Collateral RNA cleavage by CRISPR-Cas13 effectors presents a major obstacle to their application in biological research and therapeutics, yet the molecular determinants of this activity remain poorly understood. Here, we define the molecular basis of collateral activity across Cas13 nucleases and show that target RNA abundance governs potent activation of Rfx Cas13d collateral activity. Moderately expressed targets induce limited collateral cleavage, whereas highly abundant targets trigger widespread activation of cellular Rfx Cas13d, resulting in global RNA degradation, disruption of proteome homeostasis, and cellular toxicity in human cells and zebrafish embryos. In transgenic zebrafish, collateral activity produces tissue-restricted developmental defects in endothelial and neuronal tissues that mirror the site of target RNA expression, demonstrating that collateral RNA cleavage remains spatially confined. Mechanistically, target abundance determines the proportion of activated Rfx Cas13d molecules within a cell, establishing a threshold-dependent switch for collateral RNA degradation. We further identify synthetic guide-target mismatches that substantially reduce collateral activity while preserving on-target silencing, enabling partial uncoupling of these two nuclease activities. Together, our findings reveal fundamental principles governing Cas13 collateral activity and provide a framework for engineering safer and more precise RNA-targeting technologies.

The blood-brain barrier (BBB) protects the brain from circulating metabolites and plays central roles in neurological diseases. Endothelial cells (ECs) of the BBB are enwrapped by mural cells including pericytes and vascular smooth muscle cells (vSMCs) that regulate angiogenesis, vessel stability and barrier function. To explore mural cell control of the BBB, we investigated neurovascular phenotypes in zebrafish pdgfrb mutants that lack brain pericytes and vSMCs. As expected, mutants showed an altered cerebrovascular network with mispatterned capillaries. Unexpectedly, mutants displayed no BBB leakage at larval stages of development. This suggests that pericytes and vSMCs are not essential for normal BBB function in developing zebrafish. Instead, we observed juvenile and adult BBB disruption occurring at 'hotspot' focal hemorrhages at large vessel aneurysms. ECs at leakage hotspots showed induction of caveolae on abluminal surfaces and structural defects including basement membrane thickening and disruption. Our work suggests that capillary pericytes primarily regulate cerebrovascular patterning in development and vSMCs of major arteries protect from hemorrhage and BBB breakdown in older zebrafish. The fact that young zebrafish have a functional BBB in the absence of mural cells calls for renewed interrogation of mural cell control of the BBB throughout vertebrate evolution.

cis-Regulatory elements (cREs) are essential for the spatio-temporal control of gene expression during development and disease. However, cRE activity is highly dependent on cell and tissue type. The developing heart is composed of several cell-types, predominantly cardiomyocytes. Therefore, cardiomyocyte-specific modelling is required to understand the cis-regulation of the developing heart. Zebrafish are an ideal model to study heart development, as they share several physiological features with the human heart during cardiogenesis. Here, we present a comprehensive cardiomyocyte-specific repertoire of cREs isolated from zebrafish larvae. This data combines in vivo transcriptomics and epigenetic profiling, providing insights into cREs and their associated genes involved in heart development. We further perform transgenic reporter assays for the identified cREs associated with popdc2 and bmp10 genes, validating these genomic regions as cardiac regulatory elements. We share this comprehensive, reproducible cardiomyocyte-specific cREs resource as an interrogable web tool for understanding the epigenetic and transcriptomic mechanisms underlying heart development and emergence of congenital heart defects.

Caspase-8 activity is required to inhibit necroptosis during embryogenesis in mice. In vitro studies have suggested that caspase-8 directly cleaves RIPK1, CYLD and the key necroptotic effector kinase RIPK3 to repress necroptosis. However, recent studies have shown that mice expressing uncleavable RIPK1 die during embryogenesis due to excessive apoptosis, while uncleavable CYLD mice are viable. Therefore, these results raise important questions about the role of RIPK3 cleavage. To evaluate the physiological significance of RIPK3 cleavage, we generated Ripk3D333A/D333A mice harbouring a point mutation in the conserved caspase-8 cleavage site. These mice are viable, demonstrating that RIPK3 cleavage is not essential for blocking necroptosis during development. Furthermore, unlike RIPK1 cleavage-resistant cells, Ripk3D333A/D333A cells were not significantly more sensitive to necroptotic stimuli. Instead, we found that the cleavage of RIPK3 by caspase-8 restricts NLRP3 inflammasome activation-dependent pyroptosis and IL-1β secretion when Inhibitors of APoptosis (IAP) are limited. These results demonstrate that caspase-8 does not inhibit necroptosis by directly cleaving RIPK3 and further underscore a role for RIPK3 in regulating the NLRP3 inflammasome.

