Symptom-burden in people living with frailty and chronic kidney disease.

BACKGROUND: Frailty is independently associated with worse health-related quality of life (HRQOL) in chronic kidney disease (CKD). However, the relationship between frailty and symptom experience is not well described in people living with CKD. This study's aim was to evaluate the relationship between frailty and symptom-burden in CKD. METHODS: This study is a secondary analysis of a cross-sectional observational study, the QCKD study (ISRCTN87066351), in which participants completed physical activity, cardiopulmonary fitness, symptom-burden and HRQOL questionnaires. A modified version of the Frailty Phenotype, comprising 3 self-report components, was created to assess frailty status. Multiple linear regression was performed to assess the association between symptom-burden/HRQOL and frailty. Logistic regression was performed to assess the association between experiencing symptoms frequently and frailty. Principal Component Analysis was used to assess the experienced symptom clusters. RESULTS: A total of 353 patients with CKD were recruited with 225 (64%) participants categorised as frail. Frail participants reported more symptoms, had higher symptom scores and worse HRQOL scores. Frailty was independently associated with higher total symptom score and lower HRQOL scores. Frailty was also independently associated with higher odds of frequently experiencing 9 out of 12 reported symptoms. Finally, frail participants experienced an additional symptom cluster that included loss of appetite, tiredness, feeling cold and poor concentration. CONCLUSIONS: Frailty is independently associated with high symptom-burden and poor HRQOL in CKD. Moreover, people living with frailty and CKD have a distinctive symptom experience. Proactive interventions are needed that can effectively identify and address problematic symptoms to mitigate their impact on HRQOL.

Recent advances in regenerative medicine to treat enteric neuropathies: use of human cells.

As current options for treating most enteric neuropathies are either non-effective or associated with significant ongoing problems, cell therapy is a potential attractive possibility to treat congenital and acquired neuropathies. Studies using animal models have shown that following transplantation of enteric neural progenitors into the bowel of recipients, the transplanted cells migrate, proliferate, and generate neurons that are electrically active and receive synaptic inputs. Recent studies have transplanted human enteric neural progenitors into the mouse colon and shown engraftment. In this article, we summarize the significance of these recent advances and discuss priorities for future research that might lead to the use of regenerative medicine to treat enteric neuropathies in the clinic.

Design and methods of CYCLE-HD: improving cardiovascular health in patients with end stage renal disease using a structured programme of exercise: a randomised control trial.

BACKGROUND: There is emerging evidence that exercise training could positively impact several of the cardiovascular risk factors associated with sudden cardiac death amongst patients on haemodialysis. The primary aim of this study is to evaluate the effect of an intradialytic exercise programme on left ventricular mass. METHOD AND DESIGN: Prospective, randomised cluster open-label blinded endpoint clinical trial in 130 patients with end stage renal disease on haemodialysis. Patients will be randomised 1:1 to either 1) minimum of 30 min continuous cycling thrice weekly during dialysis or 2) standard care. The primary outcome is change in left ventricular mass at 6 months, assessed by cardiac MRI (CMR). In order to detect a difference in LV mass of 15 g between groups at 80 % power, a sample size of 65 patients per group is required. Secondary outcome measures include abnormalities of cardiac rhythm, left ventricular volumes and ejection fraction, physical function measures, anthropometric measures, quality of life and markers of inflammation, with interim assessment for some measures at 3 months. DISCUSSION: This study will test the hypothesis that an intradialytic programme of exercise leads to a regression in left ventricular mass, an important non-traditional cardiovascular risk factor in end stage renal disease. For the first time this will be assessed using CMR. We will also evaluate the efficacy, feasibility and safety of an intradialytic exercise programme using a number of secondary end-points. We anticipate that a positive outcome will lead to both an increased patient uptake into established intradialytic programmes and the development of new programmes nationally and internationally. TRIAL REGISTRATION NUMBER: ISRCTN11299707 (registration date 5(th) March 2015).

Genetic fate-mapping of tyrosine hydroxylase-expressing cells in the enteric nervous system.

