Pacemaker Heterogeneity in the Suprachiasmatic Nucleus: Origins and Network Implications

In mammals, the suprachiasmatic nuclei: SCN) in the ventral hypothalamus function as a circadian pacemaker, controlling daily rhythms in behavior and physiology. Together the SCN contain approximately 20,000 neurons that maintain rhythms in firing rate and gene expression. Previous studies led to the assumption that single SCN neurons are capable of self-sustained circadian rhythms. Whether and which SCN neurons can maintain cell-autonomous daily oscillations has not been extensively tested. We measured PERIOD2::LUCIFERASE expression in isolated SCN neurons over multiple days to determine if all SCN neurons were circadian. We then examined neuropeptide content of the recorded neurons. We found that when isolated physically or with a blocker of cell-cell communication, SCN neurons expressed a range of circadian periods, amplitudes, and abilities to sustain cycling. Surprisingly, most cells were sloppy oscillators, switching from rhythmic to arrhythmic or vice versa throughout their lifetime. We also found no evidence for a class of circadian-pacemaker neurons in the SCN based on neuropeptide expression. We conclude that while all SCN neurons are capable of cell-autonomous rhythms, they are intrinsically sloppy with network interactions dramatically increasing the number of circadian neurons. We next used a mathematical model of the mammalian circadian clock to determine whether rates of gene transcription, protein translation, degradation or phosphorylation might explain the ability of SCN neurons to switch between circadian and arrhythmic behaviors. We found that rhythmicity was more sensitive to the rates of protein translation and degradation. We next tested what effect having neurons with different intrinsic circadian behaviors would have on population synchrony. We simulated cells of known circadian phenotypes: e.g. arrhythmic, damped, or self-sustained) in a pattern defined by small-world network properties and varied the positions and proportions of each oscillator type. We found that increasing the number of damped oscillators or placing them in highly connected locations within the network both augmented the rate at which the network synchronized. We conclude that the SCN likely benefit from a heterogeneous population of oscillators, especially when recovering from an environmental perturbation that causes desynchrony. Finally, we generated and characterized two independent lines of transgenic mice to test the role of vasoactive intestinal polypeptide: VIP) neurons in circadian rhythmicity. These mice express Yellow Fluorescent Protein: YFP) under the control of a fragment of the VIP promoter in VIP neurons of the SCN, neocortex, olfactory bulbs, and enteric nervous system. We crossed these mice to generate a line in which VIP neurons are targeted for deletion using Cre-mediated recombination upon addition of tamoxifen. We observed successful deletion of VIP neurons in cultured SCN explants, but have no evidence to date for deletion of SCN neurons in vivo using a variety of protocols. We conclude that our construct is faithfully expressed in VIP neurons and that in vitro experiments show promising results for further study

Emergence and the human genome

Peer reviewedThe (human) genome functions as an open system within human nutritional, economic, cultural, intellectual and emotional contexts. Of profound importance is the extent of free will that emerged with our cognitive and consciousness traits. We have been instrumental in creating particular environments and semiotics according to which we live and with which our genes are expressed. The possibility exists that an information continuum between genes, brain and environment may follow quantum rules and exhibit correlated properties that result in coordinated behaviour (entanglement), even without signal transfer or interaction. With the unprecedented technological advances made during the last century, for the first time a biological organism can, in theory, purposefully design its own future evolution. This is likely to remain limited by ultimate unpredictability due to emergent novelties arising during the process. The effect(s) of a strong human strategic guiding influence, however, implies a tremendous moral responsibility to help shape future outcomes which will enhance the continued existence of quality Life on Earth. How are we doing so far, and how can we exploit knowledge of the possible structural basis of genomic memory and the principles linked with self organisation and emergence to avoid recurrence of outcomes previously shown to have had negative consequences for Life. Can we feed back crucial brain memories to the germline contrary to prevailing dogma, and does this contribute to a compound interest situation not only of intellectual ability but also of a hereditary basis for augmenting ("negative", Machiavellian type) moral behaviour previously found to be successful for pure biological survival?Research Institute for Theology and Religio

Suprachiasmatic nuclei development: A characterization of transcription factors and the influence of retinal innervation and VIP signaling

