The conserved transcriptional co-activator YAP1 is a central regulator of organ development and tissue homeostasis, integrating mechanical and biochemical cues to control cell proliferation and survival. YAP1 variants underlie a spectrum of congenital disorders, including autosomal dominant ocular coloboma with or without syndromic features. Despite this clinical significance, the functional role of YAP1 in human eye development, as well as the impact of disease-associated missense variants, remains poorly understood. We show YAP1 expression at the optic fissure in human embryos, a key structure involved in coloboma pathogenesis. Using in silico prediction, cell-based assays, and fluorescence cross-correlation spectroscopy (FCCS) to directly quantify YAP1-TEAD binding, we demonstrate that the position of YAP1 missense variants dictates their functional changes. TEAD-binding domain mutations most strongly disrupted transcriptional activity in a luciferase assay, whereas all tested variants impaired induction of endogenous YAP1-TEAD target genes. Furthermore, mimicking reduced YAP1-TEAD binding using verteporfin small molecule in retinal organoids led to reduced progenitor proliferation and survival. These findings establish defective YAP1-dependent transcription as a mechanism driving congenital eye malformations and provide a framework for interpreting the pathogenicity of human YAP1 variants. More broadly, they highlight the need for functional analyses to connect genetic variation with disease.

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, such that cells express one TF but not both. Hybrid states in which cells express both cross-repressing fate determinants have been observed, but how these arise or persist remains unclear. Here, we focus on HES1 and HES5, which are auto-repressive oscillatory TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral progenitor domains in the developing spinal cord. Knockdown experiments demonstrate that HES1 and HES5 are cross-repressing in mouse spinal cord neural progenitors, and live-cell imaging in vitro shows that they can be co-expressed, defining a hybrid state. In this state, HES proteins co-oscillate in-phase within single cells. Computational modelling indicates that modulation of cross-repression strength or relative TF abundance destabilises this state, driving resolution towards a single oscillatory HES TF. This is consistent with in vivo analysis showing transient HES1 and HES5 co-expression, followed by progressive restriction to a single TF oscillator. Our findings suggest that oscillatory expression enables the co-existence of cross-repressing TFs, allowing hybrid states within a developmental bistable motif.

ABSTRACT 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 ∼20 µm, 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.

This paper investigates the effect of altering the protein expression dynamics of the bHLH transcription factor Her6 at the single -cell level in the embryonic zebrafish telencephalon. Using a homozygote endogenous Her6:Venus reporter and 4D single -cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that the fusion of protein destabilisation (PEST) domain alters its expression dynamics, causing most cells to downregulate Her6 prematurely. However, counterintuitively, oscillatory cells increase, with some expressing Her6 at high levels, resulting in increased heterogeneity of Her6 expression in the population. These tissuelevel changes appear to be an emergent property of coupling between single -cells, as revealed by experimentally disrupting Notch signalling and by computationally modelling alterations in Her6 protein stability. Despite the profound differences in the single -cell Her6 dynamics, the size of the telencephalon is only transiently altered and differentiation markers do not exhibit significant differences early on; however, a small increase is observed at later developmental stages. Our study suggests that cell coupling provides a compensation strategy, whereby an almost normal phenotype is maintained even though single -cell gene expression dynamics are abnormal, granting phenotypic robustness.

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories 1 . Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication 2 . Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins.

MicroRNAs (miRs) have an important role in tuning dynamic gene expression. However, the mechanism by which they are quantitatively controlled is unknown. We show that the amount of mature miR-9, a key regulator of neuronal development, increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of seven distinct microRNA primary transcripts (pri-mir)-9s that produce the same mature miR-9 and show that they are sequentially expressed during hindbrain neurogenesis. Expression of late-onset pri-mir-9-1 is added on to, rather than replacing, the expression of early onset pri-mir-9-4 and-9-5 in single cells. CRISPR/Cas9 mutation of the late-onset pri-mir-9-1 prevents the developmental increase of mature miR-9, reduces late neuronal differentiation and fails to downregulate Her6 at late stages. Mathematical modelling shows that an adaptive network containing Her6 is insensitive to linear increases in miR-9 but responds to stepwise increases of miR-9. We suggest that a sharp stepwise increase of mature miR-9 is created by sequential and additive temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition of Her6 to a new dynamic regime or steady state.

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.

