Enteric Nervous System Progenitors Are Coordinately Controlled by the G Protein-Coupled Receptor EDNRB and the Receptor Tyrosine Kinase RET

AbstractThe enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals

Growth and metabolism in adult Drosophila

In mammals, dietary nutrients regulate growth and metabolism via a network of specialised organs, including the liver and adipose tissue. Nutrient availability is coupled to growth and metabolism via Insulin and amino acid signals transduced by target of Rapamycin (TOR) and phosphatidylinositol 3-­‐kinase (PI3K). Diet also has multiple complex influences upon lipid metabolism, impacting upon fat storage and numerous other processes. It is known that TOR/PI3K signalling and lipid metabolism can be misregulated in human metabolic disorders such as type 2 diabetes but the underlying mechanisms are complex and challenging to dissect. Drosophila is emerging as a useful model for studying the integrative physiology of lipid metabolism. However, despite much research on lipid metabolism in the developing larva, relatively little is known in the adult fly about which organs other than the fat body are important. To generate new tools to study this issue, I developed an amino acid-­‐defined holidic diet and identified physiological markers of age-­‐dependent metabolism. Using these, I demonstrated that an adult cell type previously implicated in cuticular hydrocarbon synthesis, the adult oenocyte, accumulates lipid droplets in response to a dietary deficiency in methionine. This response is similar to that described for the liver and requires Lpr1, an orthologue of the mammalian Low Density Lipoprotein Receptor. Although many tissues do not grow during adulthood, oenocytes undergo a surprising amino acid and TOR/PI3K dependent volume increase without nuclear DNA replication. Cell ablations and tissue-­‐specific genetic manipulations support a model in which there is bidirectional lipid metabolic cooperation between adult oenocytes and fat body. Together, these results reveal that adult oenocytes are nutrient-­‐responsive and play a hitherto unknown role in lipid metabolism. They also indicate that there are interesting parallels between the metabolic regulation of hepatocytes and adult oenocytes

Neutralisation of myoelectric interference from recorded nerve signals using models of the electrode impedance

Any form of paralysis due to spinal cord injury or other medical condition, can have a significant impact on the quality and life expectancy of an individual. Advances in medicine and surgery have offered solutions that can improve the condition of a patient, however, most of the times an individual’s life does not dramatically improve. Implanted neuroprosthetic devices can partially restore the lost functionalities by means of functional electrical stimulation techniques. This involves applying patterns of electrical current pulses to innervate the neural pathways between the brain and the affected muscles/organs, while recording of neural information from peripheral nerves can be used as feedback to improve performance. Recording naturally occurring nerve signals via implanted electrodes attached to tripolar amplifier configurations is an approach that has been successfully used for obtaining desired information in non-acute preparations since the mid-70s. The neural signal (i.e. ENG), which can be exploited as feedback to another system (e.g. a stimulator), or simply extracted for further processing, is then intrinsically more reliable in comparison to signals obtained by artificial sensors. Sadly, neural recording of this type can be greatly compromised by myoelectric (i.e. EMG) interference, which is present at the neural interface and registered by the recording amplifier. Although current amplifier configurations reduce myoelectric interference this is suboptimal and therefore there is room for improvement. The main difficulty exists in the frequency-dependence of the electrode-tissue interface impedance which is complex. The simplistic Quasi-Tripole amplifier configuration does not allow for the complete removal of interference but it is the most power efficient because it uses only one instrumentation amplifier. Conversely, the True-Tripole and its developed automatic counterpart the Adaptive-Tripole, although minimise interference and provide means of compensating for the electrode asymmetries and changes that occur to the neural interface (e.g. due to tissue growth), they do not remove interference completely as the insignificant electrode impedance is still important. Additionally, removing interference apart from being dependent on the frequency of the interfering source, it is also subject to its proximity and orientation with respect to the recording electrodes, as this affects the field. Hence neutralisation with those two configurations, in reality, is not achieved in the entire bandwidth of the neural signal in the interfering spectrum. As both are less power efficient than the Quasi-Tripole an alternative configuration offering better performance in terms of interference neutralisation (i.e. frequency-independent, insensitive to the external interference fields) and, if possible, consume less power, is considered highly attractive. The motivation of this work is based on the following fact: as there are models that can mimic the frequency response of metal electrodes it should be possible, by constructing a network of an equivalent arrangement to the impedance of electrodes, to fit the characteristic neutralisation impedance – the impedance needed to balance a recording tripole – and ideally require no adjustment for removing interference. The validity of this postulation is proven in a series of in-vitro preparations using a modified version of the Quasi-Tripole made out of discrete circuit components where an impedance is placed at either side of the outer electrodes for balancing the recording arrangement. Various models were used in place of that impedance. In particular, representing the neutralisation impedance as a parallel RC reduced interference by a factor of 10 at all frequencies in the bandwidth of the neural signal while removed it completely at a spot frequency. Conversely, modelling the effect of the constant phase angle impedance of highly polarisable electrodes using a 20 stages non-uniform RC ladder network resulted in the minimisation of interference without the initial requirement of continuous adjustment. It is demonstrated that with a model that does not perfectly fit the impedance profile of a monopolar electrochemical cell an average reduction in interference of about 100 times is achieved, with the cell arranged as a Wheatstone bridge that can be balanced in the ENG band

Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut

Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line-derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest-derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.published_or_final_versio

Multiple Roles of Ret Signalling During Enteric Neurogenesis

The majority of the enteric nervous system is formed by vagal neural crest cells which enter the foregut and migrate rostrocaudally to colonise the entire length of the gastrointestinal tract. Absence of enteric ganglia from the distal colon are the hallmark of Hirschsprung disease, a congenital disorder characterised by severe intestinal dysmotility. Mutations in the receptor tyrosine kinase RET have been identified in approximately 50% of familial cases of Hirschsprung disease but the cellular processes misregulated in this condition remain unclear. By lineage tracing neural crest cells in mice homozygous for a knock-in allele of Ret (Ret51/51), we demonstrate that normal activity of this receptor is required in vivo for the migration of enteric nervous system progenitors throughout the gut. In mutant mice, progenitors of enteric neurons fail to colonise the distal colon, indicating that failure of colonisation of the distal intestine is a major contributing factor for the pathogenesis of Hirschsprung disease. Enteric nervous system progenitors in the ganglionic proximal guts of mutant mice are also characterised by reduced proliferation and differentiation. These findings suggest that the functional abnormalities in Hirschsprung disease result from a combination of colonic aganglionosis and deficits in neuronal circuitry of more proximal gut segments. The reduced neurogenesis in the gut of Ret51/51 mutants was reproduced in the multilineage enteric nervous system progenitors isolated from these animals. Correction of the molecular defects of such progenitors fully restored their neurogenic potential in culture. These observations enhance our understanding of the pathogenesis of Hirschsprung disease and highlight potential approaches for its treatment

Dynamic Image-Based Modelling of Kidney Branching Morphogenesis

Kidney branching morphogenesis has been studied extensively, but the mechanism that defines the branch points is still elusive. Here we obtained a 2D movie of kidney branching morphogenesis in culture to test different models of branching morphogenesis with physiological growth dynamics. We carried out image segmentation and calculated the displacement fields between the frames. The models were subsequently solved on the 2D domain, that was extracted from the movie. We find that Turing patterns are sensitive to the initial conditions when solved on the epithelial shapes. A previously proposed diffusion-dependent geometry effect allowed us to reproduce the growth fields reasonably well, both for an inhibitor of branching that was produced in the epithelium, and for an inducer of branching that was produced in the mesenchyme. The latter could be represented by Glial-derived neurotrophic factor (GDNF), which is expressed in the mesenchyme and induces outgrowth of ureteric branches. Considering that the Turing model represents the interaction between the GDNF and its receptor RET very well and that the model reproduces the relevant expression patterns in developing wildtype and mutant kidneys, it is well possible that a combination of the Turing mechanism and the geometry effect control branching morphogenesis

Contribution of neural crest-derived cells in the embryonic and adult thymus

Abstract Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26eYfp reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.</jats:p

MTG8 interacts with LHX6 to specify cortical interneuron subtype identity

Cortical interneurons originating in the embryonic medial ganglionic eminence (MGE) diverge into a range of different subtypes found in the adult mouse cerebral cortex. The mechanisms underlying this divergence and the timing when subtype identity is set up remain unclear. We identify the highly conserved transcriptional co-factor MTG8 as being pivotal in the development of a large subset of MGE cortical interneurons that co-expresses Somatostatin (SST) and Neuropeptide Y (NPY). MTG8 interacts with the pan-MGE transcription factor LHX6 and together the two factors are sufficient to promote expression of critical cortical interneuron subtype identity genes. The SST-NPY cortical interneuron fate is initiated early, well before interneurons migrate into the cortex, demonstrating an early onset specification program. Our findings suggest that transcriptional co-factors and modifiers of generic lineage specification programs may hold the key to the emergence of cortical interneuron heterogeneity from the embryonic telencephalic germinal zones