Anatomy and Neuroscience - Theses
Now showing items 1-12 of 91
The role of neuronal TrkB in central nervous system myelination
Myelin, the specialised membrane surrounding many axons in the nervous system, is vital for the normal sensory and motor function as well as high order function such as learning and memory. Despite the importance of myelin in neural development and functions, little is known whether neuronally expressed molecules can promote the central nervous system (CNS) myelinating process. Through selectively deleting the expression of neurotrophin receptor TrkB in neurons in vivo (TrKB cKO), I have found that neuronal TrkB is essential for oligodendroglial cell production and lineage progression in multiple CNS regions including the lumbar spinal cord white matter tracts, cerebral cortex and corpus callosum and importantly de novo myelination, independent of axonal number and calibre in vivo. I have performed ultrastructural analysis of myelinated axons and found that TrkB cKO mice have significantly fewer myelinated axons compared to control mice during early postnatal development, indicative of delayed initiation of myelination. The analysis of myelin thickness via G ratio has revealed thinner myelin membranes in TrkB cKO mice compared to littermate control mice during development, persisting to late adulthood (n=3 mice/genotype/timepoint). This hypomyelinating phenotype has resulted in impaired myelin function, as evident by reduced optomoter responses in TrkB cKO mice compared to control mice. Moreover, I have found that TrkB cKO mice have significantly fewer oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes in both white and grey matter regions throughout postnatal development and into adulthood compared to littermate controls. A 24-hour EdU labelling experiment has demonstrated that neuronal TrkB expression is required for oligodendroglial production. Interestingly, the overall number of astrocytes and microglia has remained unchanged after neuronal TrkB deletion, suggesting a specific effect upon oligodendroglial lineage cells and myelination. In addition, assessment of neuronal morphology revealed no significant difference in overall dendritic branching including the number of primary and secondary processes. Taken together, results of this PhD study identify that TrkB is a novel neuronal signal that instructs oligodendroglial lineage development and de novo myelination within different neural circuits, indicating a new mechanism that underpins nervous system plasticity.
The KH-domain protein Psi fine-tunes transcription of developmental genes to pattern cell and tissue growth in Drosophila
Mammalian FUBP-family proteins play roles in both transcription and RNA processing, by binding single stranded nucleic acids via their KH domains. Particularly, in vitro mammalian tissue culture studies suggest that FUBP1 drives MYC transcription in response to growth stimuli. MYC is a key regulator of growth and cell cycle progression essential for normal development. Furthermore, expression of MYC is aberrantly upregulated in approximately 70% of all cancers. Understanding the mechanisms that control MYC expression may lead to insights into how MYC dysregulated in diseases such as cancer. However, the significance of the transcriptional control by FUBP1 proteins at the MYC promoter and genome-wide has remained unclear in the context of development in whole organisms. Mammalian studies have been complicated by the fact that mammals have three FUBP-family proteins that bind overlapping genomic targets. However, in Drosophila, the three mammalian FUBP proteins are represented by one ortholog, Psi. Due to a high degree of structural similarity between FUBP1 and Psi proteins, we hypothesized that Psi will also regulate transcription of MYC and other targets. We have taken advantage of the reduced functional redundancy in Drosophila to demonstrate that an essential in vivo function of Psi is control of cell and tissue growth. Analysis of published Co-IP-mass spectrometry screens positioned Psi in an interactome predominantly comprised of RNA Polymerase II transcriptional machinery, particularly the transcriptional Mediator (MED) complex, a known sensor of developmental signalling inputs. Our work indicates that a key transcriptional target of the Psi/MED network that impacts tissue growth is Myc. During development of the wing disc epithelial tissue, Psi was required to promote Myc expression in response to activated PI3K pathway signalling. In addition to Myc, Psi binds to multiple transcriptional targets in the wing epithelium in vivo. Intersection of RNA-seq to detect differentially expressed genes following Psi depletion, and wing-specific direct binding sites identified genome-wide by Targeted DamID, revealed a developmental patterning signature for Psi including Wnt, Notch and TGF-beta cell fate determinant pathways. Moreover, analysis of direct Psi targets identified several novel growth regulators, including the TGF-beta signalling regulator tolkin (tok) and the CD36 scavenger receptor-related epithelial membrane protein (emp). Together our data suggest Psi integrates cellular signalling inputs to directly modulate transcriptional outputs and fine-tune development.