MicroRNAs (miRs) have important quantitative roles in tuning dynamical gene expression. Hes/Her transcription factor dynamics are sensitive to the increasing amount of miR-9 in the cell, transitioning from noisy high-level expression to oscillatory expression and then to downregulation. However, the mechanism by which miR-9 is quantitatively controlled is not known. In vertebrates, several distinct genomic loci produce the same mature miR-9, but the functional significance of multiple primary transcripts remains unknown. Here, we show that the amount of mature miR-9 increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of 7 distinct pri-mir-9s and show that they are sequentially expressed during hindbrain neurogenesis. Quantitative analysis of expression at the single-cell level, shows that expression of late-onset pri-mir-9-1 is added on, rather than replacing the expression of early onset pri-mir-9-4 and 9-5. Mutating the late-onset pri-mir-9-1 with CRISPR/Cas9 prevents the developmental increase of mature miR-9 and reduces late neuronal differentiation. Finally, we use mathematical modelling to explore possible benefits of a stepwise increase of miR-9 over a linear increase. We find that an adaptive network containing Her6 can be insensitive to a linear increase in miR-9 and show that such adaptation can be overcome by step-wise increases of miR-9. In conclusion, our work suggests that a sharp stepwise increase of mature miR-9 is contributed by sequential temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition to a new state of Her6 dynamics or level.

Mucosal surfaces such as fish gills interface between the organism and the external environment and as such are major sites of foreign Ag encounter. In the gills, the balance between inflammatory responses to waterborne pathogens and regulatory responses toward commensal microbes is critical for effective barrier function and overall fish health. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune responses to pathogens and suppressing inflammation. Although considerable progress has been made in the field of fish immunology in recent years, whether the fish counterparts of these key mammalian cytokines perform similar roles is still an open question. In this study, we have generated IL-4/13A and IL-4/13B mutant zebrafish (Danio rerio) and, together with an existing IL-10 mutant line, characterized the consequences of loss of function of these cytokines. We demonstrate that IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills and the suppression of type 1 immune responses. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4-like cytokines and is essential for gill homeostasis. Thus, both IL-4/13 and IL-10 paralogs in zebrafish exhibit aspects of conserved function with their mammalian counterparts.

Healthy fish stocks are central to global food security. Key to fish health is robust immunity at mucosal surfaces, and especially at the gills. However, a balance must be struck between tolerating commensal microorganisms and reacting appropriately toward pathogens. In mammals, IL-4 and IL-13 in concert with IL-10 are essential for balancing immune response to pathogens and suppressing inflammation. Whether their fish counterparts perform similar roles is an open question. Here, we have generated IL-4/13A and IL-4/13B mutant zebrafish and, together with existing IL-10 mutants, characterized the consequences of loss-of-function of these cytokines. We demonstrate that these cytokines are required to suppress inflammation. Further, IL-4/13A and IL-4/13B are required for the maintenance of a Th2-like phenotype in the gills. As in mammals, IL-10 appears to have a more striking anti-inflammatory function than IL-4-like cytokines. Thus, both IL-10 and IL-4/13 paralogues in zebrafish exhibit aspects of conserved function with their mammalian counterparts.

Noise is prevalent in biology and has been widely quantified using snapshot measurements. This static view obscures our understanding of dynamic noise properties and how these affect gene expression and cell state transitions. Using aCRISPR/Cas9 Zebrafishher6::Venusreporter combined with mathematical andin vivoexperimentation, we explore how noise affects the protein dynamics of Her6, a basic helix-loop-helix transcriptional repressor. During neurogenesis, Her6 expression transitions from fluctuating to oscillatory at single-cell level. We identify that absence of miR-9 input generates high-frequency noise in Her6 traces, inhibits the transition to oscillatory protein expression and prevents the downregulation of Her6. Together, these impair the upregulation of downstream targets and cells accumulate in a normally transitory state where progenitor and early differentiation markers are co-expressed. Computational modelling and double smFISHofher6and the early neurogenesis marker,elavl3, suggest that the change in Her6 dynamics precedes the downregulation in Her6 levels. This sheds light onto the order of events at the moment of cell state transition and how this is influenced by the dynamic properties of noise. Our results suggest that Her/Hes oscillations, facilitated by dynamic noise optimization by miR-9, endow progenitor cells with the ability to make a cell state transition.

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.

Survival rates in patients with glioblastoma have shown little improvement over the last 40 years due to the heterogeneity of tumours and the difficulty of specifically targeting the tumour whilst sparing surrounding healthy tissue. Altered gene methylation is often observed in glioma cells, and methylating agents such as folate may reverse aberrant methylation. Folate treatment has shown a beneficial effect, reducing risk of certain cancers (colorectal, breast, squamous cell carcinoma), whereas other studies have shown detrimental effects following folate treatment, whereby proliferation of cancer increased (mammary, prostate). The aim of this study was to investigate the opposing roles of folate in glioma. The glioma cell lines 1321N1, U87 MG and non-cancerous glial SVGp12 cells were grown in folate deficient, folic or folinic acid supplemented media and compared to standard cell culture media. Cell viability, apoptosis and cell cycle analysis along with methylation status and protein expression of the genes of interest; PTEN, FOLR1, RFC, PCFT, and MTHFR were analysed to determine differences between cell lines following treatment. Folic and folinic acid behaved differently depending on the concentration used and the cell lines treated. Low folic acid at 5 µg/ml significantly increased cell viability and protein expression levels in the U87 MG and SVGp12 cell lines, whilst the high dose of folinic acid (35 µg/ml) resulted in significant decreased cell viability, increased apoptotic activity and down regulation of the folate transporters in the 1321N1, U87 MG and SVGp12 cell lines. Folate treatment did not significantly alter cell cycle phase. Altered methylation of genes specific for folate metabolism and transport did not explain the cytotoxic effects of folate in cell lines. In conclusion folinic acid rather than folic acid supplementation should be investigated further to elucidate the mechanism of potential cytotoxic effects in glioma.