BACKGROUND: During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized. METHODS: We combined in vivo Cre-lox-based genetic fate-mapping with phenotypic analysis to fate-map enteric neuron subtypes arising from tyrosine hydroxylase (TH)-expressing cells. KEY RESULTS: Less than 3% of the total (Hu(+) ) neurons in the myenteric plexus of the small intestine of adult mice are generated from transiently TH-expressing cells. Around 50% of the neurons generated from transiently TH-expressing cells are calbindin neurons, but their progeny also include calretinin, neurofilament-M, and serotonin neurons. However, only 30% of the serotonin neurons and small subpopulations (<10%) of the calbindin, calretinin, and neurofilament-M neurons are generated from TH-expressing cells; only 0.2% of nitric oxide synthase neurons arise from TH-expressing cells. CONCLUSIONS & INFERENCES: Transiently, catecholaminergic cells give rise to subpopulations of multiple enteric neuron subtypes, but the majority of each of the neuron subtypes arises from non-TC cells.

First Report of Sclerotinia Stem Rot Caused by Sclerotinia sclerotiorum on Brassica carinata in Florida.

Brassica carinata A. Braun, commonly referred to as Ethiopian rapeseed, a near relative of collards and mustard, has become the object of increasing interest as an oil crop. It has been reported that B. carinata adapts better and is more productive than B. napus (canola) in adverse conditions, such as clay and sandy soils and under low management cropping systems (1). In late February 2012, symptoms typical of sclerotinia stem rot were observed in B. carinata trials (cultivars 090867 EM and 080814 EM) at the University of Florida, North Florida Research and Education Center located in Quincy, FL. Approximately 20 to 30% of the B. carinata cultivar 090867 EM were observed to have symptoms and approximately 5% of cultivar 080814 EM displayed symptoms. Stems had white mycelia growing on the outside, plants were lodging and spherical to cylindrical, 3 to 8 mm, and black sclerotia were found outside and inside bleached stems. Sclerotia from diseased stems were surface sterilized and placed in 9-cm diameter petri plates on quarter strength potato dextrose agar (PDA) amended with 25% lactic acid. Fungal cultures consisting of white mycelia and medium-sized (mean 4 mm), black, irregular sclerotia were consistently recovered and identified as Sclerotinia sclerotiorum (Lib.) de Bary based on morphological characteristics (3). Sequence analyses were conducted on mycelium by extracting fungal DNA with the Qiagen DNeasy Plant Mini Kit (Valencia, CA). PCR amplification was performed using primers ITS1 and ITS4. The BLAST search revealed that the sequence (GenBank Accession No. JX307092) had 99 and 100% sequence identity with S. sclerotiorum GenBank accessions JN013184.1 and JN012606.1. Pathogenicity was determined by inoculating six 1-month-old B. carinata plants (cultivars 090867 EM and 080814 EM) that were grown in greenhouse pots (20 cm in diameter). Mycelia plugs (8 mm in diameter) were excised from the colony margin after 6 days of incubation at room temperature (approximately 25°C), and placed on stems, at the soil line, of B. carinata plants. Six control plants were inoculated with noncolonized PDA plugs. All plants were enclosed in plastic bags that had been sprayed with water on the inside to maintain high humidity and kept in the laboratory at room temperature (approximately 25°C). Symptoms similar to those observed in the field were evident after 3 days on inoculated plants and S. sclerotiorum was reisolated. In the controls, no symptoms developed and the fungus could not be isolated. The experiment was repeated with similar results. The majority of rapeseed production is in North Dakota, where sclerotinia stem rot caused by S. sclerotiorum is a major fungal disease affecting production (2). Currently, there is no significant B. carinata production in Florida; however, interest in biofuels could lead to an increase in planted acreage and sclerotinia stem rot could become a significant disease problem in areas of Florida were B. carinata is planted. To our knowledge, this is the first report of sclerotinia stem rot of B. carinata caused by S. sclerotiorum in Florida. References: (1) M. Cardone et al. Biomass and Bioenergy. 25:623, 2003. (2) L. E. del Río et al. Plant Dis. 91:191, 2007. (3) L. M. Kohn. Phytopathology 69:881, 1979.

First Report of Downy Mildew (Hyaloperonospora camelinae) on Camelina sativa in Florida.

