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S Wyatt

Publications and source records attributed to S Wyatt.

At least 37 records · Page 2Linked to original sources
PubMed1999

Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts.

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by alpha retinoic acid receptors, not beta or gamma receptors. The observation that alpha-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.

Animals↗
PubMed1999

Differences and developmental changes in the responsiveness of PNS neurons to GDNF and neurturin.

We have studied the ability of GDNF and neurturin to promote the in vitro survival of populations of embryonic chicken parasympathetic, sympathetic, and sensory neurons. We show that these neurons are more responsive to one or other of these factors at particular stages of development. Whereas the parasympathetic neurons are more sensitive to neurturin at late embryonic stages, sympathetic neurons are more sensitive to neurturin at early stages. In contrast, sensory neurons of the nodose ganglion are more sensitive to GDNF throughout embryonic development. Using competitive RT/PCR, we measured the levels of mRNAs encoding GDNF and neurturin receptors in purified neurons. All neurons expressed Ret mRNA, which encodes the common receptor tyrosine kinase for GDNF and neurturin. Neurons that were more sensitive to GDNF expressed higher levels of GFRalpha-1 mRNA than GFRalpha-2 mRNA and neurons that were more sensitive to neurturin expressed higher levels of GFRalpha-2 mRNA than GFRalpha-1 mRNA. These results show that populations of PNS neurons differ markedly in their responsiveness to GDNF and neurturin at certain stages of the development and suggest that these differences are governed in part by the relative levels of expression of members of the GFRalpha family of GPI-linked receptors.

Animals↗
PubMed1999

p75-mediated NF-kappaB activation enhances the survival response of developing sensory neurons to nerve growth factor.

We have investigated whether the transcription factor NF-kappaB plays a role in regulating neuronal survival by manipulating NF-kappaB activation in the nerve growth factor (NGF)-dependent sensory neurons of the embryonic mouse trigeminal ganglion. Overexpression of either the p65 or the p50 NF-kappaB subunits resulted in NF-kappaB activation and promoted in vitro survival as effectively as NGF. Expression of a superrepressor IkappaB-alpha protein prevented NF-kappaB activation in p65/p50-overexpressing neurons and caused the neurons to die as rapidly as NGF-deprived neurons. NGF treatment also activated NF-kappaB, and preventing this activation with superrepressor IkappaB-alpha reduced the NGF survival response. Antibodies that block binding of NGF to the p75 receptor prevented NGF-induced NF-kappaB activation and reduced the NGF survival response to the same extent as superrepressor IkappaB-alpha. Trigeminal neurons cultured from p65(-/-) embryos showed a reduced survival response to NGF compared with neurons from wild-type embryos and there was increased apoptosis of neurons in the trigeminal ganglia of p65(-/-) embryos in vivo. However, as with p75-deficient sensory neurons, p65-deficient sensory neurons showed a normal survival response to BDNF. These results reveal a role for NF-kappaB in regulating neuronal survival during embryonic development and suggest that in addition to the well-established Trk receptor tyrosine kinase signaling cascade, NGF enhances neuronal survival by signaling via a p75-mediated pathway.

Animals↗
PubMed1999

Developmental changes in the response of trigeminal neurons to neurotrophins: influence of birthdate and the ganglion environment.

Previous studies have shown that most neurons in cultures established during the early stages of neurogenesis in the embryonic mouse trigeminal ganglion are supported by BDNF whereas most neurons cultured from older ganglia survive with NGF. To ascertain to what extent these developmental changes in neurotrophin responsiveness result from separate phases of generation of BDNF- and NGF-responsive neurons or from a developmental switch in the response of neurons from BDNF to NGF, we administered BrdU to pregnant mice at different stages of gestation to identify neurons born at different times and studied the survival of labelled neurons in dissociated cultures established shortly after BrdU administration. Most early-generated neurons responded to BDNF, neurons generated at intermediate times responded to both factors and late-generated neurons responded to NGF, indicating that there are overlapping phases in the generation of BDNF- and NGF-responsive neurons and that late-generated neurons do not switch responsiveness from BDNF to NGF. To ascertain if early-generated neurons do switch their response to neurotrophins during development, we used repeated BrdU injection to label all neurons generated after an early stage in neurogenesis and studied the neurotrophin responsiveness of the unlabelled neurons in cultures established after neurogenesis had ceased. The response of these early-generated neurons had decreased to BDNF and increased to NGF, indicating that at least a proportion of early-generated neurons switch responsiveness to neurotrophins in vivo. Because early-generated neurons do not switch responsiveness from BDNF to NGF in long-term dissociated cultures, we cultured early trigeminal ganglion explants with and without their targets for 24 hours before establishing dissociated cultures. This period of explant culture was sufficient to enable many early-generated neurons to switch their response from BDNF to NGF and this switch occurred irrespective of presence of target tissue. Our findings conclusively demonstrate for the first time that individual neurons switch their neurotrophin requirements during development and that this switch depends on cell interactions within the ganglion. In addition, we show that there are overlapping phases in the generation of BDNF- and NGF-responsive neurons in the trigeminal ganglion.