Impact of urbanisation on population’s health in Mtskheta region – Georgia, from Late Bronze Age to Early Middle Ages
Abstract The emergence of urbanisation is considered to be an important period in human history as it is associated with an increase in population density, diversification of subsistence activites, and the establishment of ancient cities, often with poor sanitation. Each of these factors contributed to the spread of disease and a change in diet. Numerous investigations suggest that the impact of early urbanisation on population health and diet is not homogeneous everywhere but varies based on the particularities of the region in question. The Caucasus is one such region. Located between the Black and Caspian seas it has always been chracterized by vibrant cultural dynamism. It acted as a frontier between urban societies of the ancient Near East and the nomadic populations of the ancient Eurasian steppes. As such this is an excellent location to study the effect of urbanisstion on health and diet. This dissertation focuses on paleopathological examinations of skeletal materials from the region of Caucasus, specifically the Greater Mtskheta territories located in the central part of the south Caucasus. The anthropological material from Greater Mtskheta forms one of the largest collections in the region. Human skeletal materials from 13 cemeteries, from three important time-periods were examined: the Late Bronze/Early Iron Age pre-state society with rural-type settlements (15th–6th centuries BC); the Antique period with the emergence of urbanisation and the establishment of the Iberian kingdom (5th century BC–4th century AD); and the Early Middle Age period with developed urbanisation and the collapse of the Iberian kingdom (4th–7th centuries AD). To observe the changes in health and diet throughout these different periods, pathological conditions such as porotic hyperostosis and dental diseases—caries, periapical lesions, antemortem tooth loss and calculus—were examined. Besides these pathologies, molecular analysis of dental calculus was done for these three periods. Statistically, there were no significant differences in lesions and the degrees of activity of porotic hyperostosis. But there was a significant trend in prevalence of porotic hyperostosis across time-periods and between sex. This indicated that deterioration in health conditions during the transitional periods was gradual and did not severely affect the population. The incidences of caries, periapical lesions, antemortem tooth loss and calculus throughout the periods may be associated with changing diet. An insignificant decrease of caries lesions during the urbanisation period may suggest diversification of food production and less dependence of grain consumption, rich in hydrocarbonates, which are responsible for caries. A statistically significant difference in the oral microbiome in pre-and urbanised-state societies may be associated to an increase of frequency of porotic lesions on the skulls and the rise of abscesses over time. The decrease and low incidences of caries throughout the periods may be associated with the absence of specific bacteria in the oral microbiome of the ancient population of Georgia. This study is part of a growing body of research into the impact of early urbanisation on population health and diet. Using certain pathological changes on human skulls, together with analysis of dental diseases, and combining these with the study of oral microbiomes, it contributes to future research on similar topics. This is particularly relevant for the Caucasus, where this type of research has not yet been conducted.