Camelina sativa (L.) Crantz, Brassicaceae, whose common name is Crantz-large-seeded false flax, is an annual oilseed species. It is grown as a forage and biofuel crop in Europe and North America. False flax is an ideal low-input crop for biodiesel production, because of its low requirements for nitrogen fertilizer and pesticides. Production costs of this crop are substantially lower than those of many other oilseed crops such as rapeseed, corn, and soybean. It is an excellent rotation crop and can reduce disease and insect and weed pressure in wheat fields. During the spring of 2011, commercial and research plantings of C. sativa cultivar Calena in Liberty and Gadsden counties in north Florida developed symptoms typical of downy mildew. In spring of 2012, the same symptoms were observed in experimental plantings of false flax. A white downy mold was observed on the upper third portion of the plants, on the upper stem internodes, and on the developing seed. The affected stems exhibited a twisted growth. Conidiophores had main trunks with dichotomous branches terminating in slender curved tips. Conidia were ovoid and 28 to 45 (mean 36) µm long and 22 to 38 (mean 30) µm wide. Conidiophores were branched (three to four branches, each with six to eight curved tips) and ranged from 107 to 236 µm long and 5 to 14 µm wide. Mycelium was obtained directly from diseased plants for DNA extraction (Qiagen DNeasy Plant Mini Kit, Valencia, CA). Primers ITS1 and ITS4 were used for PCR amplification (4). The PCR product was sequenced bidirectionally with the PCR primers. A consensus nucleotide sequence (Accession JQ997103) was compared to those in the NCBI GenBank database using a BLAST search. The sequence was 99% similar to sequence from Hyaloperonospora camelinae (Gäum.) Göker, Voglmayr, Riethm, M. Weiss & Oberw. (Accession AY198249.1) with a 95% query coverage (1). Pathogenicity was established by applying white conidial masses of downy mildew from field samples to stems of 4-week-old plants grown in pots in a greenhouse maintained at 25 ± 2°C. Noninoculated plants maintained under the similar conditions served as control. Symptoms and signs of downy mildew developed after 14 days only on inoculated plants. Downy mildew constitutes a serious threat to the cultivation of C. sativa in Florida because of the humid climate favoring disease development. Diseased plants may reduce yield and disease management would increase production costs. H. camelinae was previously reported on C. sativa in Oregon, Minnesota, Montana (3), and Nebraska (2). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Florida. References: (1) M. Göker et al. Canad. J. Bot. 81:672, 2003. (2) R. M. Harveson et al. Plant Health Progress. 2011. doi: 10.1094/PHP-2011-1014-01-BR. (3) M. L. Putnam et al. Plant Health Progress. 2009. doi: 10.1094/PHP-2009-0910-01-BR. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

The role of neural activity in the migration and differentiation of enteric neuron precursors.

BACKGROUND: As they migrate through the developing gut, a sub-population of enteric neural crest-derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs. METHODS: The appearance of neuronal sub-types in the gut of embryonic mice was examined using immunohistochemistry. The effects of blocking various forms of neural activity on ENCC migration and neuronal differentiation were examined using explants of cultured embryonic gut. KEY RESULTS: Nerve fibers were present in close apposition to many ENCCs. Commencing at E11.5, neuronal nitric oxide synthase (nNOS), calbindin and IK(Ca) channel immunoreactivities were shown by sub-populations of enteric neurons. In cultured explants of embryonic gut, tetrodotoxin (TTX, an inhibitor of action potential generation), nitro-L-arginine (NOLA, an inhibitor of nitric oxide synthesis) and clotrimazole (an IK(Ca) channel blocker) did not affect the rate of ENCC migration, but tetanus toxin (an inhibitor of SNARE-mediated vesicle fusion) significantly impaired ENCC migration as previously reported. In explants of E11.5 and E12.5 hindgut grown in the presence of TTX or tetanus toxin there was a decrease in the number nNOS+ neurons close to the migratory wavefront, but no significant difference in the proportion of all ENCC that expressed the pan-neuronal marker, Hu. CONCLUSIONS & INFERENCES: (i) Some enteric neuron sub-types are present very early during the development of the enteric nervous system. (ii) The rate of differentiation of some sub-types of enteric neurons appears to be influenced by TTX- and tetanus toxin-sensitive mechanisms.

Effects of tissue age, presence of neurones and endothelin-3 on the ability of enteric neurone precursors to colonize recipient gut: implications for cell-based therapies.

