BACKGROUND:Human mutations in INVERSIN are associated with nephronophthisis, variable penetrance of situs inversus and congenital heart disease. Inversin has been shown to localize to cilia and many of the patient phenotypes are attributed to disrupted cilia function. We set out to characterize the invssa12246 allele in zebrafish to investigate its utility in understanding patient disease. RESULTS:The left-right axis is established correctly in invssa12246 mutants, kidneys appear to develop normally without any cysts and cilia appear normal, however mutants are significantly shorter. In post-embryonic stages, invssa12246 mutants display significant growth delay and signs of retinal mis-patterning together with spinal deformities reminiscent of idiopathic scoliosis. The allele is lethal in the juvenile stage. CONCLUSIONS:We show that invssa12246 allele has a distinct phenotype from other models where inversin function is disrupted, uncovering novel roles in post-embryonic development.

Aims Bicuspid aortic valve (BAV), where two valve leaflets are found instead of the usual three, affects 1-2% of the general population and is associated with significant morbidity and mortality. Despite its frequency, the majority of cases remain unexplained. This is, at least in part, because there are two types of valve leaflet primordia: endocardial cushions and intercalated valve swellings (ICVS). Moreover, multiple progenitors make distinct contributions to the formation of these primordia. Genomic studies in mouse and human have suggested a correlation between BAV and malfunctional primary cilia. However, the precise requirement for cilia during early embryonic valvulogenesis remains unknown.Methods and results Here, we disrupted primary cilia by deleting the ciliary gene Ift88 in the main progenitor cells forming the aortic valve using specific Cre drivers: Wnt1-Cre for neural crest cells, Isl1-Cre for second heart field (SHF) cells, Tie2-Cre for endocardial-derived cells, and Tnnt2-Cre for direct-differentiating SHF in the ICVS. Loss of Ift88, and thus primary cilia, from neural crest cells and endocardium did not impact aortic valve formation. However, primary cilia were essential in SHF cells for aortic valve leaflet formation, with over half of Ift88f/f;Isl1-Cre mutants presenting with BAV. As the valve leaflets were forming, 50% of the Ift88f/f;Isl1-Cre mutants had two small leaflets in the position of the usual posterior leaflet, meaning that at this stage, the aortic valve was quadricuspid, which then remodelled to BAV by E15.5. Mechanistic studies demonstrated premature differentiation of SHF cells as the ICVS formed, leading to the formation of a broadened ICVS that formed two posterior leaflet precursors. This abnormality in the formation of the ICVS was associated with disruption of Notch-Jag1 signalling pathway, with Jag1f/f;Isl1-Cre mutants presenting with a similar phenotype.Conclusion These data show that primary cilia, via the Notch-Jag1 signalling pathway, regulate differentiation of SHF cells in the aortic valve primordia. Additionally, we identify a mechanistic link between the developmental basis of quadricuspid and bicuspid arterial valve leaflets.

Aims Bicuspid aortic valve (BAV) is the most common congenital heart defect, affecting at least 2% of the population. The embryonic origins of BAV remain poorly understood, with few assays for validating patient variants, limiting the identification of causative genes for BAV. In both human and mouse, the left and right leaflets of the arterial valves arise from the outflow tract cushions, with interstitial cells originating from neural crest cells and the overlying endocardium through endothelial-to-mesenchymal transition (EndoMT). In contrast, an EndoMT-independent mechanism of direct differentiation of cardiac progenitors from the second heart field (SHF) is responsible for the formation of the anterior and posterior leaflets. Defects in either of these developmental mechanisms can result in BAV. Although zebrafish have been suggested as a model for human variant testing, their naturally bicuspid arterial valve has not been considered suitable for understanding human arterial valve development. Here, we have set out to investigate to what extent the processes involved in arterial valve development are conserved in zebrafish and, ultimately, whether functional testing of BAV variants could be carried out. Methods and results Using a combination of live imaging, immunohistochemistry, and Cre-mediated lineage tracing, we show that the zebrafish arterial valve primordia develop directly from SHF progenitors with no contribution from EndoMT or neural crest, in keeping with the human and mouse anterior and posterior leaflets. Moreover, once formed, these primordia share common subsequent developmental events with all three aortic valve leaflets. Conclusion Our work highlights a conserved ancestral mechanism of arterial valve leaflet formation from the SHF and identifies that development of the arterial valve is distinct from that of the atrioventricular valve in zebrafish. Crucially, this confirms the utility of zebrafish for understanding the development of specific BAV subtypes and arterial valve dysplasia, offering potential for high-throughput variant testing.