The suprachiasmatic nuclei: SCN) are highly specialized neural structures with an essential behavioral function; creating the rhythm of the mammalian central clock and entraining that internal clock to the external world. The nuclei each consist of approximately 10,000 neurons, each capable of creating near 24 h rhythms, organized into a highly structured network. While the molecular clockwork underlying the rhythm within neurons and network properties have been well studied, how the nuclei are initially specified and how the network develops is poorly understood. Herein, we seek to elucidate the genes and mechanisms involved in the specification and development of SCN neurons, the SCN network, and circadian function. We first identified genes expressed relatively discretely with the SCN. Using these genes we provided a detailed analysis of transcription factor: TF) and developmental-gene expression within the SCN from neurogenesis through to adulthood in mice (Mus musculus). Through this analysis we identified a genetically distinct neuroepithelium from which SCN neurons are derived and described a gene cascade through which SCN neurons progress as they become postmitotic. In addition, we observed changes in patterns of TF expression through development indicating maturation of nuclei both prenatally and postnatally. We investigated the contribution of critical circadian components in shaping SCN development by monitoring the localization of TF expression in mouse models that lacked either Atoh7, necessary for retinal ganglion cell development, or functional VIP peptide or VIP receptor 2: VPAC2, Vipr2). We found that maturation of TF expression patterns within the SCN occurred independent of retinal innervation and VIP signaling, suggesting that localizations may reflect intrinsic differences in subsets of neurons within the nuclei rather than induced changes. Finally, we began to define specific TFs necessary for SCN development using a Cre/loxP system to temporally localize TF deletion. We found that the well-conserved TF, Six3, is necessary for the initial formation and specification of SCN neurons, but not involved postmitotically in onset or localization of TF or peptide expression. This work begins to reveal aspects of the development of circadian function, by providing a characterization of SCN anatomical development and the first descriptions of TFs necessary for specification

Somatostatin Regulates Circadian Clock Function and Photic Processing

Daily and seasonal rhythms are programmed by neural circuits that use daily timing and duration of light to anticipate predictable environmental changes (i.e., day length, temperature, food, predation). Daily and annual changes in light modulate human health to produce both positive and negative effects, but neural mechanisms underlying light-driven changes in the brain remain poorly understood. In mammals, light is processed and encoded by the brain’s central clock, the suprachiasmatic nucleus (SCN). The SCN also encodes day length (i.e., photoperiod) to regulate annual fluctuations in mammalian physiology, but it’s not clear precisely how the SCN network achieves this. One signal that may contribute to SCN photoperiod encoding is the neuropeptide somatostatin (SST). In rodents, SST expression is modulated by photoperiod in hypothalamic regions regulated by the SCN, suggesting involvement of the central clock. The SCN expresses SST but its role in central clock function and photoperiodic encoding has not been examined. Here, using a range of genetic and imaging approaches, I demonstrate that SST signaling increases circadian robustness in a sexually dimorphic manner. First, I use cellular fate-mapping approaches to demonstrate that SCN SST is regulated by photoperiod in a manner that suggests de novo Sst transcription. Next, I use a battery of circadian behavioral assays to demonstrate that SST contributes to photoperiodic entrainment and circadian responses to light in a manner influenced by sex. However, lack of SST does not alter basic circadian properties, suggesting that SST signaling modulates specific circadian characteristics under particular conditions. Third, I demonstrate that SST regulates SCN neurochemistry via influence on neurons that mediate photic responses. Further, those same cells express a subtype of SST receptor capable of resetting molecular clock function. Last, I demonstrate that lack of SST enhances SCN photoperiodic encoding by modulating photic processing and network communication in a sex-dependent manner. Collectively, these results provide new insight into mechanisms that regulate seasonality and circadian clock function in mammals. The discovery of sexually divergent clock circuits may provide new insights relevant for understanding gender disparities in seasonal/circadian disease states

Mathematical modeling for pattern design in networks of mammalian cells

During the early stages of embryonic development, mammalian cells communicate, undergo morphological changes, and self-assemble into highly organized tissues, and eventually organ systems. Recently, there have been several efforts to engineer the multicellular patterning in mammalian cells to better understand early development and create organoid systems to better understand human disease and drug interactions. However, existing approaches to engineer large scale multicellular patterning in mammalian cells are limited to reproducing well known behaviors or trail-and-error based design. In this thesis, I developed mathematical models to predictively design and quantitatively validate de novo multicellular patterning in mammalian cells. First, I have developed a computational to automate self-organized multicellular organization in human pluripotent stem cells that quantitatively matches the in vitro velocity distribution, temporal dynamics of CRISPR induced perturbations to protein expression, and the resulting changes in spatial organization in human pluripotent stem cell colonies. I have also developed a mathematical model to predict the programmable self-assembly from a single cell into 3D shapes. Overall, this work offers insights into how mathematical modeling can be integrated with pattern recognition and optimization algorithms to efficiently discover and direct self-organized multicellular patterning in cell aggregates and tissues.2021-02-20T00:00:00

