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PubMed2001

BSF binds specifically to the bicoid mRNA 3' untranslated region and contributes to stabilization of bicoid mRNA.

The early stages of Drosophila melanogaster development rely extensively on posttranscriptional forms of gene regulation. Deployment of the anterior body patterning morphogen, the Bicoid protein, requires both localization and translational regulation of the maternal bicoid mRNA. Here we provide evidence that the bicoid mRNA is also selectively stabilized during oogenesis. We identify and isolate a protein, BSF, that binds specifically to IV/V RNA, a minimal form of the bicoid mRNA 3' untranslated region that supports a normal program of mRNA localization during oogenesis. Mutations that disrupt the BSF binding site in IV/V RNA or substantially reduce the level of BSF protein lead to reduction in IV/V RNA levels, indicating a role for BSF in RNA stabilization. The BSF protein is novel and lacks all of the characterized RNA binding motifs. However, BSF does include multiple copies of the PPR motif, whose function is unknown but appears in other proteins with roles in RNA metabolism.

Amino Acid Sequence↗
PubMed1999

Cytoplasmic polyadenylation in development and beyond.

Maternal mRNA translation is regulated in large part by cytoplasmic polyadenylation. This process, which occurs in both vertebrates and invertebrates, is essential for meiosis and body patterning. In spite of the evolutionary conservation of cytoplasmic polyadenylation, many of the cis elements and trans-acting factors appear to have some species specificity. With the recent isolation and cloning of factors involved in both poly(A) elongation and deadenylation, the underlying biochemistry of these reactions is beginning to be elucidated. In addition to early development, cytoplasmic polyadenylation is now known to occur in the adult brain, and there is circumstantial evidence that this process occurs at synapses, where it could mediate the long-lasting phase of long-term potentiation, which is probably the basis of learning and memory. Finally, there may be multiple mechanisms by which polyadenylation promotes translation. Important questions yet to be answered in the field of cytoplasmic polyadenylation are addressed.

Animals↗
PubMed2005

Subdural haematoma and effusion in infancy: an epidemiological study.

AIM: To determine incidence, aetiology, and clinical features of subdural haematoma and effusion (SDH/E) in infancy throughout the British Isles. METHODS: Cases were notified to the British Paediatric Surveillance Unit over 12 months by paediatricians, neurosurgeons, and paediatric and forensic pathologists. RESULTS: A total of 186 infants (121 boys, 65 girls) aged 0-2 years were identified. Annual incidence of SDH/E for the UK and Republic of Ireland is 12.54/100,000 aged 0-2 (95% CI 10.3 to 14.62) and 24.1/100,000 aged 0-1 (95% CI 20.89 to 28.18). A total of 106 infants suffered non-accidental head injury (NAHI), 7 accidental head injury, 26 a perinatal cause, 7 a non-traumatic medical condition, 23 meningitis, and in 17 the cause was undetermined; 35 infants died. Significant differences were found in injury pattern, body weight, and Townsend score between NAHI and SDH/E from other cause. There were fewer diagnostic investigations in non-NAHI cases. Delay in diagnosis of greater than a week occurred in 48/181. CONCLUSION: SDH/E is a significant cause of morbidity and mortality in infancy. NAHI is the predominant cause of SDH/E. SDH/E can present in a non-specific and varied way and must be considered in any infant who is unwell. Determining the cause of the SDH/E in some cases continues to present a diagnostic challenge.

Child Abuse↗
PubMed1993

Relationship between smoking, weight and attitudes to weight in adolescent schoolgirls.

A total of 1,932 schoolgirls aged 11-18 from seven schools in the South London area were surveyed using questionnaires which addressed eating patterns, body weight history, attitudes to body weight and shape, menstrual history and current smoking behaviour. They were also weighed and their height was measured. Twelve per cent of the girls were regular smokers and 10% smoked seven or more cigarettes over a 4 day period. Amongst girls aged 14 and over, 15% smoked regularly and a further 9% occasionally. A significant relationship was found between smoking and weight. Smokers were more likely to be moderately overweight in relation to their peers and to have been worried about their weight at some stage. There were differences between girls in state schools and those in independent schools with regard to smoking behaviour and weight. The findings have implications for anti-smoking strategies and health education generally.

