Biology subjects

Hastings, M. H.

Publications and source records attributed to Hastings, M. H..

12 recordsLinked to original sources

bioRxiv2026

PER2- and state-dependent transcriptional programs gate neural stem cell proliferation with niche-specific circadian autonomy

Adult neural stem cells (NSCs) in the mouse brain are predominantly quiescent, with activation tightly regulated to balance neurogenesis and stem cell maintenance. Circadian clocks temporally organize core cellular processes, potentially gating NSC activation. We examined adult NSC temporal dynamics across the day and observed rhythmic expression of core circadian clock components BMAL1 and PER2 in both mammalian niches, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ). While cell cultures derived from both niches show BMAL1 and PER2 protein, only SGZ-derived NSCs exhibit synchronized self-sustained core clock oscillations across the population. Comparative analyses of WT and Per2 knockout NSCs revealed state-specific, circadian clock-dependent oscillatory transcriptional programs. This identified ASCL1 and CCND1 as candidate regulators of cell-cycle coordination, with daytime accumulation preceding a nighttime S-phase peak. The temporal control of S-phase entry in NSCs was abolished in Per2 knockout mice. Together, our findings reveal state-specific, PER2-dependent circadian regulation of adult NSCs in both neurogenic niches, and a specific dependence on external stimuli for synchronization in the SVZ.

cell biology↗

bioRxiv2025

Magnetic Fields Influence Visual Responses in Mice

Many animals use the Earths magnetic field for the purposes of orientation and navigation, although the sensory mechanisms remain unclear. It has been proposed that retinal responses to light may be modulated by magnetic fields. However, to date, there is no evidence for a retinal response to magnetic fields in mammals. Here we show that magnetic fields affect expression of the neuronal activity marker c-Fos in the mouse retina in a light dependent manner. These retinal responses to magnetic fields are abolished in mice lacking the candidate magnetoreceptor cryptochrome. To characterise the signalling pathways involved, we then used RNAseq and cell-type mapping. We also show that magnetic fields increase exploratory behaviour in a visually dependent task and lengthen the period of the retinal circadian clock. Together, our data provide the first evidence for a mammalian retinal response to magnetic fields at a cellular, molecular and functional level, which may influence vision.

neuroscience↗

bioRxiv2025

Circadian and circatidal oscillations of clock gene expression in brains of Eurydice pulchra and Parhyale hawaiensis.

Intertidal organisms, such as the crustaceans Eurydice pulchra and Parhyale hawaiensis, express daily and tidal rhythms of physiology and behaviour to adapt to their temporally complex environments. Although the molecular-genetic basis of the circadian clocks driving daily rhythms in terrestrial animals is well understood, the nature and mechanism of the circatidal clocks driving tidal rhythms remain a mystery. Using in situ hybridisation, we identified discrete clusters of [~]60 putative "clock" cells co-expressing canonical circadian clock genes with comparable distributions across the protocerebrum of E. pulchra and P. hawaiensis brains. In tidally rhythmic, field-collected E. pulchra sampled under a light:dark (LD) cycle, the expression of period (per) and cryptochrome 2 (cry2) exhibited daily rhythms in particular cell groups (e.g., medioposterior cells) whereas timeless (tim) showed 12-hour rhythms in others (e.g., medial cells). In tidally rhythmic laboratory-reared P. hawaiensis, previously entrained to 12.4-hour cycles of agitation under LD and sampled under continuous darkness, several cell groups (e.g., medioposterior cells) exhibited circadian expression of per and cry2. In contrast, dorsal-lateral cells in the protocerebrum exhibited robust [~]12-hour, i.e., circatidal, rhythms of per and cry2, phased to the prior tidal agitation but not the prior LD. In P. hawaiensis exhibiting daily behaviour under LD without tidal agitation, robust daily rhythms of per and cry2 expression were evident in medioposterior and other cells whereas expression in dorsal-lateral cells was not rhythmic, underlining their intrinsic tidal periodicity. These results implicate canonical circadian mechanisms in circatidal time-keeping and reveal conserved brain networks as potential neural substrates for the generation of interactive daily and tidal rhythms appropriate to intertidal habitats.

neuroscience↗

bioRxiv2023

Loss of zinc finger homeobox-3 (ZFHX3) affects rhythmic gene transcription in mammalian central clock