Investigating the role of Seizure related gene 6 family proteins and their BACE shed products at excitatory synapses: impacts on motor and cognitive function
Correct wiring of the brain during development is a complex process that involves the precise spatial and temporal expression of a vast number of genes and proteins. Excitatory synapses in the central nervous system are formed primarily onto specialised protrusions of the dendrite called dendritic spines. During development, synaptic contacts mature into dendritic spines through a process that requires stabilisation and strengthening of the synaptic contact. Strengthening of neuronal connections is not only important for synaptic maturation during development but is also generally assumed to be the cellular process underlying learning and memory in the adult. Seizure related gene 6 (Sez6), Sez6 Like (Sez6L) and Sez6 Like 2 (Sez6L2) comprise a family of homologous proteins widely expressed throughout the brain and Sez6 knockout (KO) mice revealed a role for Sez6 in dendritic arborisation and the development of excitatory synapses. Additionally, Sez6 family proteins have been linked to neurodevelopmental disorders, with which synaptic dysfunction is commonly associated. Therefore, the broad aim of this project was to further characterise the role of Sez6 family proteins, and their shed ectodomains, in synaptic structure and function and additionally investigate their role in motor and cognitive function in the mouse. To elucidate the functions of Sez6 family proteins in the central nervous system, wild-type (WT) and Sez6 triple knockout (TKO) mice, which lack all three Sez6 family proteins, were used. Compared to WT, Sez6 TKO mice were found to have reduced spine density in the hippocampus and dendritic spines were shifted to more immature morphologies in the somatosensory cortex. Additionally, synaptic function was found to be altered in the prelimbic cortex. Sez6 TKO mice had impaired motor learning and motor coordination from as early as 6 weeks old. Cognitive testing revealed Sez6 TKO mice had enhanced stress responsiveness, impaired working and spatial short-term memory but intact spatial long-term memory in the Morris water maze that was accompanied by a reversal deficit. Sez6 family members are substrates of the Alzheimer’s protease, beta-amyloid precursor protein cleaving enzyme 1 (BACE1), and are cleaved proximal to the transmembrane domain to produce a shed ectodomain. The contribution of the shed Sez6 family protein ectodomains to cognition, and the potential for BACE inhibitor treatment of Alzheimer’s disease to be associated with cognitive side-effects as a result of impaired processing of other non-amyloid precursor protein substrates, was assessed by chronically treating WT and Sez6 TKO mice with a BACE inhibitor. In both WT and Sez6 TKO mice, BACE inhibition did not significantly impact cognition or anxiety-related behaviour in the behavioural paradigms utilised. WT mice treated with BACE inhibitor were, however, found to be hyperactive on the elevated open field and this effect was not observed in treated Sez6 TKO mice suggesting a role for the shed ectodomains of Sez6 family proteins in regulating activity levels. The results presented in this thesis further our understanding of the role of Sez6 family proteins in synaptic structure and function and contribute to the body of evidence that links Sez6 family proteins to the cognitive and motor deficits observed in patients with neurodevelopmental disorders, such as autism and schizophrenia.
Transcriptomic diversification along the monocyte-macrophage continuum.
Current models of innate immune responses describe hard wired, gene-centric signalling networks, with limited capacity to define the molecular mechanisms underpinning transcriptomic diversity. It is well established that a transcriptional spectrum of responses accompanies acute macrophage activation, however it is unclear whether this spectrum originates in monocytes and to what extent it continues throughout reinfection. The contribution of molecular mechanisms such as enhancers and alternative transcription start sites is also undetermined. Given the critical roles that myeloid cells play in directing acute infection and priming adaptive immunity, it is important the regulation of their responses be understood. This thesis employed bioinformatic analysis of Cap Analysis Gene Expression (CAGE) and microarray data to describe transcriptional diversity along the monocyte-macrophage continuum. Using CAGE to map transcription start sites for capped RNAs, this thesis has shown that pathogen-specific transcriptional diversification commences early in monocyte infection (Chapters Three and Four) and continues throughout acute macrophage infection (Chapter Five). Transcriptomic diversity during acute infection was the product of kinetic and pathogen-specific engagement of distinct transcription start sites. Engagement of multiple transcription start sites drove responsiveness by regulating expression amplitude in functionally focused inflammatory gene sets and diversifying secondary response networks via expression of distinct protein isoforms. Chapter Six extended these studies of acute infection, demonstrating that transcriptional phenotypes continue to diversify during reinfection. These findings highlight the importance of studying innate immune responses at the isoform level and prompt the need to revise current models of innate immune signalling, such that monocyte-macrophage biology should no longer be modelled as a series of static states, but rather, as a continually evolving continuum.