Animals↗
PubMed1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells.

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

Cloning, Molecular↗
PubMed1998

Bcl-2 accelerates the maturation of early sensory neurons.

Bcl-2 is a cytoplasmic protein that blocks apoptosis in a wide variety of cell types. Here we report a novel role for Bcl-2 in the early stages of neuronal development. Shortly after differentiating from progenitor cells, sensory neurons undergo a distinct morphological change; initially they have small, spindle-shaped, phase-dark cell bodies that become large, spherical, and phase-bright. Early sensory neurons cultured from the trigeminal ganglia of bcl-2-/- embryos at embryonic day 11 (E11) and E12 underwent this change more slowly than trigeminal neurons of wild-type embryos of the same ages. The delay was not attributable to the well documented role of Bcl-2 in preventing apoptosis, because Bcl-2-deficient early sensory neurons survived as well as wild-type neurons. Accordingly, there was a significantly smaller number of the more mature type of neuron in the early trigeminal ganglia of bcl-2-/- embryos, yet the number of neurons in the trigeminal ganglia of bcl-2-/- and wild-type embryos was similar. The absence of Bcl-2 did not cause a uniform delay in the developmental program of sensory neurons, because the time course of nerve growth factor receptor expression (both trkA and p75) was unaffected in the trigeminal neurons of bcl-2-/- embryos. These findings indicate that Bcl-2 expression is required for the normal progression of a particular early maturational change in embryonic sensory neurons.

Animals↗
PubMed1998

GFRalpha-4, a new GDNF family receptor.

GFRalpha-1, GFRalpha-2, and GFRalpha-3 constitute a family of structurally related, glycosyl-phosphatidylinosital-linked, cell surface proteins, two of which, GFRalpha-1 and GFRalpha-2, are components of the receptor complex for the neurotrophic factors GDNF and neurturin, respectively. By screening an embryonic chicken brain cDNA library with a GFRalpha-1 probe at low stringency, we isolated cDNAs encoding an additional member of the GFRalpha family, GFRalpha-4. The nucleotide sequence predicts a 431-amino-acid secreted protein that is more closely related to GFRalpha-1 and GFRalpha-2 than to GFRalpha-3. GFRalpha-4 mRNA is expressed in distinctive patterns in the brain and several other organs and tissues of the chicken embryo. Our findings extend the family of GFRalpha proteins and provide information about the tissues in which GFRalpha-4 may function during development.

Animals↗
PubMed1998

NT-3 regulates expression of Brn3a but not Brn3b in developing mouse trigeminal sensory neurons.

We have used a quantitative RT-PCR approach to determine the levels of Brn3a and Brn3b POU domain transcription factor mRNAs in the developing mouse trigeminal ganglion from E10 to E18. Using low density neuronal cultures, we have shown that NT-3 can regulate the expression of Brn3a mRNA in trigeminal neurons during the periods that they are differentiating and innervating their peripheral and central targets. In contrast to Brn3a, Brn3b mRNA is expressed at extremely low levels in the early trigeminal ganglion. Trigeminal neurons from early ganglia express low levels of Brn3b mRNA in culture and do not up-regulate Brn3b mRNA in response to a number of growth factors and experimental conditions. However, at later ages, when in vivo levels of Brn3b mRNA are high, FGF2, TGFbeta1 and retinoic acid all up-regulate Brn3b mRNA expression in cultured trigeminal neurons. Since NT-3 regulates the developmental expression of Brn3a, Brn3a may mediate some of the effects that NT-3 exerts on sensory neurons and their progenitors. Similarly, Brn3b may mediate some of the effects that FGF2, TGFbeta1 and retinoic acid have on neurons.

Animals↗
PubMed1998

Multiple roles for hepatocyte growth factor in sympathetic neuron development.

We have studied the role of hepatocyte growth factor (HGF)/Met signaling in the development of sympathetic neuroblasts and neurons. Anti-HGF antibodies reduced the number of sympathetic neuroblasts that differentiated into neurons, but neither anti-HGF antibodies nor HGF affected neuroblast proliferation. Anti-HGF antibodies also reduced the survival of neuroblasts but not sympathetic neurons. HGF greatly enhanced the neurite outgrowth of NGF-dependent sympathetic neurons throughout development. These in vitro effects of anti-HGF antibodies and HGF were abolished by a disabling mutation of Met, the HGF receptor tyrosine kinase. The Met mutation also increased sympathetic neuroblast apoptosis in vivo. Because Met and HGF are expressed in sympathetic ganglia throughout development, it is possible that the multiple effects of HGF/Met signaling on sympathetic neuroblasts and neurons occur in part by an autocrine mechanism.