Adolescent↗
PubMed1999

Drink composition, voluntary drinking, and fluid balance in exercising, trained, heat-acclimatized boys.

This study examined the effects of beverage composition on the voluntary drinking pattern, body fluid balance, and thermoregulation of heat-acclimatized trained boys exercising intermittently in outdoor conditions (wet bulb globe temperature 30.4 +/- 1.0 degreesC). Twelve boys (age 13.4 +/- 0.4 yr) performed two 3-h sessions, each consisting of four 20-min cycling bouts at 60% maximal aerobic power alternating with 25-min rest. One of two beverages was assigned: unflavored water (W) or flavored water plus 6% carbohydrate and 18 mmol/l Na (CNa). Drinking was ad libitum. Total intake was higher (P < 0.05) during CNa (1,943 +/- 190 g) compared with W (1,470 +/- 143 g). Euhydration was maintained with CNa (+0.18% body wt), but a mild dehydration resulted with W (-0.94% body wt; P < 0.05). Sweat loss, much higher than previously published for children of similar age, was similar between conditions (CNa = 1,644.7 +/- 117.5; W = 1,750.2 +/- 152.7 g). The increase in rectal temperature (CNa = 0.86 +/- 0.3; W = 0.76 +/- 0.1 degreesC), heart rate, and all perceptual variables did not differ between conditions. In conclusion, a flavored carbohydrate-electrolyte drink prevents voluntary dehydration in trained heat-acclimatized boys exercising in a tropical climate despite their large sweat losses. Because hydration changes were minor, the thermoregulatory strain observed was similar between conditions.

Acclimatization↗
PubMed2003

Conservation of regulatory elements between two species of Drosophila.

BACKGROUND: One of the important goals in the post-genomic era is to determine the regulatory elements within the non-coding DNA of a given organism's genome. The identification of functional cis-regulatory modules has proven difficult since the component factor binding sites are small and the rules governing their arrangement are poorly understood. However, the genomes of suitably diverged species help to predict regulatory elements based on the generally accepted assumption that conserved blocks of genomic sequence are likely to be functional. To judge the efficacy of strategies that prefilter by sequence conservation it is important to know to what extent the converse assumption holds, namely that functional elements common to both species will fall within these conserved blocks. The recently completed sequence of a second Drosophila species provides an opportunity to test this assumption for one of the experimentally best studied regulatory networks in multicellular organisms, the body patterning of the fly embryo. RESULTS: We find that 50%-70% of known binding sites reside in conserved sequence blocks, but these percentages are not greatly enriched over what is expected by chance. Finally, a computational genome-wide search in both species for regulatory modules based on clusters of binding sites suggests that genes central to the regulatory network are consistently recovered. CONCLUSIONS: Our results indicate that binding sites remain clustered for these "core modules" while not necessarily residing in conserved blocks. This is an important clue as to how regulatory information is encoded in the genome and how modules evolve.

Algorithms↗
PubMed2004

Identification of chaperonin CCT gamma subunit as a determinant of retinotectal development by whole-genome subtraction cloning from zebrafish no tectal neuron mutant.

Zebrafish no tectal neuron (ntn) mutant obtained by trimethylpsoralen (TMP) mutagenesis showed defects in tectal neuropil formation and small eyes. We carried out whole-genome subtraction between wild-type and mutant zebrafish embryos using the representational difference analysis (RDA) method. Nineteen subtraction products enabled us to construct genetic and physical maps of the ntn region. Direct selection of cDNAs using a YAC clone encompassing the ntn locus and RT-PCR analysis of transcripts identified a 143 bp deletion in the cct3 gene encoding the gamma subunit of chaperonin containing TCP-1 (CCT). Injection of antisense cct3 morpholino oligonucleotides into zebrafish embryos induced characteristic ntn phenotypes including defects in retinal ganglion cell (RGC) differentiation and tectal neuropil formation. Moreover, injection of cct3 mRNA successfully rescued ntn mutant embryos. Our results suggest that RDA is an efficient and widely applicable cloning strategy in zebrafish genetics. The strong expression of the cct3 mRNA started in the entire embryos by 12 hpf and was sustained thereafter, but there were no detectable abnormalities in body patterning and neurogenesis in ntn mutant embryos at 30 hpf. The expression patterns of transcription factor genes ath5 and brn3b that are essential for the development and maintenance of RGCs were indistinguishable between wild-type and ntn mutant embryos, but those of early and late differentiation markers of RGCs, nicotinic acetylcholine receptor beta 3 and zn5, were diminished in mutant embryos. Immunostaining of acetylated tubulin also revealed the impairment of RGC neurite extension. Thus, the ntn mutation of the cct3 gene impaired the differentiation of retinal neuroepithelial cells to RGCs. Similarly, the expression of brn3b was normal in the tectum of ntn mutants, but tectal neuropil formation was abolished. These results suggest that the gamma subunit of chaperonin CCT plays an essential role in retinotectal development.

