Biology subjects

Biga, V.

Publications and source records attributed to Biga, V..

10 recordsLinked to original sources

bioRxiv2026

Ciliary cAMP regulates Shh signal interpretation to drive polarisation of differentiating neurons

Cellular differentiation is characterised by transitions in cell-state through reinterpretation of extracellular signals. However, the mechanisms facilitating context-dependent signal interpretation remain poorly understood. Differentiating neurons remodel a molecularly distinct primary cilium as they switch from canonical to non-canonical Shh signalling. Here, using long-term live-tissue imaging, we demonstrate that the opposing Shh signalling modulators Smo and GPR161 simultaneously accumulate in the remodelled primary cilium. The correct balance of Smo and GPR161 leads to elevated ciliary cAMP levels, which suppresses Gli transcription factor activation and regulates actin dynamics to drive neuron polarisation. Notably, disrupting this balance through Smo hyperactivation or GPR161 depletion results in reduced ciliary cAMP, inappropriate activation of canonical Shh signalling, dysregulated actin dynamics and initiation of multiple unstable axon-like projections. Thus, this study identifies shifts in ciliary cAMP levels as a key regulator of cellular signal interpretation, and links primary cilium-mediated signal transduction to precise control of cytoskeletal organisation.

developmental biology↗

bioRxiv2025

Engineering protein expression dynamics with Tet-ON and dTAG degron systems: from precise control to oscillations

Precise temporal control of protein expression is essential for dissecting protein function and dynamic cellular processes. We present a framework for engineering tunable oscillatory protein expression (repeated pulses in expression) using widely adopted molecular tools, applying them to modulate NGN3 expression. Single-cell time-lapse microscopy reveals that the Tet-On system unexpectedly generates asynchronous oscillations in protein expression under continuous doxycycline administration. These oscillations are dependent on protein instability and are not tunable by doxycycline concentration. In contrast, the dTAG degron system enables precise, reversible, concentration-dependent control of protein degradation and reaccumulation. Coupled with a constitutive promoter, we achieve synchronous oscillatory protein expression (COD: Constituitive promoter driving Oscillations via Degradation). Mathematical modelling identifies optimal dTAG drug addition and removal timings using the COD system to flexibly tune NGN3 oscillation periods while maintaining other oscillatory parameters (mean level and peak-to-trough fold-change). Using microfluidics (COD+CHIPS) we reproduce model-predicted 5 and 10 hours periodicities while maintaining similar mean levels and peak-to-trough fold-changes. This work introduces a generalisable, programmable approach for generating and modulating protein oscillations, allowing investigation into how dynamic protein expression governs cellular function.

developmental biology↗

bioRxiv2025

Oscillatory Co-expression of HES1 and HES5 Enables a Hybrid State in a Bistable Transcription Factor Regulatory Motif

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, enforcing expression of one dominant TF. However, evidence of hybrid states, where cells co-express opposing fate determinants, challenges this model. We hypothesised that oscillatory expression enables co-existence of cross-repressive TFs within single cells, allowing hybrid states in bistable motifs. To test this, we focused on HES1 and HES5, oscillatory, cross-repressive TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral domains in the developing spinal cord. Using live-cell imaging of fluorescent reporters and computational modelling, we show that HES1 and HES5 co-express and oscillate in-phase within single cells. Differences in protein stability result in distinct free-running periodicity, but co-expression results in entrainment and phase-locking. Modulating cross-repression strength and/or abundance shifts the system towards bistability and dominance of a single TF oscillator. Consistent with this, we observe progressive separation of the HES expression domains in vivo, through a decrease in oscillatory co-expression. Our findings provide a mechanism for hybrid states to emerge in a developmental bistable motif.

developmental biology↗

bioRxiv2024

Cellular signalling protrusions enable dynamic distant contacts in spinal cord neurogenesis

In the developing mouse ventral spinal cord, HES5, a transcription factor downstream of Notch signalling, is expressed as evenly spaced clusters of high HES5-expressing neural progenitor cells along the dorsoventral axis. While Notch signalling requires direct membrane contact for its activation, we have previously shown mathematically that contact needs to extend beyond neighbouring cells for the HES5 pattern to emerge. However, the presence of cellular structures that could enable such long-distance signalling was unclear. Here, we report that cellular protrusions are present all along the apicobasal axis of individual neural progenitor cells. Through live imaging, we show that these protrusions dynamically extend and retract reaching lengths of up to [~]20m, enough to extend membrane contact beyond adjacent cells. The Notch ligand DLL1 was found to colocalise with protrusions, further supporting the idea that Notch signalling can be transduced at a distance. The effect of protrusions on the HES5 pattern was tested by reducing the density of protrusions using the CDC42 inhibitor ML141, leading to a tendency to decrease the distance between high HES5 cell clusters. However, this tendency was not significant and leaves an open question about their role in the fine-grained organisation of neurogenesis.

developmental biology↗

bioRxiv2024

NGN3 oscillatory expression controls the timing of human pancreatic endocrine differentiation