Circadian and cardiovascular dysfunction in the Huntington’s disease mouse models

Circadian and sleep disruptions have been shown to lead to an increased risk for cardiovascular (CV) events. Huntington’s disease (HD) patients are reported to have various symptoms including circadian and motor deficits. In addition, CV symptoms are implicated as the cause of death in over 30% of the HD patient population. However, very little is understood about the intersection between circadian disruption, CV disease, and HD. Here we utilized two mouse models, BACHD and Q175, to investigate CV pathology in HD. Reduced diurnal and circadian resting heart rate rhythms, heart rate variability, and baroreceptor reflex support that the autonomic regulation of the CV system is compromised in both HD mouse lines. In addition, age-dependent reduction in the heart function and cardiac fibrosis were observed. These results led us to test the hypothesis that reducing the mutant huntingtin (mHtt) expression in the cardiomyocytes is sufficient to rescue CV symptoms seen in BACHD mice by crossing with the cardiomyocyte-specific Cre, Myh6-Cre. Our results show that reduced mHtt expression in the cardiomyocytes help improve left ventricular ejection fraction, several heart disease markers, and grip strength, a behavioral marker of cardiovascular health in the BACHD mice. Together this data indicates that heart disease in HD patients is likely to be driven both by cardiomyocyte specific pathology as well as dysfunction in the ANS

Visual Pigments and Light Detection in the Eye

Most forms of animal vision begin with light absorption by visual pigments in the eye. A typical visual pigment consists of a G protein-coupled receptor protein – opsin – covalently conjugated to a chromophore. Sub-families of opsins show distinctive physicochemical properties and cellular expressions, often attuned to the specific visual functions that they serve. Here, we examined a number of molecular and functional features of three sub-families of opsins. We found that: (1) an active molecule of rhodopsin (a ciliary opsin expressed in rod photoreceptors for dim-light vision) amplifies the light signal by activating about 20-30 transducin molecules at the peak of the current response to single photon-absorption. (2) the thermal activation of native and some mutant rhodopsin and cone pigments (ciliary opsins in cone photoreceptors for color vision) in the dark is indeed an isomerization event, the rate of which can be quantitatively predicted by multi-vibrational-mode statistical mechanics. (3) melanopsin, a rhabdomeric opsin that underlies the intrinsic photosensitivity of a subgroup of retinal ganglion cells and is responsible for diverse non-image-forming visual functions in mammals, is also expressed in some thick, myelinated neuronal processes in the rat iris that possibly originate from the trigeminal ganglia. (4) neuropsin (OPN5), a previous orphan opsin, mediates the photoentrainment of the local circadian rhythm in the mammalian retina and cornea

Fundamental boolean network modelling for genetic regulatory pathways : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

A Boolean model is a switch-like behaviour model of which it is easy to ignore any effects at the intermediate levels. Boolean modelling has been applied in many areas, including mammalian cell cycle networks. However, little effort has been put into the consideration of activation, inhibition and protein decay networks to designate the direct roles of a gene or a synthesised protein, as an activator or inhibitor of a target gene. Hence, we proposed to split the conventional Boolean functions at the subfunction level into activation and inhibition domains, taking into account the effectiveness of protein decay. As a consequence, two novel data-driven Boolean models for genetic regulatory pathways, namely the fundamental Boolean model (FBM) and the temporal fundamental Boolean model (TFBM), have been proposed to draw insights into gene activation, inhibition, and protein decay. The novel Boolean models could reveal significant trajectories in genes and provide a new direction on Boolean modelling research. The proposed novel Boolean models are fine-grained. A novel network inference methodology named Orchard cube technology has been proposed to infer the related networks, namely fundamental Boolean networks (FBNs) and temporal fundamental Boolean networks (TFBNs) based on FBM and TFBM respectively. As a primary result of this study, an R package, called FBNNet, has been developed based on the proposed methodology and has been used to demonstrate the FBNs and TFBNs for mammalian cell cycle pathways and acute childhood leukaemia pathways respectively. Our experimental results show that the proposed FBM and TFBM could be used to explicitly reconstruct the internal networks of mammalian cell cycles and acute childhood leukaemia. Especially during the study, we produced the fundamental Boolean networks on the childhood acute lymphoblastic leukaemia gene expression data, which were produced in clinical settings. The pathways may be useful for pharmaceutical agents to identify any side effects when applying GC induced apoptosis on children