The mammalian suprachiasmatic nucleus (SCN), situated in the ventral hypothalamus, directs daily cellular and physiological rhythms across the body. The SCN clockwork is a self-sustaining transcriptional-translational feedback loop (TTFL) that in turn co-ordinates the expression of clock-controlled genes (CCGs) directing circadian programmes of SCN cellular activity. In the mouse, the transcription factor, ZFHX3 (zinc finger homeobox-3), is necessary for the development of the SCN and influences circadian behaviour in the adult. The molecular mechanisms by which ZFHX3 affects the SCN at transcriptomic and genomic levels are, however, poorly defined. Here, we used chromatin immunoprecipitation sequencing (ChIP-seq) to map the genomic localization of ZFHX3 binding sites in SCN chromatin. To test for function, we then conducted comprehensive RNA sequencing at six distinct times-of-day to compare the SCN transcriptional profiles of control and ZFHX3-conditional null mutants. We show that the genome-wide occupancy of ZFHX3 occurs predominantly around gene transcription start sites (TSS), co-localizing with known histone modifications, and preferentially partnering with clock transcription factors (CLOCK, BMAL1) to regulate clock gene(s) transcription. Correspondingly, we show that the conditional loss of ZFHX3 in the adult has a dramatic effect on the SCN transcriptome, including changes in the levels of transcripts encoding elements of numerous neuropeptide neurotransmitter systems while attenuating the daily oscillation of the clock TF Bmal1. Furthermore, various TTFL genes and CCGs exhibited altered circadian expression profiles, consistent with an advanced in daily behavioural rhythms under 12h light-12h dark conditions. Together, these findings reveal the extensive genome-wide regulation mediated by ZFHX3 in the central clock that orchestrates daily timekeeping in mammals.

neuroscience↗

bioRxiv2023

Astrocytic control of extra-cellular GABA drives circadian time-keeping in the suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity. Intercellular signals synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level time-keeping is unclear. SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counter-intuitively, runs in antiphase to neuronal activity, peaking in circadian night. Resolving this paradox, we found that [GABA]e is regulated by GABA transporters (GATs), uptake peaking during circadian day. This is mediated by the circadian-regulated, astrocytically expressed GAT3 (Slc6a11). Clearance of [GABA]e in circadian day facilitates neuronal firing, neuropeptide release and TTFL rhythmicity. Moreover, genetic complementation demonstrated that the astrocytic TTFL can alone drive [GABA]e rhythms. Thus, astrocytic clocks maintain SCN circadian time-keeping by temporally controlling GABAergic inhibition of SCN neurons.

neuroscience↗

bioRxiv2022

The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal ([~]12.4 hour) or other lunar-mediated cycles ([~]15 day, [~]29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between the positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2. Epcry2 and Epbmal1 knockdown disrupted Eurydices circadian phenotypes as expected but in addition, circatidal behaviour was also sensitive to Epbmal1 knockdown. Thus three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM, do not appear to be required for the expression of circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators BMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

genetics↗

bioRxiv2022

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus mediates daily timekeeping in mammals

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is crucial for synchronising and resetting all cellular rhythms in accordance with critical environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. Here, we sought to identify SCN enriched gene regulatory elements that enable temporal gene expression using histone-ChIP-seq. We found a vast majority of SCN enhancers not only exhibit robust 24-hour rhythmic modulation in H3K27ac occupancy, but also possess canonical E-box (CACGTG) motif, potentially influencing downstream cycling gene expression. In parallel, we conducted RNA-Seq at six distinct times to establish enhancer-gene relationships in the SCN. Surprisingly, around 35% of cycling H3K27ac abundance is seen adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass non-coding actively transcribing enhancer RNAs (eRNAs), that in turn oscillate along with cyclic histone acetylation to direct gene transcription. Taken together, these findings shed light on genome-wide pre-transcriptional regulation operative in the central clock that enables its orchestration of daily timekeeping mechanisms in mammals.

genomics↗

bioRxiv2022

Beneficial Effects of Moderate Hepatic Activin A Expression on Metabolic pathways, Inflammation, and Atherosclerosis

BACKGROUNDAtherosclerosis is an inflammatory vascular disease marked by hyperlipidemia and hematopoietic stem cell (HSC) expansion. Activin A, a member of the Activin/GDF/TGF{beta}/BMP family is broadly expressed and increases in human atherosclerosis, but its functional effects in vivo in this context remain unclear. METHODSWe studied LDLR-/- mice on a Western diet for 12 weeks and used adeno-associated viral vectors with a liver-specific thyroxine binding globulin (TBG) promoter to express Activin A or GFP (control). Atherosclerotic lesions were analyzed by oil red staining. Blood lipid profiling was performed by HPLC (High Performance Liquid Chromatography), and immune cells were evaluated by flow cytometry. Liver RNA-sequencing was performed to explore the underlying mechanisms. RESULTSActivin A expression decreased in both livers and aortae from LDLR-/- mice fed a Western diet compared with chow. AAV-TBG-Activin A increased Activin A hepatic expression ([~]10-fold at 12-weeks, p<0.0001) and circulating Activin A levels ([~]2000pg/ml vs [~]50pg/ml, p<0.001, compared with controls). Hepatic Activin A expression decreased plasma total and low-density lipoprotein (LDL) cholesterol ([~]60% and [~]40%, respectively), reduced inflammatory cells in aortae and proliferating hematopoietic stem cells (HSC) in bone marrow, and reduced atherosclerotic lesion area in the aortic arch by [~]60%. Activin A also attenuated liver steatosis and expression of the lipogenesis genes, Srebp1 and Srebp2. RNA sequencing revealed Activin A not only blocked expression of genes involved in hepatic de novo lipogenesis but also fatty acid uptake, and liver inflammation. In addition, Activin A expressed in the liver also reduced white fat tissue accumulation, decreased adipocyte size, and improved glucose tolerance. CONCLUSIONSOur studies reveal hepatic Activin A expression reduces inflammation, HSC expansion, liver steatosis, circulating cholesterol, and fat accumulation, which likely all contribute to the observed protection against atherosclerosis. The reduced Activin A observed in LDLR-/- mice on a Western diet appears maladaptive and deleterious for atherogenesis.

