loci.

To gain experience in the in vitro assay of environmental teratogens, 32 pesticides and TCDD have been tested for their ability to Inhibit tumor cells attachment to polyethylene disks coated with concanavalin A. Of 25 pesticides that inhibited attachment, 23 (92%) have been reported to be teratogenic in mammals or avians. From a qualitative viewpoint, agents that inhibit attachment in vitro are very likely to be teratogenic in vivo. There was a quantitative correlation between the inhibitory activity and the lowest reported teratogenic dose in chick embryo for nine pesticides. However, no similar correlation between inhibitory concentration, in vitro, and mammalian teratogenicity was found. Presumably maternal effects of absorption, metabolism, and distribution distort the relationship between the dose administered to the mother and the concentration of teratogenic agent at target embryonic tissues. Lack of inhibition in vitro did not indicate lack of teratogenicity, as five of eight noninhibitory agents have been reported to be teratogenic in vivo. Such false negatives of the attachment assay may be detectable in complementary in vitro tests.

In vertebrate embryos, the precursor cells of the central nervous system (CNS) are induced by signaling from the organizer region. Here we report the isolation of a novel vertebrate achaete-scute homolog, cash4, which is expressed in the presumptive posterior nervous system in response to such signaling. cash4 is first expressed in epiblast cells flanking the late-phase organizer (Hensen's node), which retains its ability to induce cash4 during regression to the caudal end of the embryo. We show that these node-derived signals can be mimicked in vivo by the activity of fibroblast growth factor (FGF). We demonstrate that cash4 can substitute for the achaete/scute genes in the fly and that it also has proneural activity in vertebrate embryos. Together these results suggest that cash4 functions as a proneural gene downstream of node-derived signals (including FGF) to promote the formation of the neural precursors that will give rise to the posterior CNS in the chick embryo.

Short muscle fibers (1.5 mm) were dissected from hindlimb muscles of frogs and voltage clamped with two microelectrodes to study phenomena related to depolarization-contraction coupling. Isometric myograms obtained in response to depolarizing pulses of durations between 10 and 500 ms and amplitudes up to 140 mV had the following properties. For suprathreshold pulses of fixed duration (in the range of 20-100 ms), the peak tension achieved, the time to peak tension, and contraction duration increased as the internal potential was made progressively more positive. Peak tension eventually saturates with increasing internal potentials. For pulse durations of greater than or equal to 50 ms, the rate of tension development becomes constant for increasing internal potentials when peak tensions become greater than one-third of the maximum tension possible. Both threshold and maximum steepness of the relation between internal potential and peak tension depend on pulse duration. The relation between the tension-time integral and the stimulus amplitude-duration product was examined. The utility of this relation for excitation-contraction studies is based on the observation that once a depolarizing pulse configuration has elicited maximum tension, further increases in either stimulus duration or amplitude only prolong the contractile response, while the major portion of the relaxation phase after the end of a pulse is exponential, with a time constant that is not significantly affected by either the amplitude or the duration of the pulse. Hence, the area under the tension-response curve provides a measure of the availability to troponin of the calcium released from the sarcoplasmic reticulum in response to membrane depolarization. The results from this work complement those obtained in experiments in which intramembrane charge movements related to contractile activation were studied and those in which intracellular Ca++ transients were measured.

Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the effects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in surface membrane is about 175/muM2 and that in the transverse tubular membrane is 41-52/mum2.

The action of theophylline on both sodium efflux and influx was measured using freshly isolated frog sartorius muscles. In normal Ringer's fluid, 2 mM theophylline increased sodium efflux by 35% whereas it decreased sodium influx by about 10%. The percent increase in sodium efflux produced by 2 mM theophylline was not significantly altered in sodium-free, lithium-containing solutions. Strophanthidin prevented the stimulation of sodium efflux by 2 mM theophylline in both normal, sodium-containing Ringer's fluid and sodium-free, lithium-containing solutions. Hence, the major effect of theophylline seems to be stimulation of active sodium transport and the enhanced rate of sodium exit induced by theophylline does not seem to require the presence of external sodium. An interesting and unexplained findings is that 2 mM theophylline, which does not produce a maximal stimulation of sodium efflux, prevents the increased sodium efflux induced by saturating doses of epinephrine.

Segmentation of the Drosophilaembryo requires expression of the pair-rule genes, mutations of which cause reiterated deletions in alternate segments along the antero-posterior body axis. We find that transcripts of one such gene, hairy,accumulate in eight distinct regions of the early embryo. This pattern of expression is compared with that of another pair-rule gene, fushi tarazu,and its dependence on maternally expressed genes is described.

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.

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.

Unidirectional Na fluxes in isolated fibers from the frog's semitendinosus muscle were measured in the presence of strophanthidin and increased external potassium ion concentrations. Strophanthidin at a concentration of 10-5 M inhibited about 80 per cent of the resting Na efflux without having any detectable effect on the resting Na influx. From this it is concluded that the major portion of the resting Na efflux is caused by active transport processes. External potassium concentrations from 2.5 to 7.5 mM had little effect on resting Na efflux. Above 7.5 mM and up to 15 mM external K, the Na efflux was markedly stimulated; with 15 mM K the Na influx was 250 to 300 per cent greater than normal. On the other hand, Na influx was unchanged with 15 mM K. The stimulated Na efflux with the higher concentrations was not appreciably reduced when choline or Li was substituted for external Na, but was completely inhibited by 10-5 M strophanthidin. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of K. It is postulated that this effect on the Na "pump" is produced as a result of the depolarization of the muscle membranes and is related to the increased metabolism and heat production found under conditions of high external K.