A powerful and commonly used technique for assessing phenotype, associated with alterations in genotype, is whole-mount in situ hybridization (WMISH), which examines the temporal and spatial patterns of gene expression. Xenopus tropicalis has emerged as a power model organism, which permits the combination of experimental embryology and genetics. Here we present a comprehensive protocol for performing WMISH on Xenopus embryos. While the main purpose of this chapter is to provide a method for WMISH in Xenopus embryos, we also present, for the first time, critical modifications to the standard WMISH protocol, which permit genotyping of single embryos after completion of the WMISH method. We believe that these modifications will prove very useful for the biomedical community investigating gene function in Xenopus, especially given the advent of new gene editing approaches, which facilitates reverse genetic approaches in this model organism.

It is becoming increasingly clear that plants ranging across the plant kingdom produce anionic host defence peptides (AHDPs) with potent activity against a wide variety of human cancers cells. In general, this activity involves membrane partitioning by AHDPs, which leads to membranolysis and / or internalization to attack intracellular targets such as DNA. Several models have been proposed to describe these events including: the toroidal pore and Shai-Matsuzaki-Huang mechanisms but, in general, the mechanisms underpinning the membrane interactions and anticancer activity of these peptides are poorly understood. Plant AHDPs with anticancer activity can be conveniently discussed with reference to two groups: cyclotides, which possess cyclic molecules stabilized by cysteine knot motifs, and other ADHPs that adopt extended and α-helical conformations. Here, we review research into the anticancer action of these two groups of peptides along with current understanding of the mechanisms underpinning this action.

The ability to diagnose cancer rapidly with high sensitivity and specificity is essential to exploit advances in new treatments to lead significant reductions in mortality and morbidity. Current cancer diagnostic tests observing tissue architecture and specific protein expression for specific cancers suffer from inter-observer variability, poor detection rates and occur when the patient is symptomatic. A new method for the detection of cancer using 1 μl of human serum, attenuated total reflection—Fourier transform infrared spectroscopy and pattern recognition algorithms is reported using a 433 patient dataset (3897 spectra). To the best of our knowledge, we present the largest study on serum mid-infrared spectroscopy for cancer research. We achieve optimum sensitivities and specificities using a Radial Basis Function Support Vector Machine of between 80.0 and 100 % for all strata and identify the major spectral features, hence biochemical components, responsible for the discrimination within each stratum. We assess feature fed-SVM analysis for our cancer versus non-cancer model and achieve 91.5 and 83.0 % sensitivity and specificity respectively. We demonstrate the use of infrared light to provide a spectral signature from human serum to detect, for the first time, cancer versus non-cancer, metastatic cancer versus organ confined, brain cancer severity and the organ of origin of metastatic disease from the same sample enabling stratified diagnostics depending upon the clinical question asked.

Motor neurone disease (MND) is a fatal neurodegenerative disease of unknown aetiology. Malnutrition is a common occurrence and an independent risk factor for worse prognosis. However, it remains unclear whether provision of enteral nutrition (EN) through a gastrostomy tube offers any survival advantage. Our aim was to describe the demographic and clinical characteristics of MND in Lancashire and South Cumbria in North West England and the impact of EN on survival in the 8year period of 2005–2012. Four hundred and seven patients with MND were identified through the Preston MND care and research centre registry giving a crude incidence rate of 3.15/100,000. Three hundred and forty patients with adequate information were included in the final analysis of whom 53.2% were male. The presentation was limb/spinal in 62.1% and bulbar in 37.9% of patients, bulbar onset being more common in elderly females. Mean age of onset was 67.28years (standard deviation 11.06; range 22.78–93.06). Median survival was 1.98years (range 1.18–3.05). Ninety-one patients received EN of whom 67% had bulbar onset disease. EN was not associated with a statistically significant survival advantage except for the subgroup who received EN more than 500days after symptom onset. In conclusion, the early requirement for EN may indicate a prognostically less favourable subgroup.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.