BACKGROUND Most enteric neurones arise from neural crest cells that originate in the post-otic hindbrain, and migrate into and along the developing gastrointestinal tract. There is currently great interest in the possibility of cell therapy to replace diseased or absent enteric neurones in patients with enteric neuropathies, such as Hirschsprung's disease. However, it is unclear whether neural crest stem/progenitor cells will be able to colonize colon (i) in which the mesenchyme has differentiated into distinct layers, (ii) that already contains enteric neurones or (iii) that lacks a gene expressed by the gut mesenchyme, such as endothelin-3 (Et-3). METHODS Co-cultures were used to examine the ability of enteric neural crest-derived cells (ENCCs) from E11.5 mouse gut to colonize a variety of recipient hindguts. KEY RESULTS Enteric neural crest-derived cells migrated and gave rise to neurones in E14.5 and E16.5 aneural colon in which the external muscle layers had differentiated, but they did not migrate as far as in younger colon. There was no evidence of altered ENCC proliferation, cell death or neuronal differentiation in older recipient explants. Enteric neural crest-derived cells failed to enter most recipient E14.5 and E16.5 colon explants already containing enteric neurones, and the few that did showed very limited migration. Finally, ENCCs migrated a shorter distance and a higher proportion expressed the pan-neuronal marker, Hu, in recipient E11.5 Et-3(-/-) colon compared to wild-type recipient colon. CONCLUSIONS & INFERENCES Age and an absence of Et-3 from the recipient gut both significantly reduced but did not prevent ENCC migration, but the presence of neurones almost totally prevented ENCC migration.

First Report of Brown Ring Patch on Poa annua Caused by Waitea circinata var. circinata in West Virginia.

In mid-November 2009, thin, yellow, and irregular-shaped scalloped rings 10 to 25 cm in diameter were observed on 5 to 10% of a golf course putting green in Charles Town, WV. The 20-year-old USGA-specification sand-based green was mowed at 3.1-mm height and consisted of 60% annual bluegrass (Poa annua L.) and 40% creeping bentgrass (Agrostis stoloniferous L. 'Putter'). Minimum and maximum daily air temperature ranged from 2 to 22°C, respectively, with 38 mm of rainfall during the appearance of rings symptoms. Only affected annual bluegrass plants exhibited a peculiar yellow chlorosis of the upper and lower leaves. A single fungal isolate was obtained from active mycelium found within symptomatic annual bluegrass leaves and grown on potato dextrose agar (PDA) amended with chloramphenicol (0.1 g/liter). Fungal colony morphology (i.e., light yellow with irregular-shaped 2- to 4-mm-diameter sclerotia first appearing off-white but progressing to brown by 21 to 28 days in culture) and sequencing of the internal transcribed spacer (ITS) 5.8S rDNA region with primers ITS1 and ITS4 confirmed the isolate as Waitea circinata var. circinata (Warcup & Talbot) with ≥99% sequence identity with GenBank Accession No. FJ755889 (1,2,4). To confirm pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 4-day-old culture grown on PDA and placed in contact with the lower leaves of 12-week-old annual bluegrass (0.001 g of surface-sterilized seed per cm2) grown in 5- × 5-cm plastic pots of autoclaved 85% sand and 15% potting soil. Six pots were inoculated with the isolate and six pots were inoculated with an isolate-free agar plug and then placed in a moist chamber at 28°C. Leaf chlorosis and aerial mycelium was observed in all six inoculated pots 8 to 10 days after inoculation, and symptoms were similar to those expressed in the field. All noninoculated plants remained healthy and asymptomatic. W. circinata var. circinata was reisolated from symptomatic leaves and again confirmed by colony traits and sequencing of the ITS-5.8S rDNA region and submitted as GenBank Accession No. HM807582. To our knowledge, this is the first report of brown ring patch in West Virginia and could be economically important because of intensive fungicide practices used to maintain high-quality putting greens on golf courses (3). References: (1) C. Chen et al. Plant Dis. 91:1687, 2007. (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Manage. 75:98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.

The migratory behavior of immature enteric neurons.

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.

Functional development of the enteric nervous system--from migration to motility.

The enteric nervous system (ENS) consists of many different types of enteric neurones forming complex reflex circuits that underlie or regulate many gut functions. Studies of humans with Hirschsprung's disease (distal aganglionosis), and of animal models of Hirschsprung's disease, have led to the identification of many of the genetic, molecular and cellular mechanisms responsible for the colonization of the gut by enteric neurone precursors. However, later events in the ENS development are still poorly understood, including the development of functioning ENS circuits. This article is a personal view of the current state of play in our understanding of the ENS development and of the future of the field.

Effects of different regions of the developing gut on the migration of enteric neural crest-derived cells: a role for Sema3A, but not Sema3F.