In this special issue on “Mechanisms of cardiovascular development,” we present a series of studies that explore key questions in cardiovascular developmental biology and regeneration. In recent decades, the broad field of cardiovascular development has expanded to encompass new areas of investigation such as organ specific vascular networks, regeneration of the heart and vessels, as well as the role of the non-coding genome. We kick off with two comprehensive review articles, one by Beisaw and Wu1 explores cardiomyocyte maturation and its regulation in cardiac regeneration. In particular, the mechanisms that control myofibril maturation, the metabolic processes underlying maturation and polyploidization of cardiomyocytes are extensively reviewed, and how these processes are altered in regeneration outlined in detail. Payne et al.,2 then provide a detailed overview of the transcription factors that control developmental angiogenesis and vasculogenesis. This helpful resource, points readers to detailed information on binding motifs, phenotypes in mice and zebrafish, and gaps in the current understanding of transcriptional control of vascular development. These reviews set the scene for a series of seven research articles. In cardiac development, Auman et al.,3 genetically map a zebrafish mutant with pleiotropic phenotypes including loss of pectoral fins and a string-like heart. They discover that smarcc1a controls heart chamber development following normal specification of the early cardiac field. In particular, smarcc1a controls the normal formation of the atrioventricular canal (AVC, which contains the future valves), identifying an unappreciated regulator of this process. Furthermore, uncovering new understanding of how the valve territories are regulated, but this time using mouse models, Okumura et al.,4 explore the role of Hey2. Hey2 knockout (KO) mice at P0 were found to have ventricular septal defects (VSDs) and tricuspid valve malformations. Conditional KO mice reveal that function of Hey2 is essential in developing myocardium for normal development of the septum and valves. The complex phenotypic description here is aided by reconstructed 3D images generated from H&E sections using freely available software, a resource that may be of value to many more groups in the future. The trabeculae of the heart form finger-like projections in development that arise from the compact myocardium and serve to thicken the maturing heart wall. This occurs while the developing heart is contracting. In this issue, Olejnickova et al.5 use simulation of electrical conduction in 3D models of wildtype and trabecular-deficient chick embryos. They combine modeling with detailed analysis of genetic and pharmacological trabecular deficient models to find that trabeculae support normal conduction and development. Maturation of the heart also involves the formation of an organ-specific vascular network to support tissue growth and function. ZFP57 is a regulator of chromatin methylation that can have distinct maternal and zygotic functions. In a study that uses elegant mutant combinations, it is shown that loss of both maternal and zygotic Zfp57 leads to a surprisingly selective loss of coronary vasculature (Zhao and Zhao).6 While it is clear that these mice also have defects in myocardial maturation, and the precise cellular interactions underpinning phenotypes remain to be fully understood, this study discovers a surprising regulator of coronary vessel development. Shifting to development of other vascular lineages, this issue reveals an unappreciated source of vasculature in the developing thyroid. Amniogenic somatopleure cells (ASCs) derived from the somatopleure and contribute to the extraembryonic membrane that surrounds the embryo: the amnion. However, they can also contribute to the embryo. Haneda et al.,7 use quail chick chimera analysis to show that intraembryonic ASCs in pharyngeal regions contribute to the developing vasculature of the thyroid. They use single-cell sequencing and explants of ASCs to suggest the presence of hemangioblast-like cells that respond to FGF and VEGF in the early but not late amnion. This work represents a new contribution to our growing understanding of diverse origins for vascular endothelial cells. Vessels are surrounded by important mural cell lineages, including the vascular smooth muscle cells (vSMCs). In the pharyngeal region of the embryo, cardiac neural crest-derived cells migrate into aortic arches to form vSMCs. Here, Alexander et al.,8 comprehensively examine conditional knockouts of SMAD4 in different pharyngeal cell types and conclude that SMAD4 is essential for normal NCC contributions to vSMCs. However, in an intriguing turn, they also suggest a compensatory cellular contribution in the absence of SMAD4. This large body of work reveals new insights into how vSMCs develop at pharyngeal arteries. Finally, a special edition on cardiovascular mechanisms would not be complete without new insights into how the heart regenerates and in this case, how cells including the endocardium interact to regulate regeneration. A study in zebrafish by Shin et al.,9 investigates an enhancer element associated with regeneration at the leptinb locus. Re-analyzed single-cell studies implicate leptinb expressing endocardial cells as producing regenerative factors. They find that the leptinb enhancer is activated in the endocardium and epicardium upon injury and essential for normal leptinb expression, but not expression of other surrounding genes. This work develops useful new tools and single-cell resources to help dissect how different cell types interact to orchestrate cardiac regeneration in the zebrafish. The group of articles and reviews submitted to this special issue serve to nicely illustrate several current questions and research directions in cardiovascular development and regeneration. This is a field with many open questions, many new and exciting tools and enormous opportunity for discovery.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

Tumour-associated angiogenesis play key roles in tumour growth and cancer metastasis. Consequently, several anti-angiogenic drugs such as sunitinib and axitinib have been approved for use as anti-cancer therapies. However, the majority of these drugs target the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) pathway and have shown mixed outcome, largely due to development of resistances and increased tumour aggressiveness. In this study, we used the zebrafish model to screen for novel anti-angiogenic molecules from a library of compounds derived from natural products. From this, we identified canthin-6-one, an indole alkaloid, which inhibited zebrafish intersegmental vessel (ISV) and sub-intestinal vessel development. Further characterisation revealed that treatment of canthin-6-one reduced ISV endothelial cell number and inhibited proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that canthin-6-one inhibits endothelial cell proliferation. Of note, canthin-6-one did not inhibit VEGFA-induced phosphorylation of VEGFR2 in HUVECs and downstream phosphorylation of extracellular signal-regulated kinase (Erk) in leading ISV endothelial cells in zebrafish, suggesting that canthin-6-one inhibits angiogenesis independent of the VEGFA/VEGFR2 pathway. Importantly, we found that canthin-6-one impairs tumour-associated angiogenesis in a zebrafish B16F10 melanoma cell xenograft model and synergises with VEGFR inhibitor sunitinib malate to inhibit developmental angiogenesis. In summary, we showed that canthin-6-one exhibits anti-angiogenic properties in both developmental and pathological contexts in zebrafish, independent of the VEGFA/VEGFR2 pathway and demonstrate that canthin-6-one may hold value for further development as a novel anti-angiogenic drug.

In recent decades, developmental biologists have come to view vascular development as a series of progressive transitions. Mesoderm differentiates into endothelial cells; arteries, veins and lymphatic endothelial cells are specified from early endothelial cells; and vascular networks diversify and invade developing tissues and organs. Our understanding of this elaborate developmental process has benefitted from detailed studies using the zebrafish as a model system. Here, we review a number of key developmental transitions that occur in zebrafish during the formation of the blood and lymphatic vessel networks.