The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development.

Congenital heart defects (CHDs), the most common congenital anomalies, are considered to have a significant genetic component. However, despite considerable efforts to identify pathogenic genes in patients with CHDs, few gene variants have been proven as causal. The complexity of the genetic architecture underlying human CHDs likely contributes to this poor genetic discovery rate. However, several other factors are likely to contribute. For example, the level of patient phenotyping required for clinical care may be insufficient for research studies focused on mechanistic discovery. Although several hundred mouse gene knockouts have been described with CHDs, these are generally not phenotyped and described in the same way as CHDs in patients, and thus are not readily comparable. Moreover, most patients with CHDs carry variants of uncertain significance of crucial cardiac genes, further complicating comparisons between humans and mouse mutants. In spite of major advances in cardiac developmental biology over the past 25 years, these advances have not been well communicated to geneticists and cardiologists. As a consequence, the latest data from developmental biology are not always used in the design and interpretation of studies aimed at discovering the genetic causes of CHDs. In this Special Article, while considering other in vitro and in vivo models, we create a coherent framework for accurately modelling and phenotyping human CHDs in mice, thereby enhancing the translation of genetic and genomic studies into the causes of CHDs in patients.

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.

eNOS (NOS3) is the enzyme that generates nitric oxide, a signalling molecule and regulator of vascular tone. Loss of eNOS function is associated with increased susceptibility to atherosclerosis, hypertension, thrombosis and stroke. Aortopathy and cardiac hypertrophy have also been found in eNOS null mice, but their aetiology is unclear. We evaluated eNOS nulls before and around birth for cardiac defects, revealing severe abnormalities in the ventricular myocardium and pharyngeal arch arteries. Moreover, in the aortic arch, there were fewer baroreceptors, which sense changes in blood pressure. Adult eNOS null survivors showed evidence of cardiac hypertrophy, aortopathy and cartilaginous metaplasia in the periductal region of the aortic arch. Notch1 and neuregulin were dysregulated in the forming pharyngeal arch arteries and ventricles, suggesting that these pathways may be relevant to the defects observed. Dysregulation of eNOS leads to embryonic and perinatal death, suggesting mutations in eNOS are candidates for causing congenital heart defects in humans. Surviving eNOS mutants have a deficiency of baroreceptors that likely contributes to high blood pressure and may have relevance to human patients who suffer from hypertension associated with aortic arch abnormalities.

Abnormalities of the arterial valves, including bicuspid aortic valve (BAV) are amongst the most common congenital defects and are a significant cause of morbidity as well as predisposition to disease in later life. Despite this, and compounded by their small size and relative inaccessibility, there is still much to understand about how the arterial valves form and remodel during embryogenesis, both at the morphological and genetic level. Here we set out to address this in human embryos, using Spatial Transcriptomics (ST). We show that ST can be used to investigate the transcriptome of the developing arterial valves, circumventing the problems of accurately dissecting out these tiny structures from the developing embryo. We show that the transcriptome of CS16 and CS19 arterial valves overlap considerably, despite being several days apart in terms of human gestation, and that expression data confirm that the great majority of the most differentially expressed genes are valve-specific. Moreover, we show that the transcriptome of the human arterial valves overlaps with that of mouse atrioventricular valves from a range of gestations, validating our dataset but also highlighting novel genes, including four that are not found in the mouse genome and have not previously been linked to valve development. Importantly, our data suggests that valve transcriptomes are under-represented when using commonly used databases to filter for genes important in cardiac development; this means that causative variants in valve-related genes may be excluded during filtering for genomic data analyses for, for example, BAV. Finally, we highlight "novel" pathways that likely play important roles in arterial valve development, showing that mouse knockouts of RBP1 have arterial valve defects. Thus, this study has confirmed the utility of ST for studies of the developing heart valves and broadens our knowledge of the genes and signalling pathways important in human valve development.