The enteric nervous system arises from vagal (caudal hindbrain) and sacral level neural crest-derived cells that migrate into and along the developing gut. Data from previous studies have suggested that (i) there may be gradients along the gut that induce the caudally directed migration of vagal enteric neural precursors (ENPs), (ii) exposure to the caecum might alter the migratory ability of vagal ENPs and (iii) Sema3A might regulate the entry into the hindgut of ENPs derived from sacral neural crest. Using co-cultures we show that there is no detectable gradient of chemoattractive molecules along the pre-caecal gut that specifically promotes the caudally directed migration of vagal ENPs, although vagal ENPs migrate faster caudally than rostrally along explants of hindgut. Exposure to the caecum did not alter the rate at which ENPs colonized explants of hindgut, but it did alter the ability of ENPs to colonize the midgut. The co-cultures also revealed that there is localized expression of a repulsive cue in the distal hindgut, which might delay the entry of sacral ENPs. We show that Sema3A is expressed by the hindgut mesenchyme and its receptor, neuropilin-1, is expressed by migrating ENPs. Furthermore, there is premature entry of sacral ENPs and extrinsic axons into the distal hindgut of fetal mice lacking Sema3A. These data show that Sema3A expressed by the distal hindgut regulates the entry of sacral ENPs and extrinsic axons into the hindgut. ENPs did not express neuropilin-2 and there was no detectable change in the timetable by which ENPs colonize the gut in mice lacking neuropilin-2.

Phenotypes of neural-crest-derived cells in vagal and sacral pathways.

Enteric neurons arise from vagal and sacral level neural crest cells. To examine the phenotype of neural-crest-derived cells in vagal and sacral pathways, we used antisera to Sox10, p75, Phox2b, and Hu, and transgenic mice in which the expression of green fluorescent protein was under the control of the Ret promoter. Sox10 was expressed prior to the emigration of vagal cells, whereas p75 was expressed shortly after their emigration. Most crest-derived cells that emigrated adjacent to somites 1-4 migrated along a pathway that was later followed by the vagus nerve. A sub-population of these vagal cells coalesced to form vagal ganglia, whereas others continued their migration towards the heart and gut. Cells that coalesced into vagal ganglia showed a different phenotype from cells in the migratory streams proximal and distal to the ganglia. Only a sub-population of the vagal cells that first entered the foregut expressed Phox2b or Ret. Sacral neural crest cells gave rise to pelvic ganglia and some neurons in the hindgut. The pathways of sacral neural crest cells were examined by using DbetaH-nlacZ mice. Sacral cells appeared to enter the distal hindgut around embryonic day 14.5. Very few of the previously demonstrated, but rare, neurons that were present in the large intestine of Ret null mutants and that presumably arose from the sacral neural crest expressed nitric oxide synthase, unlike their counterparts in Ret heterozygous mice.

The location and phenotype of proliferating neural-crest-derived cells in the developing mouse gut.

Neural crest cells that originate in the caudal hindbrain migrate into and along the developing gastrointestinal tract to form the enteric nervous system. While they are migrating, neural-crest-derived cells are also proliferating. Previous studies have shown that the expression of glial-derived neurotrophic factor (GDNF) and endothelin-3 is highest in the embryonic caecum, and that GDNF alone or in combination with endothelin-3 promotes the proliferation of enteric neural-crest-derived cells in vitro. However, whether neural proliferative zones, like those in the central nervous system, are found along the developing gut is unknown. We used a fluorescent nucleic acid stain to identify dividing cells or BrdU labelling (2 h after administration of BrdU to the mother), combined with antibodies specific to neural crest cells to determine the percentage of proliferating crest-derived cells in various gut regions of embryonic day 11.5 (E11.5) and E12.5 mice. The rate of proliferation of crest-derived cells did not vary significantly in different regions of the gut (including the caecum) or at different distances from the migratory wavefront of vagal crest-derived cells. The phenotype of mitotic enteric crest-derived cells was also examined. Cells expressing the pan-neuronal markers, neurofilament-M and Hu, or the glial marker, S100b, were observed undergoing mitosis. However, no evidence was found for proliferation of cells expressing neuron-type-specific markers, such as nitric oxide synthase (at E12.5) or calcitonin gene-related peptide (at E18.5). Thus, for enteric neurons, exit from the cell cycle appears to occur after the expression of pan-neuronal proteins but prior to the expression of markers of terminally differentiated neurons.

Guidance cues involved in the development of the peripheral autonomic nervous system.