Development of in vitro and in vivo models for the study of myelin plasticity
The central nervous system (CNS) constantly responds to changes in environmental stimuli by undergoing structural and functional modifications. Some stimuli induce persistent CNS changes which in turn underpin adaptive behaviours that enable individual animals to function in their unique environmental circumstances. This phenomenon, referred to as neuroplasticity, has been studied predominantly with respect to adaptive neuronal changes, and has focused primarily on synaptic changes and the molecular transduction mechanisms that mediate them. It is increasingly recognised, however, that glial cells can also be modified by external stimuli. Oligodendrocytes – the myelinating glia of the CNS which facilitate efficient nerve impulse conduction and support axonal metabolism – have also been demonstrated to undergo long term changes in response to environmental stimuli. Experience-dependent changes in oligodendrocyte number or myelination could underpin adaptive behaviours via modifications to neuronal metabolism and nerve impulse conduction. The emerging consensus is that stimulation – whether indirectly through modulating sensory, motor, or social experience, or directly through modulating neuronal activity – increases oligodendroglial lineage progression and myelin production. It has further been demonstrated that myelin plasticity is an axon-specific phenomenon whereby, when given the choice, myelin segments preferentially form on axons that are more highly active relative to those that are nearby but less active. The molecular mechanisms that mediate myelin plasticity are not well understood, and studies addressing this question have predominantly focused on the role of extracellular, pro-myelinating signals released by neurons in an activity-dependent manner. Comparatively little is known about the oligodendroglial intrinsic molecular transduction mechanisms that mediate myelin plasticity. This thesis aimed to develop a model system for studying myelin plasticity, including in particular to investigate the molecular transduction mechanisms that are triggered within oligodendroglia to mediate myelin plasticity. In developing such a model, two approaches were employed. First, an in vitro myelinating co-culture model was developed. A standard co-culture protocol was adopted and refined to produce robustly myelinating co-cultures of dorsal root ganglion (DRG) neurons and oligodendrocyte precursor cells (OPCs). To stimulate neuronal activity, both the hM3Dq pharmacogenetic and the channelrhodopsin-2 (ChR2) optogenetic techniques were explored. The pharmacogenetic stimulation technique was ineffective at driving DRG neurons to the levels of activity reportedly required for inducing myelin plasticity. In contrast, the optogenetic stimulation technique reliably drove DRG neurons to fire at the required frequency. Contrary to expectations, optogenetic stimulation did not increase myelin production in co-cultures, nor did it increase the propensity of myelin segments to preferentially form on optogenetically stimulated relative to control axons. The reasons for this are unclear, but are unlikely to be related to phototoxicity and are more likely to be explained by a negative effect of high ChR2 expression on myelination in these co-cultures. Second, an in vivo pharmacogenetic model was employed to drive activity of cortical neurons in juvenile hM3Dq transgenic mice. Contrary to expectations, there was no evidence for an activity-dependent increase in oligodendroglial lineage progression. The reasons for this are unclear, however they could relate to the young age of the animals in this relative to other studies of myelin plasticity or to the large population of neurons undergoing activity manipulation in this relative to other studies of myelin plasticity. The implications for glial plasticity, and for how it is studied, are discussed.