Animals↗
PubMed1997

Sympathetic neuron survival and TrkA expression in NT3-deficient mouse embryos.

Several in vitro and in vivo studies have led to the widely accepted view that NT3 is required for sympathetic neuroblast survival, induction of TrkA expression and the acquisition of NGF dependence. However, we show that the number of neurons and the levels of trkA and p75 mRNAs in the superior cervical sympathetic ganglion (SCG) of NT3-/- mouse embryos increase normally up to E16, 2 days after SCG neurons start responding to NGF. At E18 and in the postnatal period, there are significant reductions in the number of SCG neurons and in the levels of trkA and p75 mRNAs. These results show that the neurotrophin survival requirements of SCG neurons do not switch from NT3 to NGF during development and that NT3 is not required for the expression of TrkA and p75 and the acquisition of NGF dependence. Rather, some sympathetic neurons have a late requirement for NT3 at the time when they also depend on NGF for survival. The expression of transcripts encoding catalytic TrkC is negligible at this stage, suggesting that NT3 acts mainly via TrkA.

Animals↗
PubMed1997

NGF binding to p75 enhances the sensitivity of sensory and sympathetic neurons to NGF at different stages of development.

To clarify the role of the common neurotrophin receptor p75 in modulating the survival response of sensory and sympathetic neurons to NGF at different stages of development, we compared the actions of wild-type NGF with a mutated NGF protein that binds normally to TrkA, the NGF receptor tyrosine kinase, but has greatly reduced binding to p75. At saturating concentrations, the NGF mutant promoted the survival of similar numbers of trigeminal sensory and sympathetic neurons as NGF. At subsaturating concentrations, the NGF mutant was less effective than wild-type NGF in promoting the survival of embryonic sensory neurons and postnatal sympathetic neurons but was equally effective as wild-type NGF in promoting the survival of embryonic sympathetic neurons. Whereas the levels of trkA and p75 were similar in embryonic sensory neurons and postnatal sympathetic neurons, the level of p75 was significantly lower than that of trkA in embryonic sympathetic neurons. These results indicate that binding of NGF to p75 enhances the sensitivity of NGF-dependent neurons to NGF at stages in their development when the levels of p75 and TrkA are similar.

Animals↗
PubMed1996

Prospective evaluation of eligibility for thrombolytic therapy in acute myocardial infarction.

OBJECTIVES: To determine the proportion of patients presenting with acute myocardial infarction who are eligible for thrombolytic therapy. DESIGN: Cohort follow up study. SETTING: The four coronary care units in Auckland, New Zealand. SUBJECTS: All 3014 patients presenting to the units with suspected myocardial infarction in 1993. MAIN OUTCOME MEASURES: Eligibility for reperfusion with thrombolytic therapy (presentation within 12 hours of the onset of ischaemic chest pain with ST elevation > or = 2 mm in leads V1-V3, ST elevation > or = 1 mm in any other two contiguous leads, or new left bundle branch block); proportions of (a) patients eligible for reperfusion and (b) patients with contraindications to thrombolysis; death (including causes); definite myocardial infarction. RESULTS: 948 patients had definite myocardial infarction, 124 probable myocardial infarction, and nine ST elevation but no infarction; 1274 patients had unstable angina and 659 chest pain of other causes. Of patients with definite or probable myocardial infarction, 576 (53.3%) were eligible for reperfusion, 39 had definite contraindications to thrombolysis (risk of bleeding). Hence 49.7% of patients (537/1081) were eligible for thrombolysis and 43.5% (470) received this treatment. Hospital mortality among patients eligible for reperfusion was 11.7% (55/470 cases) among those who received thrombolysis and 17.0% (18/106) among those who did not. CONCLUSIONS: On current criteria about half of patients admitted to coronary care units with definite or probable myocardial infarction are eligible for thrombolytic therapy. Few eligible patients have definite contraindications to thrombolytic therapy. Mortality for all community admissions for myocardial infarction remains high.

Age Factors↗
PubMed1996

Trigeminal ganglion neurons are protected by the heat shock proteins hsp70 and hsp90 from thermal stress but not from programmed cell death following nerve growth factor withdrawal.

A prior mild thermal stress (heat shock) can protect neuronal cells against a subsequent exposure to either severe thermal stress or the induction of programmed cell death (apoptosis). By micro-injecting trigeminal ganglion neurons with expression constructs we show that over-expression of the individual heat shock proteins hsp70 and hsp90 can protect these cells against severe thermal stress but not against apoptosis. However, the protective effect of prior heat shock against subsequent apoptosis is dependent upon its ability to induce heat shock protein (hsp) synthesis rather than, for example, the inhibition of other protein synthesis associated with heat shock. The significance of these effects is discussed in terms of the role of different hsps in protecting neuronal cells from distinct stresses.

Animals↗