Amino Acid Sequence↗
PubMed2004

Temporal complexity within a translational control element in the nanos mRNA.

Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3' untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms.

3' Untranslated Regions↗
PubMed1988

Anterior determinants in embryos of Chironomus samoensis: characterization by rescue bioassay.

Embryos of Chironomus samoensis are programmed, by anterior u.v. irradiation, to form the abnormal body pattern 'double abdomen'. Most double abdomen embryos show a mirror-image duplication of abdominal segments in the absence of cephalic or thoracic segments. Such embryos can be 'rescued', i.e. restored to normal development, by microinjection of cytoplasm or RNA from unirradiated donor embryos. Most of the rescued embryos look completely normal and many of them hatch spontaneously. The rescuing activity decreases from the anterior to the posterior pole in the donor cytoplasm and must be delivered near the anterior pole of the recipient for maximum efficiency. Rescuing activity is present in total RNA extracted from whole, unirradiated embryos. Upon fractionation, the activity is associated with poly(A)+ RNA, with LiCl precipitate depleted of RNA smaller than 250 nucleotides (nt) and with a sucrose gradient fraction depleted of RNA larger than 500 nt. Corresponding fractions of RNA from Xenopus oocytes have no rescuing activity. The activity of Chironomus RNA is sensitive to u.v. irradiation with low fluence affecting less than 2% of the pyrimidine bases. Rescuing activity is present in cytoplasm until the blastoderm stage but disappears earlier from poly(A)+ RNA. Rescuing activity is also present, and localized, in cytoplasm of embryos from two related dipterans, Smittia sp. and Drosophila melanogaster, although the extent of rescue observed in Chironomus decreases with the phylogenetic distance between donor and recipient. The results of these and previous experiments indicate that dipteran embryos contain localized RNP particles acting as anterior determinants. In Chironomus, the activity of these particles seems to depend on the integrity of polyadenylated RNA of about 250 to 500 nt length.

Abdomen↗
PubMed1989

Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor.

We have cultured explants of Xenopus blastular animal cap tissue from embryos that had received an earlier treatment with LiCl and from their untreated siblings, in various concentrations of XTC-cell-derived mesoderm-inducing factor (XTC-MIF, Smith, 1987; Smith et al. 1988). The pretreatment with lithium that we used transforms later morphogenesis in the whole embryo to give radialized body forms with anterior/dorsal levels of structure grossly over-represented. In addition, animal caps from 'Li+' embryos were allowed to develop without exposure to in vitro MIF (Li+ controls) and compared with normal uninduced control explants, and explants were made from normal early blastulae but given various initial treatments with LiCl in culture. The results confirm that the lithium ion itself will not induce mesoderm in competent, animal cap tissue of Xenopus. It does, however, enhance the responsiveness of this tissue to XTC-MIF, in a way that parallels its recently reported effect in the case of another mesoderm inducer of different character, bFGF (Slack et al. 1988). The effects observed are sufficient to imply that the altered body pattern that follows lithium treatment, in whole embryos, could be caused by modulation of the responses to an unaltered pattern of in situ inductive stimuli. We also observe evidence that appreciable inductive signals reach animal pole tissue beyond the limits of mesoderm formation in normal development. Relatively low concentrations of MIF prevent the development of an epidermis-specific marker in dissociated blastular animal cap cells (Symes et al. 1988). When such experiments are repeated in relation to the lithium pretreatment of embryos, such treatment is seen to have sensitized the cell population, so that the MIF concentration range that assures complete suppression of the marker is reduced. The results are discussed in relation to induction considered as pattern formation.