ABSTRACT The establishment of the left-right axis is crucial for the placement, morphogenesis and function of internal organs. Left-right specification is proposed to be dependent on cilia-driven fluid flow in the embryonic node. Planar cell polarity (PCP) signalling is crucial for patterning of nodal cilia, yet downstream effectors driving this process remain elusive. We have examined the role of the JNK gene family, a proposed downstream component of PCP signalling, in the development and function of the zebrafish node. We show jnk1 and jnk2 specify length of nodal cilia, generate flow in the node and restrict southpaw to the left lateral plate mesoderm. Moreover, loss of asymmetric southpaw expression does not result in disturbances to asymmetric organ placement, supporting a model in which nodal flow may be dispensable for organ laterality. Later, jnk3 is required to restrict pitx2c expression to the left side and permit correct endodermal organ placement. This work uncovers multiple roles for the JNK gene family acting at different points during left-right axis establishment. It highlights extensive redundancy and indicates JNK activity is distinct from the PCP signalling pathway.

Abnormalities in the arterial valves are some of the commonest congenital malformations, with bicuspid aortic valve (BAV) occurring in as many as 2% of the population. Despite this, most of what we understand about the development of the arterial (semilunar; aortic and pulmonary) valves is extrapolated from investigations of the atrioventricular valves in animal models, with surprisingly little specifically known about how the arterial valves develop in mouse, and even less in human. In this review, we summarise what is known about the development of the human arterial valve leaflets, comparing this to the mouse where appropriate.

Hypoplastic left heart syndrome (HLHS) is a collective term applied to severe congenital cardiac malformations, characterised by a combination of abnormalities mainly affecting the left ventricle, associated valves, and ascending aorta. Although in clinical practice HLHS is usually sub-categorised based on the patency of the mitral and aortic (left-sided) valves, it is also possible to comprehensively categorise HLHS into defined sub-groups based on the left ventricular morphology. Here, we discuss the published human-based studies of the ventricular myocardium in HLHS, evaluating whether the available evidence is in keeping with this ventricular morphology concept. Specifically, we highlight results from histological studies, indicating that the appearance of cardiomyocytes can be different based on the sub-group of HLHS. In addition, we discuss the histological appearances of endocardial fibroelastosis (EFE), which is a common feature of one specific sub-group of HLHS. Lastly, we suggest investigations that should ideally be undertaken using HLHS myocardial tissues at early stages of HLHS development to identify biological pathways and aid the understanding of HLHS aetiology.

Exercise may ameliorate the eventual heart failure inherent in human aging. In this study, we use zebrafish to understand how aging and exercise affect cardiomyocyte turnover and myocardial remodelling. We show that cardiomyocyte proliferation remains constant throughout life but that onset of fibrosis is associated with a late increase in apoptosis. These findings correlate with decreases in voluntary swimming activity, critical swimming speed (Ucrit), and increases in biomarkers of cardiac insufficiency. The ability to respond to severe physiological stress is also impaired with age. Although young adult fish respond with robust cardiomyocyte proliferation in response to enforced swimming, this is dramatically impaired in older fish and served by a smaller proliferation-competent cardiomyocyte population. Finally, we show that these aging responses can be improved through increased activity throughout adulthood. However, despite improvement in Ucrit and the proliferative response to stress, the size of the proliferating cardiomyocyte population remained unchanged. The zebrafish heart models human aging and reveals the important trade-off between preserving cardiovascular fitness through exercise at the expense of accelerated fibrotic change.

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.

DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin-fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil-N-glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin-fixed human tissues. When compared with a leading commercial formalin-fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub-cloning and Sanger sequencing from UNG-treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte-derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin-fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four-fold to 56% and doubled specificity to 68% when compared with a standard hard-filtering approach. Thus, high-quality DNA can be extracted from excessively formalin-fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub-cloned amplicons is an important methodological step in assessing DNA quality.