The effect of genetic susceptibility and immunosuppressant treatment on mononuclear cell phenotype in multiple sclerosis
Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The immune system plays a significant role in the pathophysiology of the disease and research into the mechanisms that may be potentially contributing to the disease is essential to help advance knowledge in the field and ultimately seek better outcomes for MS patients. A key set of cells that are believed to play a role in MS immunopathology are mononuclear cells which consist of B-cells, T-cells, monocytes and their derivatives. The aims of this thesis was to assess the effects of 1) genetic susceptibility and 2) immunosuppressant treatment, on mononuclear cell phenotype in MS. The first aim involved interrogating a key co-stimulatory molecule, cluster of differentiation 40 (CD40), expressed on mononuclear cells which is known to play a fundamental role in the regulation of the humoral immune response. CD40 is a risk gene for the development of MS and I sought to assess the effects of the single nucleotide polymorphism (SNP) rs1883832 on the phenotype of CD40-expressing mononuclear cells. Utilising a range of bimolecular methods and samples taken from MS individuals and healthy matched-controls (HC), I showed that SNP rs1883832, particularly the ‘risk’ genotype (homozygous TT) was correlated with reduced soluble CD40 (sCD40) levels in HC, but not MS patients. I did not observe any phenotype-dependent differences in sCD40 levels between MS (in quiescent state) and during active demyelination compared to HC. Upon assessment of key cytokines produced by B-cell and monocytes, I observed elevated gene expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in the MS population linked to the CD40 risk TT-genotype. In the second component, I showed that the immunosuppressant drug Cladribine, is highly toxic to mononuclear cells, and that it down-regulates the expression of the key phagocytic marker MERTK at the protein level (preliminary data, study is ongoing). In summary, the data presented in this thesis suggests that the CD40 risk gene and the immunosuppressant Cladribine play roles in altering the phenotype in mononuclear cells. The understanding from this body of work provides some insights into how genetic susceptibility can drive changes in mononuclear cell phenotype which may impact on the modulation of co-stimulatory mechanisms in humoral immunity. Moreover, the findings from the Cladribine study may assist the field in the discovery of alternative mechanisms of the drug on mononuclear cells and therefore inform a refined approach to treatment of MS.
Regulation of Drosophila Myc transcription by the single stranded DNA/RNA binding proteins Psi and AGO1
MYC has wide-ranging functions, with potential to amplify transcriptional output to activate cell growth, metabolism and cell cycle progression, thus driving oncogenic programs. As even small increases in MYC abundance are sufficient to drive the proliferative cell growth fundamental to tumour progression, understanding the molecular mechanisms controlling MYC expression will provide insight into mechanisms of MYC dysregulation in cancer. In mammalian in vitro and ex vivo systems, the KH domain single stranded DNA/RNA binding protein FUBP1 binds the Far Upstream Sequence Element (FUSE) in the activated MYC promoter to modulate transcription. Using genetic models, this thesis demonstrates that the sole FUBP family member in Drosophila, Psi, is essential for Myc transcription, cell and tissue growth in the wing epithelium, in vivo. Psi is not only required to maintain endogenous levels of Myc mRNA abundance, but depletion results in a significant increase in RNA Pol II activity on the Myc gene. Consistent with Psi regulating Myc at the level of transcription, we demonstrate Psi interacts physically and genetically with the transcriptional Mediator complex (MED), thus providing a mechanism for integration of developmental signals for patterning Myc transcription, cell and tissue growth in the wing imaginal disc epithelium. We further report physical and genetic interaction between Psi and AGO1, the RNA binding protein component of the RNA-induced silencing complex (RISC). AGO1 loss-of-function mutations restored growth in the Psi knockdown wing, suggesting negative roles for AGO1 in Psi-dependent cell and tissue growth. AGO1 depletion was not only sufficient to increase Myc mRNA and protein abundance in the wing, but also increased Myc function i.e. transcriptional activity as measured by increased abundance of Myc targets required for ribosome biogenesis and cell growth (e.g. Ribosomal Proteins, rRNA and RNA Pol I subunits). Myc knockdown returned AGO1-depleted nucleolar compartments to the normal range, demonstrating dependency of increased growth on Myc i.e. rather than direct effects of AGO1 on ribosomal components. Interestingly, the following observations suggest AGO1 represses Myc at the level of transcription: 1) significant AGO1 enrichment on Myc promoter by ChIP, 2) physical interaction between AGO1 and MED, and 3) that AGO1 depletion activates the Myc promoter and the increased Myc mRNA requires RNA Pol II transcriptional activity. Together, these observations suggest a novel role for AGO1 as a transcriptional repressor of Myc, which underlies the tumour suppressor behaviour observed for AGO1 in the Drosophila wing.