During angiogenesis, vascular tip cells guide nascent vascular sprouts to form a vascular network. Apelin, an agonist of the G protein-coupled receptor Aplnr, is enriched in vascular tip cells, and it is hypothesized that vascular-derived Apelin regulates sprouting angiogenesis. We identify an apelin-expressing neural progenitor cell population in the dorsal neural tube. Vascular tip cells exhibit directed elongation and migration toward and along the apelin-expressing neural progenitor cells. Notably, restoration of neural but not vascular apelin expression in apelin mutants remedies the angiogenic defects of mutants. By functional analyses, we show the requirement of Apelin signaling for tip cell behaviors, like filopodia formation and cell elongation. Through genetic interaction studies and analysis of transgenic activity reporters, we identify Apelin signaling as a modulator of phosphoinositide 3-kinase and extracellular signal-regulated kinase signaling in tip cells in vivo. Our results suggest a previously unidentified neurovascular cross-talk mediated by Apelin signaling that is important for tip cell function during sprouting angiogenesis.

During development, the lymphatic vasculature forms as a second, new vascular network derived from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is the first step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet downstream mechanisms remain to be elucidated. We present a single cell transcriptomic atlas of lymphangiogenesis in zebrafish revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a VEC-LEC fate reversion during differentiation. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new transcriptional targets and regulatory regions of the genome downstream of Prox1 in LEC maintenance, as well as showing that Prox1 specifies LEC fate primarily by limiting blood vascular and hematopoietic fate. This extensive single cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

The Hippo signaling pathway regulates developmental organ growth, regeneration, and cell fate decisions. Although the role of the Hippo pathway, and its transcriptional effectors YAP and TAZ, has been well documented in many cell types and species, only recently have the roles for this pathway come to light in vascular development and disease. Experiments in mice, zebrafish, and in vitro have uncovered roles for the Hippo pathway, YAP, and TAZ in vasculogenesis, angiogenesis, and lymphangiogenesis. In addition, the Hippo pathway has been implicated in vascular cancers and cardiovascular diseases, thus identifying it as a potential therapeutic target for the treatment of these conditions. However, despite recent advances, Hippo's role in the vasculature is still underappreciated compared with its role in epithelial tissues. In this review, we appraise our current understanding of the Hippo pathway in blood and lymphatic vessel development and highlight the current knowledge gaps and opportunities for further research.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The nucleoporin (NUP) ELYS, encoded by AHCTF1, is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using both larval and adult zebrafish models of hepatocellular carcinoma (HCC), in which the expression of an inducible mutant kras transgene (krasG12V) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly decreases liver volume, while non-hyperplastic tissues are unaffected. We demonstrate that in the context of cancer, ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly, and chromosome segregation, leading to DNA damage and activation of a Tp53-dependent transcriptional programme that induces cell death and cell cycle arrest. Heterozygous expression of both ahctf1 and ranbp2 (encoding a second nucleoporin), or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocks krasG12V-driven hepatocyte hyperplasia. Gene expression analysis of patient samples in the liver hepatocellular carcinoma (LIHC) dataset in The Cancer Genome Atlas shows that high expression of one or more of the transcripts encoding the 10 components of the NUP107–160 subcomplex, which includes AHCTF1, is positively correlated with worse overall survival. These results provide a strong and feasible rationale for the development of novel cancer therapeutics that target ELYS function and suggest potential avenues for effective combinatorial treatments. Editor's evaluation We believe that the study demonstrates the importance of nuclear pore complex components for Kras/p53 driven liver tumors. The findings made here in zebrafish may stimulate additional preclinical and mechanistic studies to test the role of nuclear pore components in cancer. https://doi.org/10.7554/eLife.73407.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Synthetic lethality describes the death of cells in response to individual mutations in two separate genes, neither of which is lethal alone. The phenomenon has emerged as a promising framework for cancer drug development (Gao and Lai, 2018). Inherent to the approach is the capacity to induce the death of a vulnerable cell population, such as oncogene-expressing cancer cells, and leave healthy cells unaffected. In the clinic, the use of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors to successfully treat tumours carrying mutations in the breast cancer susceptibility genes, BRCA1/BRCA2 (Lord and Ashworth, 2017), has validated the approach and driven the search for other clinically relevant gene pairings, including those that confer synthetic lethality in cancer cells expressing oncogenic mutations in KRAS (Luo et al., 2009; Wang et al., 2017). In this paradigm, the interacting gene is neither mutated nor oncogenic in its own right. Rather, its function is essential to maintain the tumourigenic state, inspiring the concept of non-oncogene addiction (Solimini et al., 2007). In this study, we tested whether AHCTF1 exhibits the properties of a synthetic lethal interacting gene with mutant KRAS. We became interested in AHCTF1 when we and others showed that homozygous inheritance of an ENU-induced nonsense mutation in the zebrafish ahctf1 gene (ahctf1ti262) (de Jong-Curtain et al., 2009; Davuluri et al., 2008) disrupted nuclear pore formation and caused catastrophic levels of cell death in the intestinal epithelium and other highly proliferative cell compartments during zebrafish development (de Jong-Curtain et al., 2009; Davuluri et al., 2008). Meanwhile, cells in relatively quiescent tissues survived and remained healthy. AHCTF1 encodes ELYS, a 252-kDa multidomain nucleoporin (NUP) that was first discovered in mice where it was shown to be required for the proliferation and survival of inner mass cells during embryonic development (Okita et al., 2004). ELYS is one of 10 components of the large NUP107–160 subunit of nuclear pore complexes (NPCs). These huge (110 MDa) multi-subunit complexes comprise approximately 34 different NUPs in octameric array (Beck and Hurt, 2017; Petrovic et al., 2022), forming cylindrical channels in the nuclear envelope that regulate nucleocytoplasmic transport and intracellular localisation of large (>40 kDa) molecules. ELYS is also indispensable for NPC reassembly after mitosis (Rasala et al., 2006; Gillespie et al., 2007; Franz et al., 2007) and carries out a broad range of activities during the cell cycle, including chromatin decompaction, mitotic spindle assembly, and chromosome segregation (Gillespie et al., 2007; Kuhn et al., 2019; Güttinger et al., 2009; Chatel and Fahrenkrog, 2011; Mishra et al., 2010; Yokoyama et al., 2014; Kobayashi et al., 2019; Rasala et al., 2008). Having shown previously that cells that are rapidly growing and dividing during zebrafish development are highly vulnerable to ELYS depletion, we hypothesised that cancer cells fuelled by powerful oncogenes would be vulnerable too. To test this, we took advantage of a genetically tractable zebrafish model of hepatocellular carcinoma (HCC) in which a doxycycline-inducible, hepatocyte-specific EGFP-krasG12V transgene drives hepatocyte hyperplasia, liver enlargement, and morphological changes characteristic of human HCC (Chew et al., 2014). We chose this model of HCC because the RAS/RAF/MAPK signalling pathway is almost always hyperactivated in human HCC (Calvisi et al., 2006). We found that reducing the expression of ahctf1 mRNA by 50% disrupted multiple functions in krasG12V-expressing hepatocytes and markedly impaired their growth and survival. Further studies showed that accumulation of DNA damage and robust Tp53 activation contributed to this response. These findings suggest that ahctf1 and mutant kras participate in a synthetic lethal interaction that is selective for Kras oncogene-expressing cells, providing a rationale to investigate whether ELYS function could be targeted effectively and selectively by a new class of anti-cancer drugs. Results Molecular characterisation of mutant kras-driven zebrafish models of HCC To generate both larval and adult models of HCC (Chew et al., 2014), we varied the timing of doxycycline (dox) treatment. In our larval model, on a wildtype (WT) ahctf1 background, we induced the expression of a single EGFP-krasG12V transgene, denoted TO(krasG12V)T/+ in developing livers by treating with dox between 2 and 7 days post-fertilisation (dpf) (Figure 1a, b). This led to the accumulation of a constitutively active, EGFP-tagged, potently oncogenic form of Kras (KrasG12V) specifically in hepatocytes, causing hepatocyte hyperplasia and a substantial