Unidirectional Na fluxes from frog's striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10-5 M strophanthidin abolished the azide effect on Na24 efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN3. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.

The influence of strophanthidin, ouabain, and the removal of external sodium on the sodium efflux from frog sartorius muscle was measured. In freshly dissected muscles strophanthidin and ouabain in maximally effective concentrations reduced the efflux of sodium by about 50%. Of the sodium efflux which is strophanthidin-insensitive about 75% is inhibited after complete replacement of external sodium by lithium. In the absence of strophanthidin replacement of external sodium by lithium, calcium, or magnesium produces an initial rise in the sodium efflux, followed by a fall in the efflux as the exposure of the muscles to sodium-free media is continued. When the muscles are exposed for prolonged periods in sodium-free media, the fraction of internal sodium lost per minute is higher when returned to normal Ringer fluid than it was initially. The activation of sodium efflux by external sodium after long periods in sodium-free solutions is partly strophanthidin-sensitive and partly strophanthidin-insensitive. The internal sodium concentration is an important factor in these effects. The effects of temperature on the sodium efflux were also measured. Above 7°C the Q10 of both the strophanthidin-sensitive and strophanthidin-insensitive sodium efflux is about 2.0. Below 7°C the strophanthidin-insensitive sodium efflux has a Q10 of about 7.4.

We demonstrate enhanced loading of a vapor-cell magneto-optical trap for sodium atoms operating on a combination of atomic transitions, known in the literature as type I tuning, by using a second, nonoverlapping laser beam that captures atoms through a different combination of transitions (type II tuning). This two-color trap configuration has allowed us to achieve an ~10-fold improvement in the number and density of trapped atoms.

The functional analysis of cloned Drosophila genes in vivo has so far not been possible because of the lack of an appropriate cultured cell transformation system. We have developed a transformation method which uses the Escherichia coli gene coding for xanthine guanine phosphoribosyl transferase (gpt)1, inserted into a cloned copia2transposable element, and a medium selective for transformed cells expressing the gpt gene. We show that the resulting plasmids (pCVgpt) propagate extrachromosomally in transformed cells, apparently without integrating into the genome. This suggests that the extrachromosomal copia circles found in Drosophila tissue-culture cells may also replicate semi-conservatively.

AimsTo explore why and how genital-normalising surgery is considered as legitimate medical treatment. To explore the moral and legal issues that influence genital-normalising surgery in infancy to develop an alternative approach.MethodologyDoctrinal and socio-legal methodology, with analysis of bioethical argument.ResultsA symbiotic relationship was established between medicine and law that has directly influenced the consideration of intersex as Disorders of Sex Development (DSD) that at its most controversial justifies genital-normalisation surgery. The central focus is biological diagnosis that requires intervention in order to conform to the binary sex and gender expectations in society. Decision-making for intersex children facing genital-normalising surgery was addressed, in recognition of the concepts of parental and the child’s future autonomy. I argue that the current approach to decision-making for genital-normalising surgery is flawed and should be regarded as unlawful when determining a child’s best interests. To address this I propose a shared decisionmaking approach that involves the child, parents and a multi-disciplinary healthcare team to promote a holistic approach. I finally argue that the human rights of intersex children are being breached in genital-normalising surgery.ConclusionsGenital-normalising surgery has the potential for catastrophic harm to the intersex child’s physical and psychological welfare. Fundamental to my concerns is detriment to the physical integrity of the intersex child, where that child is not able to consent to an irreversible intervention. The legal preference for certainty of binary biological sex is symbiotic to the view of medicine that intersex is an abnormality requiring correction. This symbiotic relationship promotes an approach to the child’s best interests that is based on parental welfare and fails to consider the child’s future autonomous interests. Furthermore, the breach of the child’s rights in infant genital-normalising surgery requires that the law has an implicit obligation to safeguard these rights. I propose that a shared decision-making approach be legally and professionallyrequired to allow the child to make the decision to undergo or refuse genital-normalising surgery, with support from parents and healthcare professionals through a process based on education, information and protection of the child’s physical integrity.

Meeting Wallace Fenn for the first time could sometimes be an unsettling experience; he had little in the way of small talk and his reserve was formidable. Yet, after a few encounters one began to realize that here was a distinguished scientist, an inherently shy man but one who was strongly in favor of people.

We have introduced a Drosophila 87A heat shock locus into the genome of Rat-1 cells. A brief heat shock of a resultant transformed line, Rhsll, induces the transcription of the Drosophila heat shock genes from the Drosophila heat shock promoter. This suggests that the induction mechanism and regulatory sequences for the heat shock response have been conserved between Drosophila and mammals.

We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJB8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosphorylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1μg of target Drosophila DNA gives about 5×105 clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.