Using induced pluripotent stem cells to model primary open-angle glaucoma
Glaucoma is a group of optic neuropathies that may be characterized by gradual degeneration of retinal ganglion cells (RGCs) and their axons leading to irreversible vision loss [1, 2]. Glaucoma is the second leading cause of blindness worldwide [3-5] and it is estimated that the number of people affected by the disease will reach 80 million by 2020, while more than 11 millions will be bilaterally blind . In this project I focus on primary open-angle glaucoma (POAG), as it accounts for the majority of glaucoma cases worldwide. So far, multiple risk factors for glaucoma have been identified; however, the exact mechanism causing RGC loss in patients remains elusive. Furthermore, examination of RGCs affected in POAG is difficult pre-mortem due to their anatomical location. To overcome this problem, somatic cells can be reprogrammed into patient-specific induced pluripotent stem cells (iPSCs), which can be then differentiated into cell type of interest, i.e. RGCs. This PhD project consisted of several steps. First, I assessed the feasibility of transferring the iPSC culture into the automated platform. Using automation was essential to generate large number of samples required for analysis. The transition to automation was successful, as evidenced by maintenance of iPSC morphology, expression of pluripotency markers and ability to differentiate into derivatives of three germ layers. I also demonstrated that incorporating automation into human (h) iPSC culture allows standardization of maintenance and passaging procedures reducing inter-sample variability and human error. I subsequently used the platform to generate over 300 hiPSC lines for POAG modelling. In parallel, I optimized RGC differentiation protocol to obtain sufficient number of cells for their examination with single cell RNA sequencing (scRNA-seq). Next, iPSC-derived RGCs were subjected to scRNA-seq to gain in-depth information about transcriptomic differences between healthy controls and POAG patients. Understanding mechanisms underlying RGC function, maintenance of homeostasis and those conferring susceptibility to POAG is crucial to discover new therapeutic targets and commence the process of drug discovery.
Cellular and subcellular co-storage of gastrointestinal hormones
Gastrointestinal hormones regulate a diverse range of physiological processes including digestion, metabolism, and food intake, as well as maintaining mucosal integrity and mounting defensive mechanism in response to pathogens or toxins. Disruptions of these regulatory processes are associated with disorders that include diabetes, obesity, irritable bowel syndrome, and coeliac disease. Despite this clinical significance, many aspects of the cells that contain gut hormones, enteroendocrine cells (EEC), remain poorly characterised. Historically, EEC were categorised based on their hormone content, with the assumption that each cell contained a single hormone. However, hidden in some early studies were examples that the simple ‘one cell‐one hormone’ classification system was not correct. A major focus of this thesis is the characterising of coexpression patterns of hormones in mouse and human intestine. Remarkably, examples of overlap could be found for all hormones investigated, demonstrating a need for a revised classification system for EEC. Moreover, species differences in EEC expression patterns occur, highlighting a further level of complexity in the field of research. The coexpression of EEC hormones raises new questions about how these hormones are stored at a subcellular level. Super‐resolution microscopy allowed the visualisation and quantification of vesicular stores in EEC. Overlapping and non‐overlapping vesicular stores were observed. Furthermore, the relative abundance of hormone expression varied considerably between cells and, consequentially, small amounts of hormones sometimes could not be detected with low‐resolution methods. In addition, a monoclonal antioxyntomodulin antibody was characterised, expanding the examination of hormone colocalisation patterns to the investigation of proglucagon‐derived peptides. Several hormones, including 5‐HT, influence electrolyte transport, which can be measured as the shortcircuit current (Isc) in an Ussing chamber. The effects of 5‐HT and secretin, which are frequently colocalised, on electrolyte transport across the intestinal epithelium were examined. Both hormones stimulated a secretory response in all regions of the mouse intestine, including the colon where secretin was not thought to be expressed. However, secretin gene transcripts were identified and secretin immunoreactivity was localised to EEC in the mouse colon. The functional effects of TRPA1, which is an ion channel that has previously been localised to EEC containing both 5‐HT and CCK, was also examined using an Ussing chamber. While TRPA1 agonists stimulated a secretory response, this was not mediated by 5‐HT. This research has advanced knowledge of the colocalisation patterns of gastrointestinal hormones at a cell and subcellular level and explored some aspects of the functions of costored hormones in relation to water and electrolyte movement across the gut lining.