Understanding protein expression dynamics is crucial for the mechanistic understanding of cell differentiation. We investigate the dynamics of NGN3, a transcription factor critical for pancreatic endocrine development, including their function and decoding mechanisms. A knock-in endogenous reporter shows that the expression of NGN3 protein oscillates with a 13-hour periodicity in human iPS-derived endocrine progenitors and is switched off as cells differentiate to {beta}-like and -like cells. Increasing the stability of NGN3 protein results in one broad peak of expression instead of oscillations, with a larger peak to trough fold-change. This leads to precocious endocrine differentiation to both {beta}-like and -like cells and precocious expression of key NGN3 target genes. Single-cell analysis of dynamics, mathematical modelling and bioinformatics suggest that decoding of NGN3 oscillations occurs by fold-change detection via an incoherent feedforward motif that explains both normal and precocious differentiation. Our findings suggest that oscillatory NGN3 dynamics control the timing of differentiation, but not fate specification.

developmental biology↗

bioRxiv2022

Cell coupling compensates for changes in single-cell Her6 dynamics and provides phenotypic robustness

her6 is a zebrafish ortholog of Hes1, known for its role in maintaining neural progenitors during neural development. Here, we characterise the population-level effect of altering Her6 protein expression dynamics at the single-cell level in the embryonic zebrafish telencephalon. Using an endogenous Her6:Venus reporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that fusion of a protein destabilisation domain (PEST) to Her6:Venus alters its expression dynamics causing most cells to downregulate Her6 prematurely. However, in PEST mutants, a higher proportion of cells exhibit Her6 oscillations and while expression is reduced in most cells, some cells express Her6 at wild-type levels resulting in increased heterogeneity of Her6 expression in the population. Despite the profound differences in the single-cell Her6 dynamics, differentiation markers do not exhibit major differences early on, while an increase in differentiation is observed at later developmental stages (vglut2a, gad1 and gad2). At the same time, at late stage the overall size of the telencephalon remains the same. Computational modelling that simulates changes in Her6 protein stability reveals that the increase in population Her6 expression heterogeneity is an emergent property of finely tuned Notch signalling coupling between single cells. Our study suggests that such cell coupling provides a compensation strategy whereby a normal phenotype is maintained while single-cell dynamics are abnormal, although the limit of this compensation is reached at late developmental stages. We conclude that in the neural progenitor population, cell coupling controls Her6 expression heterogeneity and in doing so, it provides phenotypic robustness when individual cells lose Her6 expression prematurely.

developmental biology↗

bioRxiv2021

HES1 protein oscillations are necessary for neural stem cells to exit from quiescence

Quiescence is a dynamic process of reversible cell-cycle arrest. High-level sustained expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or in dynamics. Here, we induce quiescence in NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express sustained HES1 under a moderate-strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that sustained HES1 does not affect proliferation or entry into quiescence, however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.

cell biology↗

bioRxiv2020

A dynamic and spatially periodic micro-pattern of HES5 expression underlies the probability of neuronal differentiation in the mouse spinal cord

Ultradian oscillations of HES Transcription Factors (TFs) at the single cell level, enable cell state transitions. However, the tissue level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex-vivo in the developing mouse ventral spinal cord and identify microclusters of 4-6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signaling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin-2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organization of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single cell dynamics. SynopsisLive imaging of HES5 expression in the ventral mouse spinal cord together with computational modelling is used to identify and analyse spatially periodic HES5 micro-patterns that emerge from the synthesis of single cell dynamics. O_LIHES5 is expressed in spatially periodic microclusters along the dorsal-ventral axis in spinal cord that are dynamically maintained by Notch signalling. C_LIO_LIMicroclusters can arise, in part, from single cell oscillators that are synchronous and weakly coupled via Notch. C_LIO_LISpatial patterns are different between motorneuron and interneuron progenitor domains and the probability for progenitor differentiation is regulated by the coupling strength between cells. C_LIO_LINGN2 is also spatially periodic along the dorso-ventral axis and microclusters of HES5 may act to pick a single NGN2 high cell for differentiation. C_LI

developmental biology↗

bioRxiv2019

miR-9 mediated noise optimization of the her6 oscillator is needed for cell state progression in the Zebrafish hindbrain

Ultradian oscillations of key transcription factors, such as members of the Hes family, are thought to be important in Neural Progenitor Cell (NPC) maintenance and miR-9 acts as a tuner of these oscillations in vitro. However, the existence and the role of such dynamic oscillatory expression in vivo is poorly understood. Here, we have generated a Zebrafish CRISPR knock-in Her6::venus fusion (Hes1 orthologue) to study endogenous dynamic gene expression in the embryonic hindbrain. We show that Her6 undergoes a transition from irregular, noisy, fluctuations to periodic oscillations as neurogenesis proceeds. In the absence of miR-9 input, noise in the Her6 oscillator increases and NPCs are unable to transit away from an intermediary state where they co-express progenitor and early differentiation markers. Thus, Her6 oscillations are facilitated by noise optimization mediated by miR-9 and this noise-tuning step is functionally important for cells to transition to differentiation.

developmental biology↗

bioRxiv2018

Quantitative, real-time, single cell analysis in tissue reveals expression dynamics of neurogenesis

During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.

developmental biology↗