Development of 3D leaf shape: utricularia gibba as a model system

The development of diverse organ shapes involves genetically specified growth patterns which may differ across a tissue in rate and/ or orientation. Understanding specified growth is not intuitive since observed (resultant) growth rates and orientations are the result of specified growth combined with the effects of mechanical constraints in a connected tissue. Growth dynamics in leaves of Arabidopsis have previously been studied experimentally and modelled using a polarity field to orient growth, and regional factors which control local specified growth rates parallel and perpendicular to the polarity. It is unclear whether the mechanisms invoked for the development of 2D leaf shape can be applied to more complex 3D leaf shapes. In this work, I developed Utricularia gibba as a new model system and studied the development of U. gibba 3D epiascidiate (cup-shaped) leaves (known as bladders). I investigated bladder shape changes through development and modelled these transitions using isotropic (equal in all directions) or anisotropic (preferentially in one orientation) specified growth, showing that specified anisotropy is required to generate the full mature bladder shape. The shape of the main body of the bladder could be accounted for by both specified isotropic or anisotropic models. I tested predictions on growth dynamics and polarity made by each model using sector analysis and by investigating markers of tissue cell polarity in bladders. Sector analysis supported an anisotropic specified growth model, while quadrifid gland and UgPIN1 analysis provided evidence of a polarity field in U. gibba. Together, these observations suggest a common underlying mechanism for the generation of 3D and 2D leaves. This work shows how computational modelling can be combined with experimentation in a biological system to allow for a better understanding of the specified growth patterns underlying the generation of an organ shape

Looking out for Lottie an inter-professional training pack

A 37 page inter-professional training pack with a literature review and worksheet

A novel role for GSK3β as a modulator of Drosha microprocessor activity and MicroRNA biogenesis

Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here, we identify the kinase GSK3β as an important modulator of miR biogenesis at Microprocessor level. Repression of GSK3β activity reduces Drosha activity toward pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisation of miR biogenesis proteins. Conversely, GSK3β activation increases Drosha activity and mature miR accumulation. GSK3β achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent upon presence of RNA. Indeed, GSK3β itself can immunoprecipitate pri-miRs, suggesting possible RNA-binding capacity. Kinase assays identify the mechanism for GSK3β-enhanced Drosha activity, which requires GSK3β nuclear localisation, as phosphorylation of Drosha at S300 and/or S302; confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature miRs in the presence of phospho-mimic Drosha. Functional implications of GSK3β-enhanced miR biogenesis are illustrated by increased levels of GSK3β-upregulated miR targets following GSK3β inhibition. These data, the first to link GSK3β with the miR cascade in humans, highlight a novel pro-biogenesis role for GSK3β in increasing miR biogenesis as a component of the Microprocessor complex with wide-ranging functional consequences

Evolution of carnivorous traps from planar leaves through simple shifts in gene expression

Leaves vary from planar sheets and needle-like structures to elaborate cup-shaped traps. Here, we show that in the carnivorous plant Utricularia gibba, the upper leaf (adaxial) domain is restricted to a small region of the primordium that gives rise to the trap's inner layer. This restriction is necessary for trap formation, because ectopic adaxial activity at early stages gives radialized leaves and no traps. We present a model that accounts for the formation of both planar and nonplanar leaves through adaxial-abaxial domains of gene activity establishing a polarity field that orients growth. In combination with an orthogonal proximodistal polarity field, this system can generate diverse leaf forms and account for the multiple evolutionary origins of cup-shaped leaves through simple shifts in gene expression

Shaping of a three-dimensional carnivorous trap through modulation of a planar growth mechanism

Leaves display a remarkable range of forms, from flat sheets with simple outlines to cup-shaped traps. Although much progress has been made in understanding the mechanisms of planar leaf development, it is unclear whether similar or distinctive mechanisms underlie shape transformations during development of more complex curved forms. Here, we use 3D imaging and cellular and clonal analysis, combined with computational modelling, to analyse the development of cup-shaped traps of the carnivorous plant Utricularia gibba. We show that the transformation from a near-spherical form at early developmental stages to an oblate spheroid with a straightened ventral midline in the mature form can be accounted for by spatial variations in rates and orientations of growth. Different hypotheses regarding spatiotemporal control predict distinct patterns of cell shape and size, which were tested experimentally by quantifying cellular and clonal anisotropy. We propose that orientations of growth are specified by a proximodistal polarity field, similar to that hypothesised to account for Arabidopsis leaf development, except that in Utricularia, the field propagates through a highly curved tissue sheet. Independent evidence for the polarity field is provided by the orientation of glandular hairs on the inner surface of the trap. Taken together, our results show that morphogenesis of complex 3D leaf shapes can be accounted for by similar mechanisms to those for planar leaves, suggesting that simple modulations of a common growth framework underlie the shaping of a diverse range of morphologies