Although in many ways the arterial and atrioventricular valves are similar, both being derived for the most part from endocardial cushions, we now know that the arterial valves and their surrounding structures are uniquely dependent on progenitors from both the second heart field (SHF) and neural crest cells (NCC). Here, we will review aspects of arterial valve development, highlighting how our appreciation of NCC and the discovery of the SHF have altered our developmental models. We will highlight areas of research that have been particularly instructive for understanding how the leaflets form and remodel, as well as those with limited or conflicting results. With this background, we will explore how this developmental knowledge can help us to understand human valve malformations, particularly those of the bicuspid aortic valve (BAV). Controversies and the current state of valve genomics will be indicated.

The arterial roots are important transitional regions of the heart, connecting the intrapericardial components of the aortic and pulmonary trunks with their ventricular outlets. They house the arterial (semilunar) valves and, in the case of the aorta, are the points of coronary arterial attachment. Moreover, because of the semilunar attachments of the valve leaflets, the arterial roots span the anatomic ventriculo‐arterial junction. By virtue of this arrangement, the interleaflet triangles, despite being fibrous, are found on the ventricular aspect of the root and located within the left ventricular cavity. Malformations and diseases of the aortic root are common and serious. Despite the mouse being the animal model of choice for studying cardiac development, few studies have examined the structure of their arterial roots. As a consequence, our understanding of their formation and maturation is incomplete. We set out to clarify the anatomical and histological features of the mouse arterial roots, particularly focusing on their walls and the points of attachment of the valve leaflets. We then sought to determine the embryonic lineage relationships between these tissues, as a forerunner to understanding how they form and mature over time. Using histological stains and immunohistochemistry, we show that the walls of the mouse arterial roots show a gradual transition, with smooth muscle cells ( SMC ) forming the bulk of wall at the most distal points of attachments of the valve leaflets, while being entirely fibrous at their base. Although the interleaflet triangles lie within the ventricular chambers, we show that they are histologically indistinguishable from the arterial sinus walls until the end of gestation. Differences become apparent after birth, and are only completed by postnatal day 21. Using Cre‐lox ‐based lineage tracing technology to label progenitor populations, we show that the SMC and fibrous tissue within the walls of the mature arterial roots share a common origin from the second heart field ( SHF ) and exclude trans‐differentiation of myocardium as a source for the interleaflet triangle fibrous tissues. Moreover, we show that the attachment points of the leaflets to the walls, like the leaflets themselves, are derived from the outflow cushions, having contributions from both SHF ‐derived endothelial cells and neural crest cells. Our data thus show that the arterial roots in the mouse heart are similar to the features described in the human heart. They provide a framework for understanding complex lesions and diseases affecting the aortic root.

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

Background Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy disorder with 18 known causative genes ( BBS1-18 ). The primary clinical features are renal abnormalities, rod-cone dystrophy, post-axial polydactyly, learning difficulties, obesity and male hypogonadism. Results We describe the clinical phenotype in three Saudi siblings in whom we have identified a novel mutation in exon 12 of BBS5 (c.966dupT; p.Ala323CysfsX57). This single nucleotide duplication creates a frame shift results in a predicted elongated peptide. Translation blocking Morpholino oligonucleotides were used to create zebrafish bbs5 morphants. Morphants displayed retinal layering defects, abnormal cardiac looping and dilated, cystic pronephric ducts with reduced cilia expression. Morphants also displayed significantly reduced dextran clearance via the pronephros compared to wildtype embryos, suggesting reduced renal function in morphants. The eye, kidney and heart defects reported in morphant zebrafish resemble the human phenotype of BBS5 mutations. The pathogenicity of the novel BBS5 mutation was determined. Mutant mRNA was unable to rescue pleiotropic phenotypes of bbs5 morphant zebrafish and in cell culture we demonstrate a mislocalisation of mutant BBS5 protein which fails to localise discretely with the basal body. Conclusions We conclude that this novel BBS5 mutation has a deleterious function that accounts for the multisystem ciliopathy phenotype seen in affected human patients.