Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

From Drosophila segmentation to human cancer therapy

First described in Drosophila, Hedgehog signalling is a key regulator of embryonic development and tissue homeostasis and its dysfunction underlies a variety of human congenital anomalies and diseases. Although now recognised as a major target for cancer therapy as well as a mediator of directed stem cell differentiation, the unveiling of the function and mechanisms of Hedgehog signalling was driven largely by an interest in basic developmental biology rather than clinical need. Here, I describe how curiosity about embryonic patterning led to the identification of the family of Hedgehog signalling proteins and the pathway that transduces their activity, and ultimately to the development of drugs that block this pathway.Published versio

Musculoskeletal regeneration: a zebrafish perspective

Musculoskeletal injuries are common in humans. The cascade of cellular and molecular events following such injuries results either in healing with functional recovery or scar formation. While fibrotic scar tissue serves to bridge between injured planes, it undermines functional integrity. Hence, faithful regeneration is the most desired outcome; however, the potential to regenerate is limited in humans. In contrast, various non-mammalian vertebrates have fascinating capabilities of regenerating even an entire appendage following amputation. Among them, zebrafish is an important and accessible laboratory model organism, sharing striking similarities with mammalian embryonic musculoskeletal development. Moreover, clinically relevant muscle and skeletal injury zebrafish models recapitulate mammalian regeneration. Upon muscle injury, quiescent stem cells - known as satellite cells - become activated, proliferate, differentiate and fuse to form new myofibres, while bone fracture results in a phased response involving hematoma formation, inflammation, fibrocartilaginous callus formation, bony callus formation and remodelling. These models are well suited to testing gene- or pharmaco-therapy for the benefit of conditions like muscle tears and fractures. Insights from further studies on whole body part regeneration, a hallmark of the zebrafish model, have the potential to complement regenerative strategies to achieve faster and desired healing following injuries without any scar formation and, in the longer run, drive progress towards the realisation of large-scale regeneration in mammals. Here, we provide an overview of the basic mechanisms of musculoskeletal regeneration, highlight the key features of zebrafish as a regenerative model and outline the relevant studies that have contributed to the advancement of this field.AKKP is supported by a Nanyang Technological University (Lee Kong Chian School of Medicine) Research Scholarship. PWI is supported by the Toh Kian Chui Foundation

Hedgehog signalling

The Hedgehog (Hh) signalling pathway is one of the key regulators of metazoan development. Hh proteins have been shown to play roles in many developmental processes and have become paradigms for classical morphogens. Dysfunction of the Hh pathway underlies a number of human developmental abnormalities and diseases, making it an important therapeutic target. Interest in Hh signalling thus extends across many fields, from evo-devo to cancer research and regenerative medicine. Here, and in the accompanying poster, we provide an outline of the current understanding of Hh signalling mechanisms, highlighting the similarities and differences between species.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Fibrodysplasia ossificans progressiva : current concepts from bench to bedside

Heterotopic ossification (HO) is a disorder characterised by the formation of ectopic bone in soft tissue. Acquired HO typically occurs in response to trauma and is relatively common, yet its aetiology remains poorly understood. Genetic forms, by contrast, are very rare, but provide insights into the mechanisms of HO pathobiology. Fibrodysplasia ossificans progressiva (FOP) is the most debilitating form of HO. All patients reported to date carry heterozygous gain-of-function mutations in the gene encoding activin A receptor type I (ACVR1). These mutations cause dysregulated bone morphogenetic protein (BMP) signalling, leading to HO at extraskeletal sites including, but not limited to, muscles, ligaments, tendons and fascia. Ever since the identification of the causative gene, developing a cure for FOP has been a focus of investigation, and studies have decoded the pathophysiology at the molecular and cellular levels, and explored novel management strategies. Based on the established role of BMP signalling throughout HO in FOP, therapeutic modalities that target multiple levels of the signalling cascade have been designed, and some drugs have entered clinical trials, holding out hope of a cure. A potential role of other signalling pathways that could influence the dysregulated BMP signalling and present alternative therapeutic targets remains a matter of debate. Here, we review the recent FOP literature, including pathophysiology, clinical aspects, animal models and current management strategies. We also consider how this research can inform our understanding of other types of HO and highlight some of the remaining knowledge gaps.Nanyang Technological UniversityPublished versionA.-K.K.-P. is supported by a Nanyang Technological University (Lee Kong Chian School of Medicine) Research Scholarship. P.W.I. is supported by the Toh Kian Chui Foundation.

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio

Deep sequencing of small RNA facilitates tissue and sex associated microRNA discovery in zebrafish

Background: The role of microRNAs in gene regulation has been well established. The extent of miRNA regulation also increases with increasing genome complexity. Though the number of genes appear to be equal between human and zebrafish, substantially less microRNAs have been discovered in zebrafish compared to human (miRBase Release 19). It appears that most of the miRNAs in zebrafish are yet to be discovered. Results: We sequenced small RNAs from brain, gut, liver, ovary, testis, eye, heart and embryo of zebrafish. In brain, gut and liver sequencing was done sex specifically. Majority of the sequenced reads (16–62 %) mapped to known miRNAs, with the exception of ovary (5.7 %) and testis (7.8 %). Using the miRNA discovery tool (miRDeep2), we discovered novel miRNAs from the unannotated reads that ranged from 7.6 to 23.0 %, with exceptions of ovary (51.4 %) and testis (55.2 %). The prediction tool identified a total of 459 novel pre-miRNAs. We compared expression of miRNAs between different tissues and between males and females to identify tissue associated and sex associated miRNAs respectively. These miRNAs could serve as putative biomarkers for these tissues. The brain and liver had highest number of tissue associated (22) and sex associated (34) miRNAs, respectively. Conclusions: This study comprehensively identifies tissue and sex associated miRNAs in zebrafish. Further, we have discovered 459 novel pre-miRNAs (~30 % seed homology to human miRNA) as a genomic resource which can facilitate further investigations to understand miRNA-mRNA gene regulatory networks in zebrafish which will have implications in understanding the function of human homologs.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio