BACKGROUND:Truncating variants in the TTN gene (TTNtv), encoding the giant sarcomeric protein titin, cause a range of human cardiac and skeletal muscle disorders of varying penetrance and severity. The effects of variant location on clinical manifestations are incompletely understood. METHODS:We generated 6 zebrafish lines carrying truncating ttn.2 variants in the Z-disk, I-band, A-band, and M-band titin regions. Expression of titin transcripts and protein levels was evaluated using quantitative polymerase chain reaction and proteomics. Phenotype analysis was performed during embryonic development and in adult hearts. RESULTS:Homozygous embryos from all lines except the C-terminal line, e232, showed a significant reduction of Z-disk and I-band ttn.2 transcripts, but A-band and M-band transcript levels were reduced only in lines with truncations distal to the cronos promoter. These homozygous embryos uniformly died by 7 to 10 days postfertilization with marked impairment of cardiac morphology and function. Skeletal muscle motility and sarcomere organization were more disrupted in mutants with truncations distal to the cronos promoter compared with those proximal. In contrast, homozygous e232 embryos, which lacked only the titin kinase and M-band regions, had relatively preserved cardiac function with incorporation of truncated Ttn.2/Cronos protein and normal sarcomere assembly, but selective degradation of fast skeletal muscle sarcomeres. All heterozygous embryos were phenotypically indistinguishable from wild type. High-frequency echocardiography in adult heterozygous fish showed reduced ventricular contraction under resting conditions in A-band mutants. Heterozygous Z-disk and I-band mutants had no significant baseline impairment but were unable to augment ventricular contraction in response to acute adrenaline exposure, indicating a lack of cardiac reserve. CONCLUSIONS:Our data suggest that cardiac and skeletal muscle dysfunction associated with truncating ttn.2 variants is influenced by age, variant location, and the amount of functional titin protein. The distinctive phenotype associated with distal C-terminal truncations may reflect different requirements for C-terminal titin for maintenance of fast, slow, and cardiac muscle sarcomeres.

The amyloid precursor protein (APP) is heavily studied as the source of amyloid beta in Alzheimer's disease (AD), however, the complex functions of APP remain poorly understood, as does the impact of APP dosage on neurodevelopment. Here we study APP specifically in the context of Trisomy 21. In an effort to reduce APP dosage in trisomy 21 iPSCs, we generated trisomic isogenic lines which vary in APP dosage, including a full APP knock-out line, as well as lines carrying mutations of the APP extracellular domain. We used a panel of these lines to study potential impacts of APP dosage or structure on two distinct steps of neurogenesis in trisomic cells: 1) terminal differentiation of human neuro-progenitor cells (NPC) to post-mitotic neurons and 2) neuron structure as reflected in neurite outgrowth. Complete loss of APP causes marked defects in each of these two distinct steps, reducing both the terminal differentiation of NPCs to neurons, and proper neurite development for extended neuron structure. Hence, APP is necessary for both aspects of normal neurogenesis. Further analyses of the null and other mutant lines indicate that APP likely impacts these two distinct steps by two different mechanisms. Collective results suggest that the reduced terminal differentiation of NPCs reflects an effect of APP dosage, whereas the defects in neurite extension are due to structural mutation of the APP extracellular domain. Absence of APP or reduced (monosomic) APP dosage prolonged the cycling of trisomic NPCs, which is known to be regulated by Notch signaling. APP and Notch are the main targets of gamma-secretase cleavage, hence we hypothesized that APP dosage may impact neurogenesis indirectly, potentially via effects on Notch signaling. To test this, we treated NPCs with Compound E which inhibits gamma-secretase (and Notch signaling); results show this restored levels of neurogenesis in APP depleted lines, supporting an indirect effect of APP dosage . In contrast, results indicate that disruption of APP extracellular domain integrity impacts neurite extension via a more direct role of APP in neuron structural maturation. This study describes a resource of well-characterized APP mutant isogenic DS iPSC lines, implicates a dynamic interplay between APP dosage and Notch signaling, and raises new questions about the impact of APP dosage in orchestrating neural progenitor fate decisions during human brain development, specifically in the context of trisomy 21.

Gene expression in cardiac development is regulated through complex epigenetic mechanisms. Histone deacetylases (HDACs) are one of the many layers of epigenetic modulation, whereby they remove acetylation marks on histone tails, prompting chromatin tightening and therefore bring about gene repression. The most extensively characterised HDACs in cardiac development are HDACs 1-3, all belonging to the Class I HDAC family. Global as well as tissue-specific knockout models in mice have provided insight into the phenotypes generated by loss of these key molecular regulators. In some instances, molecular processes that individual HDACs regulate within cardiac development have also been revealed, although the epigenetic targets and binding partners of HDACs within cardiac development are still relatively understudied. Knowledge has also been contributed from in vitro studies using stem cell-derived models as well as burgeoning research using the zebrafish model. The aim of this review is to summarise the current knowledge of class I HDAC function during key stages of cardiac development, including cardiac specification and differentiation, looping morphogenesis, and second heart field development. The role of class I HDACs in non-cardiomyocyte populations, such as the endocardium, valves, and epicardium is also discussed.

As clinical genomics is now embedded in healthcare systems, formal guidelines for disease gene associations and variant classification are essential for standardisation of reporting. However, genes of uncertain significance (GUS, often novel disease genes) and variants of uncertain significance (VUS) continue to present clinical and scientific challenges limiting access to targeted treatments and tailored support, thereby prolonging the diagnostic odyssey. The Australian Functional Genomics Network (AFGN) improves rare disease diagnostics by connecting clinicians and researchers via a registry and provides funding ($5 million over 5 years: 2021–2026) for projects initially in two streams: Stream 1 (S1, single variants) and Stream 2 (S2, multiple VUS). As of October 2023, 24 projects that have undergone clinical and scientific review have been awarded funding with amounts ranging from $30,000 (S1) to $200,000 (S2). These projects encompass generation of organoids and animal models, in vitro and RNA assays, and higher throughput functional assays. Data from these projects has already provided evidence of disease causality and impacted gene and variant classifications. AFGN aspires to empower clinically relevant research and accelerate our knowledge of the impact of genetic variants on health for more effective diagnoses, interventions, and treatments for patients.

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.

Protein phosphatase 2A (PP2A) regulatory subunit B55α has been implicated in the transcriptional regulation of cardiac growth and fibrosis by suppressing HDAC5/MEF2 signalling in cardiomyocytes. We created and characterised two mouse models with global or cardiomyocyte-specific disruption of the gene encoding B55α (Ppp2r2a) to conduct the first detailed exploration of B55α in the heart. Global homozygous B55α knockout mice died in utero, while heterozygous mice had thinner left ventricular walls at 12 months, an effect more pronounced in males. At 10-12 weeks of age, cardiomyocyte-specific B55α knockout mice displayed normal cardiac morphology with increased left ventricular collagen deposition, identifying B55α as a negative regulator of cardiac fibrosis. Gene expression analyses demonstrated extensive remodelling of the cardiac transcriptome in male but not female mice, revealing a sexually dimorphic role for B55α in cardiac transcriptional regulation. These findings provide a basis for future work investigating B55α in cardiac stress settings.

BACKGROUND:Down syndrome, or Trisomy 21, is the leading genetic cause of cognitive disability in children and is associated with a high risk of several comorbidities, particularly congenital heart defects, early onset Alzheimer's disease, leukaemia, and autoimmune disorders. OBJECTIVE:This study describes the design, methods, and operational procedures employed to establish a biobank dedicated to Down syndrome that can support research projects investigating the effects of various genetic and environmental factors on this complex disease. METHODS:Blood was collected from all recruited subjects, processed, aliquoted and immediately frozen at -80 °C in the Interinstitutional Multidisciplinary BioBank (BioBIM) facilities. A small aliquot of the sample was used to perform blood tests for which analysis would not be feasible at a later date, such as blood cell counts. Each biological sample was coded, assigned a Standard PREanalytical Code, and registered in the oloBIOBANK software connected to a medical card containing all the donor's anamnestic data. All samples were stored under continuous real-time temperature recording using a freezer connected to a T-GUARD alarm system. In addition, a radiofrequency identification tracking system strictly monitored each cryopreservation operation performed throughout the sample lifecycle. RESULTS:Biological samples were collected from 454 individuals with Down syndrome from 2007 to 2023. A total of 2233 biological samples were available for research purposes, including whole blood in different anticoagulants, serum, plasma, and frozen peripheral blood mononuclear cells. The quality of the nucleic acids obtained through specific standard operating procedures demonstrated that these samples were appropriate for clinical and basic research. CONCLUSION:By establishing this biobank, we have gathered a significant number of biological samples and clinical data from individuals with Down syndrome, thereby fostering collaboration between different research groups in an open and transparent manner. Sharing expertise and resources among scientists will ultimately facilitate the transfer of knowledge to clinical practice, leading to the development of more effective therapeutic treatments to improve the outcomes and quality of life of patients with Down syndrome.

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.

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.

Hydrogen’s potential as a clean energy vector has created increased research interest in recent years. The properties of hydrogen coupled with increased storage, transport and production volumes present safety hurdles which need to be understood for hydrogen to become an integral part of a clean energy system. Learning from past incidents is one method used by industry to prevent future incidents. Therefore, this work looked to published literature to decipher what can be learned from historical hydrogen incidents. The resultant systematic literature review found much published work referring to hydrogen and incidents or accidents. To gain insights from this literature, an unsupervised clustering algorithm was used to analyse the abstracts and uncover underlying patterns and groups. The algorithm is unsupervised because no prior knowledge is assumed with regards to the potential groups or patterns in the dataset. The mean silhouette co-efficient was calculated for each abstract to improve the algorithms ability to detect patterns in the short, relatively similar pieces of text. The work revealed that literature referring to incidents is quite diverse, with less than 10% of sources focusing on actual incidents. Manual coding of literature that reviewed actual incidents was also completed. Reviews of actual incidents included works which analyse incident databases and works which review single case studies. The single case studies predominantly offer a narrative of events without formal analysis using risk management or incident investigation techniques. The sparsity of published literature on actual incidents suggests that an opportunity exists to improve the reporting of hydrogen incidents. Arguably, not all incidents will have research merit. However, quality published data could be used by industry and academics to guide hydrogen safety improvement endeavours. This points to a need for further work on which incidents would be candidates for further research and how to get industry to engage.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demon-strated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning elec-tron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

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

Dilated cardiomyopathy (DCM) is a common heart muscle disorder that frequently leads to heart failure, arrhythmias, and death. While DCM is often heritable, disease-causing mutations are identified in only ~30% of cases. In a forward genetic mutagenesis screen, we identified a novel zebrafish mutant, heart and head (hahvcc43), characterized by early-onset cardiomyopathy and craniofacial defects. Linkage analysis and next-generation sequencing identified a nonsense variant in the highly conserved scfd1 gene, also known as sly1, that encodes sec1 family domain-containing 1. Sec1/Munc18 proteins, such as Scfd1, are involved in membrane fusion regulating endoplasmic reticulum (ER)/Golgi transport. CRISPR/Cas9-engineered scfd1vcc44 null mutants showed severe cardiac and craniofacial defects and embryonic lethality that recapitulated the phenotype of hahvcc43 mutants. Electron micrographs of scfd1-depleted cardiomyocytes showed reduced myofibril width and sarcomere density, as well as reticular network disorganization and fragmentation of Golgi stacks. Furthermore, quantitative PCR analysis showed upregulation of ER stress response and apoptosis markers. Both heterozygous hahvcc43 mutants and scfd1vcc44 mutants survived to adulthood, showing chamber dilation and reduced ventricular contraction. Collectively, our data implicate scfd1 loss-of-function as the genetic defect at the hahvcc43 locus and provide new insights into the role of scfd1 in cardiac development and function.

Transmembrane protein 161b (Tmem161b) was recently identified in multiple high-through-put phenotypic screens, including in fly, zebrafish, and mouse. In zebrafish, Tmem161b was identified as an essential regulator of cardiac rhythm. In mouse, Tmem161b shows conserved function in regulating cardiac rhythm but has also been shown to impact cardiac morphology. Homozygous or heterozygous missense mutations have also recently been reported for TMEM161B in patients with structural brain malformations, although its significance in the human heart remains to be determined. Across the three model organisms studied to date (fly, fish, and mouse), Tmem161b loss of function is implicated in intracellular calcium ion handling, which may explain the diverse phenotypes observed. This review summarises the current knowledge of this conserved and functionally essential protein in the context of cardiac biology.

ABSTRACT The endocardium plays important roles in the development and function of the vertebrate heart; however, few molecular markers of this tissue have been identified and little is known about what regulates its differentiation. Here, we describe the Gt(SAGFF27C); Tg(4xUAS:egfp) line as a marker of endocardial development in zebrafish. Transcriptomic comparison between endocardium and pan-endothelium confirms molecular distinction between these populations and time-course analysis suggests differentiation as early as eight somites. To investigate what regulates endocardial identity, we employed npas4l, etv2 and scl loss-of-function models. Endocardial expression is lost in npas4l mutants, significantly reduced in etv2 mutants and only modestly affected upon scl loss-of-function. Bmp signalling was also examined: overactivation of Bmp signalling increased endocardial expression, whereas Bmp inhibition decreased expression. Finally, epistasis experiments showed that overactivation of Bmp signalling was incapable of restoring endocardial expression in etv2 mutants. By contrast, overexpression of either npas4l or etv2 was sufficient to rescue endocardial expression upon Bmp inhibition. Together, these results describe the differentiation of the endocardium, distinct from vasculature, and place npas4l and etv2 downstream of Bmp signalling in regulating its differentiation.

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 heart is laterally asymmetric. Not only is it positioned on the left side of the body but the organ itself is asymmetric. This patterning occurs across scales: at the organism level, through left–right axis patterning; at the organ level, where the heart itself exhibits left–right asymmetry; at the cellular level, where gene expression, deposition of matrix and proteins and cell behaviour are asymmetric; and at the molecular level, with chirality of molecules. Defective left–right patterning has dire consequences on multiple organs; however, mortality and morbidity arising from disrupted laterality is usually attributed to complex cardiac defects, bringing into focus the particulars of left–right patterning of the heart. Laterality defects impact how the heart integrates and connects with neighbouring organs, but the anatomy of the heart is also affected because of its asymmetry. Genetic studies have demonstrated that cardiac asymmetry is influenced by left–right axis patterning and yet the heart also possesses intrinsic laterality, reinforcing the patterning of this organ. These inputs into cardiac patterning are established at the very onset of left–right patterning (formation of the left–right organiser) and continue through propagation of left–right signals across animal axes, asymmetric differentiation of the cardiac fields, lateralised tube formation and asymmetric looping morphogenesis. In this review, we will discuss how left–right asymmetry is established and how that influences subsequent asymmetric development of the early embryonic heart. In keeping with the theme of this issue, we will focus on advancements made through studies using the zebrafish model and describe how its use has contributed considerable knowledge to our understanding of the patterning of the heart.

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems: mouse and zebrafish. In both models Cavin4 localized to T-tubules and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein, Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T- tubule domain lacking caveolae that is essential for T-tubule function.

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.