All peripheral autonomic neurons arise from neural crest cells that migrate away from the neural tube and navigate to the location where ganglia will form. After differentiating into neurons, their axons then navigate to a variety of targets. During the development of the enteric nervous system, GDNF appears to play a role in inducing vagal neural crest cells to enter the gut, in retaining neural crest cells within the gut and in promoting the migration of neural crest cells along the gut. Sema3A regulates the entry of extrinsic axons into the distal hindgut, netrin-DCC signaling is responsible for the centripetal migration of cells to form the submucosal ganglia within the gut, Slit-Robo signaling prevents trunk level neural crest cells from entering the gut, and neurturin plays a role in the innervation of the circular muscle layer. During the development of the sympathetic nervous system, the migration of trunk neural crest cells through the somites is influenced by ephrin-Bs, Sema3A and F-spondin. The migration of neural crest cells ventrally beyond the somites requires neuregulin signaling and the clumping of cells into columns adjacent to the dorsal aorta is regulated by Sema3A. The rostral migration of cells to form the superior cervical ganglion (SCG) and the extension of axons along blood vessels involves artemin signaling through Ret and GFRalpha3, and the entry of sympathetic axons into target tissues involves neurotrophins and GDNF. Relatively little is known about the development of parasympathetic ganglia, but GDNF appears to play a role in the migration of some cranial ganglion precursors to their correct location, and both GDNF and neurturin are involved in the growth of parasympathetic axons into particular targets.

Dynamics of neural crest-derived cell migration in the embryonic mouse gut.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Enteric neural crest-derived cells and neural stem cells: biology and therapeutic potential.

The enteric nervous system arises from two regions of the neural crest; the vagal neural crest which gives rise to the vast majority of enteric neurones throughout the gastrointestinal tract, and the sacral neural crest which contributes a smaller number of cells that are mainly distributed within the hindgut. The migration of vagal neural crest cells into, and along the gut is promoted by GDNF, which is expressed by the gut mesenchyme and is the ligand for the Ret/GFRalpha1 signalling complex present on migrating vagal-derived crest cells. Sacral neural crest cells enter the gut after it has been colonized by vagal neural crest cells, but the molecular control of sacral neural crest cell development has yet to be elucidated. Under the influence of both intrinsic and extrinsic cues, neural crest cells differentiate into glia and different types of enteric neurones at different developmental stages. Recently, the potential for neural stem cells to form an enteric nervous system has been examined, with the ultimate aim of using neural stem cells as a therapeutic strategy for some gut disorders where enteric neurones are reduced or absent.

Developmental changes in neurite outgrowth responses of dorsal root and sympathetic ganglia to GDNF, neurturin, and artemin.

The ability of glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin to induce neurite outgrowth from dorsal root, superior cervical, and lumbar sympathetic ganglia from mice at a variety of development stages between embryonic day (E) 11.5 and postnatal day (P) 7 was examined by explanting ganglia onto collagen gels and growing them in the presence of agarose beads impregnated with the different GDNF family ligands. Artemin, GDNF, and neurturin were all capable of influencing neurite outgrowth from dorsal root and sympathetic ganglia, but the responses of each neuron type to the different ligands varied during development. Neurites from dorsal root ganglia responded to artemin at P0 and P7, to GDNF at E15.5 and P0, and to neurturin at E15.5, P0, and P6/7; thus, artemin, GDNF, and neurturin are all capable of influencing neurite outgrowth from dorsal root ganglion neurons. Neurites from superior cervical sympathetic ganglia responded significantly to artemin at E15.5, to GDNF at E15.5 and P0, and to neurturin at E15.5. Neurites from lumbar sympathetic ganglia responded to artemin at all stages from E11.5 to P7, to GDNF at P0 and P7 and to neurturin at E11.5 to P6/7. Combined with the data from previous studies that have examined the expression of GDNF family members, our data suggest that artemin plays a role in inducing neurite outgrowth from young sympathetic neurons in the early stages of sympathetic axon pathfinding, whereas GDNF and neurturin are likely to be important at later stages of sympathetic neuron development in inducing axons to enter particular target tissues once they are in the vicinity or to induce branching within target tissues. Superior cervical and lumbar sympathetic ganglia showed temporal differences in their responsiveness to artemin, GDNF, and neurturin, which probably partly reflects the rostrocaudal development of sympathetic ganglia and the tissues they innervate.