increase (4-fold) in liver volume (Figure 1c, d). To establish the clinical relevance of this phenotype to human HCC, we used RNA sequencing to analyse the gene expression patterns of livers expressing the krasG12V transgene compared to livers expressing no transgene. We detected more than 6000 significantly upregulated genes in dox-treated TO(krasG12V)T/+ livers compared WT livers, and a further 6000+ genes were significantly downregulated (Figure 1—figure supplement 1a, b). Gene set enrichment analysis identified a positive correlation between the differential gene expression data from the dox-treated TO(krasG12V)T/+ versus WT livers and the differential gene expression data obtained from the HCC (LIHC) and healthy liver subsets available in The Cancer Genome Atlas (TCGA) (Figure 1—figure supplement 1c). We also found a positive correlation between the diffentially expressed genes (DEGs) from the dox-treated TO(krasG12V)T/+ versus WT livers and a small HCC expression signature based on four patient samples carrying KRAS G12 or KRAS G13 mutations (Figure 1—figure supplement 1d). Of the upregulated genes, many were significantly enriched in KEGG pathways associated with highly proliferative cancers, including DNA replication, cell cycle regulation, and DNA damage repair (Figure 1—figure supplement 1e). These observations build on previous reports that dox-treated TO(krasG12V)T/+ zebrafish provide an authentic model of human HCC (Zheng et al., 2014b; Huo et al., 2019). Figure 1 with 3 supplements see all Download asset Open asset ahctf1 heterozygosity restricts liver volume in a zebrafish model of krasG12V-driven hepatocellular carcinoma (HCC). (a) Protocol used to induce TO(krasG12V)T/+ expression in the livers of developing zebrafish larvae. (b) RT-quantitative PCR (RT-qPCR) analysis of ahctf1 mRNA levels in pooled micro-dissected larval livers (n = 3 biological replicates). (c) Representative three-dimensional reconstructions of 2-CLiP and dox-treated TO(krasG12V)T/+ larval livers of the indicated ahctf1 genotype. Scale bar 25 µm. (d) Impact of ahctf1 heterozygosity on liver volume in 2-CLiP and TO(krasG12V)T/+ larvae (n ≥ 20). (e) Impact of ahctf1 heterozygosity on liver-to-body mass ratio of adult TO(krasG12V)T/+ zebrafish in the presence or absence of dox treatment (n = 10). (f) Histological sections of adult male TO(krasG12V)T/+ zebrafish livers of the indicated ahctf1 genotype and dox treatment, stained with haematoxylin and eosin. In vehicle-treated adults, hepatocytes are densely packed and well differentiated. White arrows point to sections through blood vessels containing red blood cells. Meanwhile, the hepatocytes in dox-treated animals are poorly differentiated and exhibit multiple cytological abnormalities, including pyknotic nuclei (arrowheads) and vacuolation (black arrows). Scale bar 25 µm. (g) Western blot of Ras and Gapdh protein signals in total input lysates (50 μg) of TO(krasG12V)T/+ larvae of the indicated ahctf1 genotype and dox treatment. (h) Western blot of active Ras-GTP protein signals in lysates following active Ras pull-down. Figure 1—source data 1 Figure 1b: Heterozygous ahctf1 mRNA expression in the livers of doxycycline (dox)-treated TO(krasG12V)T/+ zebrafish larvae, relative to wildtype (WT). Figure 1d: Liver volume [µm3] of 2-CLiP and TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 1e: Liver/body mass ratio (%) of adult male and female TO(krasG12V)T/+ zebrafish with and without dox treatment: role of ahctf1 genotype. https://cdn.elifesciences.org/articles/73407/elife-73407-fig1-data1-v2.xlsx Download elife-73407-fig1-data1-v2.xlsx Figure 1—source data 2 Figure 1g: Uncropped unlabelled western blot. https://cdn.elifesciences.org/articles/73407/elife-73407-fig1-data2-v2.zip Download elife-73407-fig1-data2-v2.zip Figure 1—source data 3 Figure 1g: Uncropped western blot (labelled). https://cdn.elifesciences.org/articles/73407/elife-73407-fig1-data3-v2.zip Download elife-73407-fig1-data3-v2.zip Figure 1—source data 4 Figure 1h: Uncropped unlabelled western blot. https://cdn.elifesciences.org/articles/73407/elife-73407-fig1-data4-v2.zip Download elife-73407-fig1-data4-v2.zip Figure 1—source data 5 Figure 1h: Uncropped western blot (labelled). https://cdn.elifesciences.org/articles/73407/elife-73407-fig1-data5-v2.zip Download elife-73407-fig1-data5-v2.zip ahctf1 heterozygosity reduces liver overgrowth in a zebrafish model of krasG12V-driven HCC We investigated the requirement for Elys in this in vivo cancer setting by introducing a mutant ahctf1 allele (floti262) (Chen et al., 1996) into the genome of the TO(krasG12V)T/+ model. This produced a 57% reduction in ahctf1 mRNA expression in ahctf1+/− larvae at 7 dpf, compared to larvae expressing WT ahctf1 (Figure 1b). This is consistent with the nonsense mutation in ahctf1ti262 triggering nonsense mediated decay of mRNA transcribed from the affected allele, rather than it being translated and expressed as a truncated Elys protein. As a control for our experiments, we used another transgenic line, denoted 2-CLiP (2-Colour Liver Pancreas), in which hepatocytes express dsRed fluorescence constitutively but no oncogenic transgene (Korzh et al., 2008). On this background, heterozygous (HET) ahctf1ti262 zebrafish develop normally (Figure 1—figure supplement 2a), reach sexual maturity, and exhibit a normal lifespan, as do HET ahctf1 mice (Okita et al., 2004). Similarly, hepatocytes on the TO(krasG12V)T/+ background receiving no dox treatment developed normally (Figure 1—figure supplement 2b). Mean liver volume in control 2-CLiP larvae at 7 dpf was 1.95 × 106 ± 4.99 × 104 μm3 and was unaffected by ahctf1 genotype (Figure 1—figure supplement 3a, b). Dox-induced expression of oncogenic KrasG12V in the TO(krasG12V)T/+ model, produced a striking (4-fold) increase in liver volume (7.97 × 106 ± 1.21 × 105 μm3) over the 5 days of dox treatment (Figure 1c, d). Remarkably, liver volume was pared back to 5.92 × 106 ± 8.83 × 104 µm3 in ahctf1 HETS, equating to a 35% reduction in excess liver volume. ahctf1 heterozygosity also reduced liver enlargement in krasG12V-expressing adult zebrafish. To induce HCC in these experiments, sexually mature zebrafish aged 3 months post-fertilisation were treated with dox (final concentration 20 mg/L) for 7 days with fresh water and dox administered daily. After 7 days, the impact of forced krasG12V expression was assessed in both male and female adults by measuring the mass of the liver and expressing this as a percentage of the total mass of the animal prior to liver dissection (Figure 1e). Compared to vehicle-treated animals, we found that dox induction of krasG12V expression produced a robust (9-fold) increase in liver mass expressed as a percentage of total body mass of male zebrafish expressing WT ahctf1. This ratio was reduced by 28% in ahctf1 HETS and we obtained similar results with females (Figure 1e). Histological sections of livers from vehicle-treated ahctf1+/+; TO(krasG12V)T/+ males stained with haematoxylin and eosin (H&E) revealed an orderly arrangement of polygonal hepatocytes interspersed with blood vessels containing red blood cells. In the absence of dox, this appearance was unaffected by ahctf1 genotype (Figure 1f, top row). In contrast, dox-treated ahctf1+/+; TO(krasG12V)T/+-expressing male livers exhibited severely disrupted architecture, and a general loss of hepatocyte organisation (Figure 1f, bottom left). Hepatocytes were generally poorly differentiated and some exhibited features such as pyknotic nuclei, condensed