Determining the role of the paratrigeminal nucleus (Pa5) in airway defence
Respiratory sensations conveyed by airway sensory nerve fibres are poorly understood, yet they contribute significantly to morbidity in pulmonary disease. Our viral tracing studies identified a previously unknown airway sensory circuit in the brain projecting through an obscure medullary site known as the paratrigeminal nucleus (Pa5). The Pa5 is located in the dorsal lateral medulla and is commonly defined as a collection of interstitial cells in the dorsal tip of the spinal trigeminal tract. The Pa5 has been previously implicated in baroreceptor function and somatosensory processing. Astonishingly, very little investigations have been made to understand its role in airway afferent processing. We therefore investigated the anatomical connectivity of the Pa5, its involvement in initiating and controlling respiratory reflexes as well as in more complex respiratory behaviours including evoked cough. Conventional neuroanatomical tracing was conducted to determine the input and output connectivity of the Pa5. Firstly, retrograde tracing confirmed, in the guinea pig, that vagal afferents from the jugular versus the nodose ganglia project specifically into the brainstem primary afferent termination sites. That is the nodose vagal ganglia project almost exclusively to the nucleus of the solitary tract (nTS), while the jugular vagal ganglia project predominately to the Pa5. Furthermore, anterograde tracing was employed from the guinea pig Pa5, showing that this nucleus has extensive projections throughout the cardiorespiratory column, including the nTS, reticular nuclei, pre-bötzinger nucleus and in particular the parabrachial and Kölliker-Fuse nuclei in the pons. We have shown functional significance of these projections by implicating the Pa5 in the initiation and control of laryngeal-evoked respiratory reflexes. Initially we found that laryngeal-evoked respiratory slowing was significantly attenuated by blocking the Pa5 using the GABAA agonist muscimol (stimulus evoked change in breaths/min pre-muscimol was 40.9±3.5 breaths/min pre-muscimol vs. 21.4±2.9 post-muscimol, p=0.001), while this pharmacological modification in the nTS had no effect on the laryngeal-evoked respiratory slowing. Importantly, this novel finding showed that the Pa5 has a role in respiratory reflexes that is independent of the nTS. Furthermore, injection of glutamate receptor antagonists into the Pa5 were able to prevent respiratory slowing evoked by laryngeal stimulation (change in respiratory rate at the maximum response 40.3±4.1 breaths/min vs. 17.3±3.1 breaths/min; p=0.0002), while peptide receptor antagonists resulted in significantly smaller effects. Intriguingly, while injection of glutamate into the Pa5 resulted in respiratory slowing and in some cases apnoea, mimicking the laryngeal-evoked reflex, the direct release of neuropeptides by capsaicin microinjection into the Pa5 resulted in the paradoxical increase in respiratory rate. This unique response could be blocked by Substance P receptor antagonists but not by glutamate receptor antagonists, suggestive of two types of Pa5 postsynaptic neurons. Additional anatomical characterisation of the Pa5 revealed the existence of two phenotypically distinct subtypes of Pa5 neurons. That is, one expressing the neurokinin 1 receptor and the other expressing calbindin. Furthermore, dual retrograde tracing from nuclei important for respiratory rhythm and modulation and were shown to receive direct projections from the Pa5 (i.e. the ventrolateral medulla and pontine nuclei) revealed that less than 7% of Pa5 neurons were dual labelled and therefore revealed that Pa5 neurons project differentially throughout the cardiorespiratory column. Given the complex processing capability of the Pa5, it seemed plausible that this nociceptive region may be important in complex respiratory behaviours, such as evoked cough and the perception of airway irritations. We developed an assay in which we can study the conscious perception of an airway irritation using behaviours (enhanced grooming, moving and chewing), in addition to the standard assessment of evoked cough. Conscious unrestrained guinea pigs were aerosolised bradykinin (a jugular and nodose C-fibre stimulant) and adenosine 5’-triphosphate (ATP, a nodose selective stimulant). While bradykinin evoked both dose dependent increases in cough (8.9±2.7 at 1mg/ml and 25.7±3.7 at 3mg/ml) and behaviours (20.7±45.8 behaviour duration during saline vs., 350.4±46.7 total behaviour duration, p=0.04), ATP only evoked responses at the highest dose (22.7±5.6 coughs and 81.3 behaviour duration during saline vs. 404±109.5 total behaviour duration). These distinct cough and behavioural profiles are suggestive that jugular and nodose airway afferents differentially process respiratory sensations. This was further supported by showing that targeted toxin lesions (substance P-Saporin, 10ng/100nl) of the Pa5 resulted in decreased bradykinin evoked cough compared to controsl (i.e. 7.2±2.8 coughs control vs. 0±0 coughs lesion at 1mg/ml and 19.2±4.1 coughs control vs. 10.4±3 coughs lesion at 3mg/ml, p=0.02 and p=0.009 respectively). Alternately, lesioning NK1 receptor expressing neurons in the Pa5 had no effect on ATP evoked cough, in turn implicating NK1 receptor expressing Pa5 neurons specifically process jugular C-fibre evoked cough. Together these data are the first to reveal a complexity of airway afferent processing in the Pa5. Better understanding this putative airway somatosensory system may help identify therapeutic targets to alleviate respiratory discomfort in disease.
The transcription factor grainy head regulates intestinal stem cells
The constant renewal of the intestinal epithelium is achieved through the activity of a population of intestinal stem cells (ISC). These cells maintain tissue homeostasis by ensuring balance between cell division and the production of the differentiated cell types of the intestine. This process is under strict molecular control with ISC maintenance and function controlled by the expression of a particular subset of genes and their production of multiple protein isoforms. However, the mechanism by which different protein isoforms are able to regulate intestinal regeneration remains poorly understood. Using the adult intestine of Drosophila melanogaster, we identified grainy head (grh) as a candidate gene that is able to influence regeneration using different protein isoforms. The Drosophila genome contains a single grh gene which is alternatively spliced to produce 8 mRNA transcripts and two major isoform groups, the N-isoforms and the neural specific, O-isoforms. In this study, I show using droplet digital PCR that transcripts for both isoform groups including the neural specific isoforms are expressed in the midgut, albeit at low cellular levels. Moreover, cell lineage tracing studies using grh null mutants result in a reduction of progeny arising from a single ISC. Interestingly, cell lineage tracing using an O-specific mutant resulted in a more severe reduction in progeny number compared to grh null mutants. Additionally, lineage traced ISCs mutant for the O-isoforms were lost over time. In the opposite experiment, an O-isoform, GRH.O’ was ectopically expressed in ISCs and its immediate daughter, the enteroblast (EB). Over expression solely in ISCs did not lead to a change in ISC number while over expression in EBs led to a block in differentiation and an increase in EB number. Conversely, ectopic expression of two GRH N-isoforms, GRH.N and GRH.N’ led to a loss of ISC and EBs through forced differentiation. I therefore hypothesize that GRH O-isoforms are required for ISC maintenance while the N-isoforms are required for differentiation in the Drosophila midgut. In the final part of my study, I conducted expression analysis for GRHL-2 and GRHL-3, the mammalian orthologues of GRH in the mouse small intestine. Expression studies using qPCR and immunohistochemistry reveal that both GRHL-2 and GRHL-3 are expressed in intestinal crypt cells suggesting that the function of GRH is conserved the mammalian small intestine.