Animals↗
PubMed1990

The biological effects of XTC-MIF: quantitative comparison with Xenopus bFGF.

Mesoderm in Xenopus and other amphibian embryos is induced by signals from the vegetal hemisphere acting on equatorial or animal hemisphere cells. These signals are diffusible and two classes of candidate signal molecule have been identified: the fibroblast growth factor (FGF) and transforming growth factor beta (TGF-beta) types. In this paper, we compare the effects of cloned Xenopus basic FGF (XbFGF) and electophoretically homogeneous XTC-MIF (a TGF-beta-like factor obtained from a Xenopus cell line) on animal pole explants. We find that they have a similar minimum active concentration (0.1-0.2 ng ml-1) but that, nonetheless, XTC-MIF is at least 40 times more active in inducing muscle. In general, we find that the two factors cause inductions of significantly different characters in terms of tissue type, morphology, gene expression and timing. At low concentrations (0.1-1.0 ng ml-1) both factors induce the differentiation of 'mesenchyme' and 'mesothelium' as well as blood-like cells. These latter cells do not, however, react with an antibody to Xenopus globin. This raised the possibility that the identification of red blood cells in other studies on mesoderm induction might have been mistaken, but combinations of animal pole regions with ventral vegetal pole regions confirmed that genuine erythrocytes are formed. The identity of the blood-like cells formed in response to the inducing factors remains unknown. At higher concentrations XTC-MIF induces neural tissue, notochord, pronephros and substantial and often segmented muscle. By contrast, XbFGF only induces significant amounts of muscle above 24 ng ml-1 and even then this is much less than that induced by XTC-MIF. For both factors an exposure of less than 30 min is effective. Competence of animal pole cells to respond to XbFGF is completely lost by the beginning of gastrulation (stage 10) while competence to XTC-MIF is detectable until somewhat later (stage 11). Since animal pole tissue is known to be able to respond to the natural inducer at least until stage 10, and perhaps until stage 10.5, this suggests that bFGF cannot be the sole inducer of mesoderm in vivo. Taken together, these results are consistent with XTC-MIF being a dorsoanterior inducer and XbFGF a ventroposterior inducer, suggesting that body pattern is established by the interaction of two types of inducing signal. This model is discussed in view of the qualitative and quantitative differences between the factors.

Animals↗
PubMed1993

Control of segmental asymmetry in Drosophila embryos.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.

Animals↗
PubMed1995

Mitotic delay dependent survival identifies components of cell cycle control in the Drosophila blastoderm.

The Drosophila body pattern is laid down by maternal and zygotic factors which act during the early phase of embryonic development. During this period, nascent zygotic transcripts longer than about 6 kilobases are aborted between the rapid mitotic cycles. Resurrector1 (Res1) and Godzilla1 (God1), two newly identified dominant zygotic suppressor mutations, and a heterozygous maternal deficiency of the cyclin B locus, complement the partial loss of function of the segmentation gene knirps (kni) by extending the length of mitotic cycles at blastoderm. The mitotic delay caused by Res1 and God1 zygotically and by the deficiency of the cyclin B locus maternally allows the expression of a much longer transcript of a kni cognate gene normally aborted between the short mitotic cycles and consequently allows survival of kni mutant progeny. In addition to the practical benefits of identifying mutations in Drosophila cell cycle regulatory genes as suppressors of kni, our results have evolutionary implications regarding the flexibility of the genome to meet sudden selective pressures by recruiting cognate genes to function.

Animals↗
PubMed1999

Translational regulation of oskar mRNA occurs independent of the cap and poly(A) tail in Drosophila ovarian extracts.

Translational regulation plays a prominent role in Drosophila body patterning. Progress in elucidating the underlying mechanisms has been limited by the lack of a homologous in vitro system that supports regulation. Here we show that extracts prepared from Drosophila tissues are competent for translation. Ovarian extracts, but not embryonic extracts, support the Bruno response element- and Bruno-dependent repression of oskar mRNA translation, which acts in vivo to prevent protein synthesis from transcripts not localized to the posterior pole of the oocyte. Consistent with suggestive evidence from in vivo experiments, regulation in vitro does not involve changes in poly(A) tail length. Moreover, inhibition studies strongly suggest that repression does not interfere with the process of 5' cap recognition. Translational regulation mediated through the Bruno response elements is thus likely to occur via a novel mechanism.