nucleoli, and vacuolation (Figure 1f, bottom left). Sections of krasG12V-expressing livers from ahctf1 HETS showed hepatocytes with improved nuclear integrity and less vacuolation (Figure 1f, bottom right). These data show that in both larval and adult TO(krasG12V)T/+-expressing zebrafish, a modest (50%) decrease in ahctf1 mRNA expression exerts a robust and selective reduction in hepatocyte hyperplasia and liver overgrowth. Level of activated (GTP-bound) Ras in dox-treated TO(krasG12V)T/+ larvae is unaffected by ahctf1 heterozygosity To determine how heterozygous ahctf1 mRNA expression restricts mutant kras-driven liver overgrowth, we investigated whether the Elys protein could interfere with the activation of KrasG12V directly. To do this, we first quantitated the abundance of the EGFP-KrasG12V protein using western blot analysis. Dox treatment markedly increased the abundance of the EGFP-KrasG12V protein in lysates of TO(krasG12V)T/+ zebrafish larvae compared to vehicle-treated larvae (Figure 1g), and this was unaffected by ahctf1 genotype. We then used an active (GTP-bound) Ras pull-down assay to isolate the active GTP-bound fraction of Ras proteins in lysates of TO(krasG12V)T/+ larvae, followed by western blot analysis (Baker and Rubio, 2021). In vehicle-treated zebrafish larvae, signals corresponding to GTP-bound endogenous Ras proteins were weak, whereas lysates from dox-treated larvae gave robust signals corresponding to activated EGFP-KrasG12V proteins. The intensity of these signals was not affected by ahctf1 genotype (Figure 1h). Thus, the impact of heterozygous ahctf1 mutation on the growth, proliferation, and survival of TO(krasG12V)T/+ expressing hepatocytes did not occur by directly or indirectly interfering with the production of GTP-bound Ras proteins. ahctf1 heterozygosity disrupts the abundance of NPCs in dox-treated TO(krasG12V)T/+ hepatocytes Having demonstrated that ahctf1 heterozygosity restricts krasG12V-driven liver enlargement, we sought to understand the biological mechanisms underlying this. As previously mentioned, Elys is a multifunctional protein with several roles in the cell cycle (Figure 2—figure supplement 1). We started by examining whether its canonical role in post-mitotic nuclear pore assembly (Figure 2—figure supplement 1a, b) was disrupted. To measure the abundance and distribution of NPCs, we stained thick sections of larval livers (200 μm), with an antibody (mAb414) that recognises FG-repeat NUPs (NUP358, 214, 153, and 62) in mature NPCs. Using Airyscan confocal laser-scanning microscopy, we showed that hepatocytes not carrying the TO(krasG12V) transgene (denoted TO(kras)+/+), exhibit fluorescent puncta corresponding to NPCs at the nuclear rim with negligible staining in the cytoplasm, a pattern that was unaffected by ahctf1 genotype (Figure 2a; left two columns). In dox-treated larvae harbouring the TO(krasG12V)T/+ transgene, fluorescence intensity was markedly increased (Figure 2a, third column) and there was an increase in the ratio of nuclear:cytoplasmic staining (Figure 2b). By comparison, fluorescence intensity at the nuclear rim was diminished in ahctf1 HETS, concomitant with the appearance of fluorescent puncta in the cytoplasm (Figure 2a, fourth column; arrows) and a significant reduction in the ratio of nuclear:cytoplasmic fluorescence intensity (Figure 2b). Figure 2 with 1 supplement see all Download asset Open asset ahctf1 heterozygosity disrupts the density of nuclear pore complexes and reduces nuclear volume in dox-treated TO(krasG12V)T/+ hepatocytes. (a) Representative Airyscan imaging of liver sections stained with mAb414 (white) marking FG-NUPs, Hoechst 33342 (cyan) marking DNA, and rhodamine phalloidin (magenta) marking the F-actin cytoskeleton in non-TO(krasG12V)-expressing cells and EGFP-KrasG12V (magenta) marking the cell membrane in dox-treated TO(krasG12V)-expressing cells of the indicated ahctf1 and TO(krasG12V) genotypes. Arrows in right-hand panel point to mAb414/FG-nucleoporin staining in the cytoplasm. Scale bar 2 µm. (b) Quantification of mean nuclear/cytoplasmic fluorescence intensity of mAb414 staining after 3D segmentation and morphological filtering of nuclear and cytoplasmic areas (n > 18). (c) Representative Airyscan images of mAb414 staining at the nuclear surface of sections of the indicated ahctf1 and TO(krasG12V) genotype. Scale bar 1 µm. (d) Quantification of nuclear pore density (n ≥ 25). (e) Quantification of nuclear volume (n ≥ 25). Data are expressed as mean ± standard error of the mean (SEM). Significance was assessed using a one-way analysis of variation (ANOVA) with Tukey’s multiple comparisons test. Figure 2—source data 1 Figure 2b: Ratio of nuclear to cytoplasmic intensity of mAb414 immunostaining in livers of dox-treated TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 2d: Abundance of nuclear pores per µm2 in livers of dox-treated TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 2e: Mean volume of nuclei per µm3 in dox-treated livers of TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. https://cdn.elifesciences.org/articles/73407/elife-73407-fig2-data1-v2.xlsx Download elife-73407-fig2-data1-v2.xlsx To determine the abundance of NPCs, we analysed the pattern and density of fluorescent puncta observed at the nuclear surface of non-krasG12V expressing hepatocytes (Figure 2c; left two columns). The induced expression of the krasG12V transgene in the presence of WT ahctf1 resulted in 59% more fluorescent puncta/NPCs at the nuclear surface of hyperplastic hepatocytes (Figure 2c, d; third column). These signals were reduced by 21% in ahctf1 HETS (Figure 2c, d; fourth column). Induced krasG12V expression also produced a 28% increase in nuclear volume compared to non-krasG12V-expressing cells (Figure 2e), and this increase in size was reduced to 13% in ahctf1 HETS. We infer from these data that hyperplastic hepatocytes expressing the krasG12V oncogene require highly efficient rates of nucleocytoplasmic transport to support their rapid proliferation, which they fulfil by increasing NPC density and the size of their nuclei. This adaptation required a full complement of ahctf1 expression, suggesting non-oncogene addiction to ahctf1. We found that in hyperplastic hepatocytes that were heterozygous for ahctf1 this adaptation was partially restricted, creating a condition likely to amplify oncogenic stress. ahctf1 heterozygosity impairs mitotic spindle assembly and chromosome segregation in dox-treated TO(krasG12V)T/+ hepatocytes Next, we examined the impact of reduced Elys expression on spindle formation and chromosome segregation during mitosis (Figure 2—figure supplement 1g and h). We assessed these features in cryosections of liver using α-tubulin and 4′,6-diamidino-2-phenylindole (DAPI) to stain microtubules and chromatin, respectively. Metaphase cells in ahctf1+/+;TO(krasG12V)T/+ livers exhibited normal bipolar spindle formation followed by complete chromosome segregation during anaphase (Figure 3a). In contrast, metaphase cells in ahctf1+/−;TO(krasG12V)T/+ hepatocytes displayed abnormal multipolar spindles and misaligned chromosomes (Figure 3b). Proper chromosome segregation was disrupted with multiple anaphase bridges formed. While the number of cells observed at different mitotic stages was similar in ahctf1+/+ and ahctf1+/− (Figure 3c), mitotic abnormalities were observed in 50% of ahctf1+/−;TO(krasG12V)T/+ hepatocytes during metaphase and anaphase but not at all in ahctf1+/+;TO(krasG12V)T/+ hepatocytes (Figure 3d). These