The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Funder: Department of HealthMalignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease

Regulators of male and female sexual development are critical for the transmission of a malaria parasite

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite

Inositol trisphosphate receptor contribution to ventricular cardiac fibroblast function in healthy and hypertrophied mouse hearts

Cardiac Fibroblasts play a critical role in pathological cardiac remodelling. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we investigated whether Inositol trisphosphate receptor type 2 (IP3R2) expression and activity contributes not only to cardiac contractile dysfunction, but also to changes in cardiac fibroblast function. Cardiac contractility was assessed in vivo using echocardiography and in vitro using stimulation of isolated tissue preparations in organ baths. Cardiac fibroblasts were isolated from sham-operated and MTAB hearts under sterile conditions using chunk digestion and maintained in short-term culture. Quantitative immunoblot assessment of whole ventricular homogenates showed that IP3R2 expression was significantly increased in MTAB hearts (IP3R2:GAPDH ratio: 0.378±0.06 vs 0.130±0.03, MTAB vs sham respectively, n=7, p<0.001). Further individual analysis of samples comparing in vivo contractile function with corresponding IP3R protein expression showed a clear distinction between the sham and MTAB groups. MTAB animals with <40% Fractional Shortening showed a clear correlation with higher expression of IP3R2 (Spearman r=−0.79, p<0.05, n=15). The contribution of IP3R activity to contractile function was further assessed in vitro using isolated ventricular preparations stimulated with 1uM isoprenaline in the presence and absence of the Ca2+ release modulator 2-aminoethyl diphenylborinate (2-APB) (10uM). Preparations from both MTAB and sham-operated hearts showed reduced contractile activity in the presence of 2-APB (~35% and 25% reduction respectively, n=2). Analysis of intracellular Ca2+ release channel expression in isolated cardiac fibroblasts revealed a lack of any ryanodine receptor (RyR) expression but significant expression of IP3R2. In fibroblasts isolated from MTAB hearts, significantly higher expression of IP3R2 was evident (IP3R2:GAPDH ratio: 0.252±0.02 vs 0.101±0.008, MTAB vs sham respectively, n=3, p<0.001) along with higher phosphorylation of IP3R at Ser1756. Fibroblasts from MTAB hearts also exhibited hyperproliferative growth characteristics as assessed following 1uM Angiotensin II stimulation of cells over a period of 48h (average cell number per area, 59.6±2.5 vs 41.8±2.3, MTAB vs sham, n=3, p=0.006). Treatment with 2-APB significantly reduced both proliferation and Angiotensin II-mediated Ca2+ release in these cells. In conclusion and for the first time, we have shown increased IP3R2 expression and IP3R phosphorylation specifically in ventricular cardiac fibroblasts isolated from hypertrophied hearts. Expression of IP3R2 shows a strong correlation with cardiac contractility, where increased IP3R2 expression not only parallels decreased contractile performance but may also influence fibroblast hyperproliferation. This highlights the possibility that altered Ca2+ handling at the level of the cardiac fibroblast could be mechanistically important in pathological remodelling of the heart

NUAK1 governs centrosome replication in pancreatic cancer via MYPT1/PP1β and GSK3β‐dependent regulation of PLK4

The AMP‐activated protein kinase (AMPK)‐related kinase NUAK1 (NUAK family SNF1‐like kinase 1) has emerged as a potential vulnerability in MYC‐dependent cancer but the biological roles of NUAK1 in different settings are poorly characterised, and the spectrum of cancer types that exhibit a requirement for NUAK1 is unknown. Unlike canonical oncogenes, NUAK1 is rarely mutated in cancer and appears to function as an obligate facilitator rather than a cancer driver per se. Although numerous groups have developed small‐molecule NUAK inhibitors, the circumstances that would trigger their use and the unwanted toxicities that may arise as a consequence of on‐target activity are thus undetermined. Reasoning that MYC is a key effector of RAS pathway signalling and the GTPase KRAS is almost uniformly mutated in pancreatic ductal adenocarcinoma (PDAC), we investigated whether this cancer type exhibits a functional requirement for NUAK1. Here, we show that high NUAK1 expression is associated with reduced overall survival in PDAC and that inhibition or depletion of NUAK1 suppresses growth of PDAC cells in culture. We identify a previously unknown role for NUAK1 in regulating accurate centrosome duplication and show that loss of NUAK1 triggers genomic instability. The latter activity is conserved in primary fibroblasts, raising the possibility of undesirable genotoxic effects of NUAK1 inhibition