pathology↗

bioRxiv2021

Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in RBCs cultured in vitro or taken from freely behaving mice or humans exhibit and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via NO signaling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

cell biology↗

bioRxiv2021

Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock

The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

systems biology↗

bioRxiv2021

The molecular clockwork of the suprachiasmatic nucleus is sufficient to co-ordinate phasing and stabilisation of sleep-wake cycles and enhance memory deficits in a clockless mouse

The timing and quality of sleep-wake cycles are regulated by interacting circadian and homeostatic mechanisms. Although the suprachiasmatic nucleus (SCN) is the principal circadian clock, local clocks are active across the brain and the respective sleep-regulatory roles of SCN and extra-SCN clocks are unclear. To determine the specific contribution(s) of the SCN, we used virally mediated genetic complementation, expressing Cryptochrome1 (Cry1) to restore circadian molecular competence to the SCN of globally clockless Cry1/Cry2-null mice. Under free-running conditions, the rest/activity behaviour of Cry1/Cry2-null controls which received EGFP (SCNCon) was arrhythmic, whereas Cry1-complemented mice (SCNCry1) had circadian behaviour comparable to that of Cry1,2-competent wild-types (WT). In SCNCon mice, sleep-wakefulness, assessed by electroencephalography/electromyography, also lacked circadian organisation. In SCNCry1 mice, however, it was comparable to WT, with consolidated vigilance states (wake, REM and NREM sleep) and rhythms in NREMS delta power and expression of REMS within total sleep. Wakefulness in SCNCon mice was more fragmented than in WT, with more wake-NREMS-wake transitions. This disruption was corrected in SCNCry1 mice. Following sleep deprivation, all mice showed an initial homeostatic increase in NREMS delta power. The SCNCon mice, however, had reduced, non-consolidated NREMS during the inactive phase of the recovery period. In contrast, the dynamics of homeostatic responses in the SCNCry1 mice were equivalent to WT. Finally, SCNCon mice exhibited poor sleep-dependent memory but this was corrected in SCNCry1mice. Therefore, the SCN clock is sufficient for circadian control of sleep-wake, facilitating initiation and maintenance of wake, promoting sleep consolidation, homeostatic dynamics, and sleep-dependent memory. Significance statementThe circadian timing system regulates sleep-wake cycles. The hypothalamic suprachiasmatic nucleus (SCN) is the principal circadian clock, but local clocks are also active across the brain and the respective roles of SCN and local clocks in regulating sleep are unclear. To determine, explicitly, the contribution of the SCN, we used virally mediated genetic complementation to restore SCN molecular circadian functions in otherwise genetically clockless mice. This initiated circadian activity-rest cycles, accompanied by circadian sleep-wake cycles, circadian patterning to the intensity of NREM sleep and circadian control of REM sleep as a proportion of total sleep. Consolidation of sleep-wake established normal dynamics of sleep homeostasis and enhanced sleep-dependent memory. Thus, the SCN is the principal and sufficient circadian regulator of sleep-wake.

neuroscience↗

bioRxiv2020

The cell-autonomous clock of VIP receptor VPAC2 cells drives circadian behaviour

Circadian ([~]daily) rhythms pervade mammalian behaviour. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFL), active in all tissues. This distributed clock network is co-ordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN, and in the absence of VIP, or its cognate receptor VPAC2, circadian behaviour is compromised because SCN cells cannot synchronise. The contributions to SCN pacemaking and circadian behaviour of other cell types, not least the VPAC2-expressing target cells of VIP, are, however, not understood. We therefore employed intersectional genetics to manipulate the cell-autonomous TTFL of VPAC2-expressing cells, creating temporally chimaeric mice. We could then determine whether and how VPAC2-expressing cells (a minority [~]35% of SCN cells) contribute to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1{varepsilon}Tau allele concomitantly lengthened the period of circadian behavioural rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo. Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioural rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.

neuroscience↗