data are consistent with hyperplastic hepatocytes requiring a full complement of ahctf1 expression to maintain rapid rounds and integrity of mitosis, and provide another facet of non-oncogene addiction to ahctf1 that is likely to contribute to oncogenic stress. Figure 3 Download asset Open asset ahctf1 heterozygosity impairs mitotic spindle assembly and chromosome segregation in dox-treated TO(krasG12V)T/+ hepatocytes. (a) Representative Airyscan imaging of liver cryosections stained with α-tubulin antibody (magenta) marking spindle microtubules and DAPI (cyan) marking DNA in mitotic cells of TO(krasG12V)T/+ larvae on a wildtype ahctf1+/+ background. (b) Mitotic cells in liver cryosections of TO(krasG12V)T/+ larvae that are heterozygous for ahctf1+/− exhibit multiple defects, including multipolar spindles, misaligned chromosomes, and anaphase bridges (arrows). Scale bar 2 µm. (c) Distribution of cells observed at different mitotic stages (n = 92 livers, 326 mitotic cells). (d) Quantification of the percentage of mitotic hepatocytes exhibiting an aberrant phenotype (n = 14–57). Significance was assessed using a Chi-square test. Figure 3—source data 1 Figure 3c: Number of cells at different stages of mitosis in livers of TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 3d: Percentage of mitotic cells expressing an aberrant phenotype in livers of TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. https://cdn.elifesciences.org/articles/73407/elife-73407-fig3-data1-v2.xlsx Download elife-73407-fig3-data1-v2.xlsx ahctf1 heterozygosity causes DNA damage and accumulation of Tp53 protein in dox-treated TO(krasG12V)T/+ hepatocytes Other features of oncogene-induced stress in response to robust and persistent overexpression of RAS oncoproteins include stalled DNA replication, DNA damage, and genome instability. To determine whether expression of krasG12V causes DNA damage in our model, we stained cryosections of larval livers with DAPI and γ-H2AX, which is a sensitive marker for stalled DNA replication forks and DNA double-strand breaks (Rogakou et al., 1998). We found that 1% of ahctf1+/+;TO(krasG12V)T/+ hepatocyte nuclei were positive for γ-H2AX (Figure 4a, c), compared to 6% in the nuclei of ahctf1 HETS (Figure 4b, c). Figure 4 Download asset Open asset ahctf1 heterozygosity causes DNA damage and accumulation of Tp53 protein in dox-treated TO(krasG12V)T/+ hepatocytes. (a) Representative Airyscan imaging of cryosections of liver from dox-treated ahctf1+/+;TO(krasG12V)T/+ larvae stained with γ-H2AX antibody (white) marking DNA double-strand breaks, DAPI (cyan) marking DNA and EGFP-KrasG12V (magenta) marking the cell membrane. Scale bar 5 µm. (a’) Inset of γ-H2AX-positive nuclei in ahctf1+/+;TO(krasG12V)T/+ hepatocytes. Scale bar 2 µm. (b) Representative images of cryosections of liver from dox-treated ahctf1+/−;TO(krasG12V)T/+ larvae. Scale bar 5 µm. (b’) Inset of γ-H2AX-positive nuclei in cryosections of liver from ahctf1+/−;TO(krasG12V)T/+ larvae. Scale bar 2 µm. (c) Quantification of the percentage of hepatocytes positive for γ-H2AX (n ≥ 31). (d) Representative western blot of Tp53 protein signals in lysates of TO(krasG12V) larvae of the indicated ahctf1 genotype. (e) Quantification of Tp53 protein levels normalised by reference to the Gapdh loading control (n = 3 independent experiments). (f) Representative three-dimensional reconstructions of dox-treated TO(krasG12V)T/+ livers of the indicated ahctf1 and tp53 genotypes. Scale bar 25 µm. (g) Impact of ahctf1 heterozygosity and homozygous tp53 mutation on liver volume in 2-CLiP and TO(krasG12V)T/+ larvae (n ≥ 20). Data are expressed as mean ± standard error of the mean (SEM). Significance was calculated using a Student’s t-test or one-way analysis of variation (ANOVA) with Tukey’s multiple comparisons test. Figure 4—source data 1 Figure 4c: Percentage of hepatocytes positive for γ-H2AX immunostaining in livers of dox-treated TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 4e: Quantitation of Tp53 protein intensity in western blots of lysates from TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 genotype. Figure 4g: Quantitation of liver volume (µm3) of TO(krasG12V)T/+ zebrafish larvae; role of ahctf1 and tp53 mutation. https://cdn.elifesciences.org/articles/73407/elife-73407-fig4-data1-v2.xlsx Download elife-73407-fig4-data1-v2.xlsx Figure 4—source data 2 Figure 4d: Uncropped unlabelled western blot. https://cdn.elifesciences.org/articles/73407/elife-73407-fig4-data2-v2.zip Download elife-73407-fig4-data2-v2.zip Figure 4—source data 3 Figure 4d: Uncropped western blot (labelled). https://cdn.elifesciences.org/articles/73407/elife-73407-fig4-data3-v2.zip Download elife-73407-fig4-data3-v2.zip In the presence of WT Tp53, DNA damage is limited by activation of Tp53 transcription-dependent pathways that can perform DNA damage repair, and, if necessary, induce cell cycle arrest, senescence, and/or apoptosis. To determine whether the increase in DNA damage that occurred in our model in response to ahctf1 heterozygosity stimulated Tp53 accumulation, we measured the levels of Tp53 protein in pooled lysates of micro-dissected krasG12V-expressing livers, krasG12V-nonexpressing livers and the larval remains after the dissection (Figure 4d, e). No Tp53 signal was obtained from non-krasG12V-expressing livers, or the body remains after liver removal. However, we detected a weak Tp53 signal in extracts of dox-treated ahctf1+/+;TO(krasG12V)T/+ livers, consistent with induced krasG12V expression causing mild cellular stress. This low level of stress was amplified significantly in heterozygous ahctf1 livers, where we obtained a strong (>3.5-fold) increase in the intensity of the Tp53 signal (Figure 4e), which correlated well with the higher density of γ-H2AX staining. To test whether Tp53 accumulation was responsible for limiting liver volume in TO(krasG12V)T/+ larvae, we introduced the zebrafish tp53M214K allele encoding a transactivation dead Tp53 variant (Berghmans et al., 2005). Abrogating Tp53 function by homozygous expression of this allele (denoted tp53m/m) in ahctf1+/+;TO(krasG12V)T/+ larvae increased liver volume by 50%, to 12.5 × 106 ± 1.10 × 105 μm3, compared to livers on a WT (tp53+/+) background (Figure 4f, g), demonstrating that Tp53 function normally places great restraint on the growth of TO(krasG12V)T/+ livers in this model. ahctf1 heterozygosity combined with loss of Tp53 function also produced an increase in liver volume by 50% compared to ahctf1+/−;TO(krasG12V)T/+ livers on a WT Tp53 background. Comparing the volume of ahctf1 HET livers and WT ahctf1 livers on a Tp53 mutant background, showed that heterozygous ahctf1 still achieved a reduction in liver volume, albeit significantly less than in the presence of WT Tp53. ahctf1 heterozygosity amplifies cell death of dox-treated TO(krasG12V)T/+ hepatocytes in the presence and absence of WT Tp53 The tumour suppressive properties of Tp53 lie in its capacity to activate the transcription of genes that participate in processes that restrict tumour growth, including cell cycle arrest, senescence, apoptosis, DNA repair, and metabolic adaptation. To test whether disruption of these processes contributed to the reduction in liver volume we observe between WT and heterozygous ahctf1 larvae, we first looked at apoptosis. To do this, we introduced an apoptosis reporter transgene, Tg(actb2:SEC-Hsa.ANXA5-mKate2,cryaa:mCherry)uq24rp (hereafter denoted Annexin 5-mKate) into the HCC model. This transgene constitutively expresses a fusi