Animals↗
PubMed2002

Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway.

Subcellular localization of mRNAs within the Drosophila oocyte is an essential step in body patterning. Yps, a Drosophila Y-box protein, is a component of an ovarian ribonucleoprotein complex that also contains Exu, a protein that plays an essential role in mRNA localization. Y-box proteins are known translational regulators, suggesting that this complex might regulate translation as well as mRNA localization. Here we examine the role of the yps gene in these events. We show that yps interacts genetically with orb, a positive regulator of oskar mRNA localization and translation. The nature of the genetic interaction indicates that yps acts antagonistically to orb. We demonstrate that Orb protein is physically associated with both the Yps and Exu proteins, and that this interaction is mediated by RNA. We propose a model wherein Yps and Orb bind competitively to oskar mRNA with opposite effects on translation and RNA localization.

Animals↗
PubMed2002

EGFR signalling inhibits Capicua-dependent repression during specification of Drosophila wing veins.

Localised activation of the Ras/Raf pathway by Epidermal Growth Factor Receptor (EGFR) signalling specifies the formation of veins in the Drosophila wing. However, little is known about how the EGFR signal regulates transcriptional responses during the vein/intervein cell fate decision. We provide evidence that EGFR signalling induces expression of vein-specific genes by inhibiting the Capicua (Cic) HMG-box repressor, a known regulator of embryonic body patterning. Lack of Cic function causes ectopic expression of EGFR targets such as argos, ventral veinless and decapentaplegic and leads to formation of extra vein tissue. In vein cells, EGFR signalling downregulates Cic protein levels in the nucleus and relieves repression of vein-specific genes, whereas intervein cells maintain high levels of Cic throughout larval and pupal development, repressing the expression of vein-specific genes and allowing intervein differentiation. However, regulation of some EGFR targets such as rhomboid appears not to be under direct control of Cic, suggesting that EGFR signalling branches out in the nucleus and controls different targets via distinct mediator factors. Our results support the idea that localised inactivation of transcriptional repressors such as Cic is a rather general mechanism for regulation of target gene expression by the Ras/Raf pathway.

Animals↗
PubMed1992

Transcriptional control by Drosophila gap genes.

The segmented body pattern along the longitudinal axis of the Drosophila embryo is established by a cascade of specific transcription factor activities. This cascade is initiated by maternal gene products that are localized at the polar regions of the egg. The initial long-range positional information of the maternal factors, which are transcription factors (or are factors which activate or localize transcription factors), is transferred through the activity of the zygotic segmentation genes. The gap genes act at the top of this regulatory hierarchy. Expression of the gap genes occurs in discrete domains along the longitudinal axis of the preblastoderm and defines specific, overlapping sets of segment primordia. Their protein products, which are DNA-binding transcription factors mostly of the zinc finger type, form broad and overlapping concentration gradients which are controlled by maternal factors and by mutual interactions between the gap genes themselves. Once established, these overlapping gap protein gradients provide spatial cues which generate the repeated pattern of the subordinate pair-rule gene expression, thereby blue-printing the pattern of segmental units in the blastoderm embryo. Our results show different strategies by which maternal gene products, in combination with various gap gene proteins, provide position-dependent sets of transcriptional activator/repressor systems which regulate the spatial pattern of specific gap gene expression. Region-specific combinations of different transcription factors that derive from localized gap gene expression eventually generate the periodic pattern of pair-rule gene expression by the direct interaction with individual cis-acting "stripe elements" of particular pair-rule gene promoters. Thus, the developmental fate of blastoderm cells is programmed according to their position within the anterior-posterior axis of the embryo: maternal transcription factors regulate the region-specific expression of first zygotic transcription factors which, by their specific and unique combinations, control subordinate zygotic transcription factors, thereby subdividing the embryo into increasingly smaller units later seen in the larva.

Animals↗