Over recent decades, shifts toward translational or applied research in many countries have come at the expense of fundamental discovery research. Here we discuss the historical importance of basic science in the cardiovascular field, the risks in its decline and the ongoing need for a strong foundation in fundamental discovery research.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

SUMMARYThis study establishes the homeodomain only protein, HOPX, as a determinant controlling the molecular switch between cardiomyocyte progenitor and maturation gene programs. Time-course single-cell gene expression with genome-wide footprinting reveal that HOPX interacts with and controls core cardiac networks by regulating the activity of mutually exclusive developmental gene programs. Upstream hypertrophy and proliferation pathways compete to regulate HOPX transcription. Mitogenic signals override hypertrophic growth signals to suppress HOPX and maintain cardiomyocyte progenitor gene programs. Physiological studies show HOPX directly governs genetic control of cardiomyocyte cell stress responses, electro-mechanical coupling, proliferation, and contractility. We use human genome-wide association studies (GWAS) to show that genetic variation in the HOPX-regulome is significantly associated with complex traits affecting cardiac structure and function. Collectively, this study provides a mechanistic link situating HOPX between competing upstream pathways where HOPX acts as a molecular switch controlling gene regulatory programs underpinning metabolic, signaling, and functional maturation of cardiomyocytes.

The lymphatic vasculature is a vital component of the vertebrate vascular system that mediates tissue fluid homeostasis, lipid uptake and immune surveillance. The development of the lymphatic vasculature starts in the early vertebrate embryo, when a subset of blood vascular endothelial cells of the cardinal veins acquires lymphatic endothelial cell fate. These cells sprout from the veins, migrate, proliferate and organize to give rise to a highly structured and unique vascular network. Cellular cross-talk, cell-cell communication and the interpretation of signals from surrounding tissues are all essential for coordinating these processes. In this chapter, we highlight new findings and review research progress with a particular focus on LEC migration and guidance, expansion of the LEC lineage, network remodeling and morphogenesis of the lymphatic vasculature.

Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1 , encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1 / Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1 −/− mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a −/− mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

ABSTRACT The nucleoporin ELYS, encoded by AHCTF1 , is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using a zebrafish model of hepatocellular carcinoma, in which the expression of an inducible mutant kras transgene (kras G12V ) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly shrinks tumour burden, while non-hyperplastic tissues are unaffected. We demonstrate that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of TP53-dependent and independent mechanisms of cell death and cell cycle arrest. This selective vulnerability of cancer cells to mild disruption of Elys function uncovers a novel synthetic lethal interaction between ahctf1 and kras mutations that could be exploited therapeutically. Heterozygous expression of both ahctf1 and ranbp2 , or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocked kras G12V -driven hepatocyte hyperplasia, revealing promising avenues for combinatorial treatments.

The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair, and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation, and migration. Extracellular signal-regulated kinase (Erk) is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca 2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response, however, involves a Vegfr-dependent mechanism that initiates concomitantly with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.