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.

BACKGROUND:The precise steps involved in development of the atrioventricular conduction axis remain incompletely understood, despite over a century of anatomical and histological studies. Recent advances in imaging, and access to human embryonic and fetal heart specimens, offer new insights into this process. OBJECTIVE:This study aimed to clarify the morphological remodeling and insulation of the atrioventricular conduction axis during late embryonic and early fetal human heart development, with emphasis on the fate of nodoventricular and fasciculo-ventricular pathways. METHODS:Serial histological sections and micro-computed tomographic datasets encompassing Carnegie stage 17 through 23, until the end of the fetal period, including a newborn (42 weeks of development), from the Human Developmental Biology Resource, and additional fetal heart archives, were analyzed. Reproducibility was assessed by blinded interobserver evaluation. RESULTS:The atrioventricular conduction axis arises, in part, from a drape of trabecular cardiomyocytes on the septal crest, which remodels into the branching and non-branching bundles. It also includes the so-called "primary ring." During fetal development, expansion of the atrioventricular junctions, producing the inferior pyramidal space, centralizes the compact atrioventricular node, and disrupts most nodoventricular pathways. Fasciculo-ventricular connections, in contrast, largely persist, with some myocardial connections remaining throughout gestation. Part of the primary ring becomes the retro-aortic node. CONCLUSION:Remodeling of the atrioventricular conduction axis is characterized by dynamic reorganization and selective insulation of specialized cardiomyocytes. Persistence of fasciculo-ventricular pathways, and loss of nodoventricular connections, has obvious implications for the understanding of the postnatal anatomy of the conduction system anatomy and its arrhythmogenic substrates.

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.

Congenital anomalies are structural or functional abnormalities present at birth, which can be caused by genetic or environmental influences. The availability of genome sequencing has significantly increased our understanding of congenital anomalies, but linking variant identification to functional relevance and definitive diagnosis remains challenging. Many genes have unknown or poorly understood functions, and with a lack of clear genotype-to-phenotype correlations, it can be difficult to move from variant discovery to diagnosis. Thus, for most congenital anomalies, there still exists a "diagnostic odyssey" which presents a significant burden to patients, families and society. Animal models are essential in the gene discovery process because they allow researchers to validate candidate gene function and disease progression within intact organisms. However, use of advanced model systems continues to be limited due to the complexity of efficiently generating clinically relevant animals. Here we focus on the use of precisely engineered mice in variant-to-function studies for resolving molecular diagnoses and creating powerful preclinical models for congenital anomalies, covering advances in genomics, genome editing and phenotyping approaches as well as the necessity for future initiatives aligning animal modelling to deep patient multimodal datasets.

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.

A proper appreciation of cardiac development can now provide the necessary background to understand the anatomical findings in the congenitally malformed heart. We recently presented an account of human cardiac development based on reconstructions of histological datasets from human embryos aged between 3.5 and 8 weeks subsequent to conception. In this review, we summarize the changes observed relative to the findings when the heart is congenitally malformed. Beginning at the stage when it is first possible to recognize the primary heart tube, we describe the looping of its ventricular component, which occurs in the 5th week. We proceed with discussion of the formation of the atrial and ventricular chambers in the 6th week. The phases are successive, albeit partially overlapping. Separation of the circulations at the venous pole is completed at stage 17, equivalent to almost 6 weeks of development. During stages representing the 7th week of development, we concentrate on the remodeling of the outflow tract. This involves initially septation, but then separation of the developing circulations. The changes involve incorporation of the proximal outflow tract into the ventricles, with formation of the arterial roots in its middle part, and addition of a distal non-myocardial component to produce the intrapericardial arterial trunks. We pay particular attention to the changes occurring during remodeling of the interventricular foramen. We show that an understanding of this process provides the basis for understanding the functionally univentricular heart, as well as the arrangement found in double outlet right ventricle.

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.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

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.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

EDITORIAL article Front. Cell Dev. Biol., 17 January 2023Sec. Cellular Biochemistry Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1133159

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.

Summary To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA- and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders. Key findings scRNA-Seq of adult human cornea and conjunctiva reveals the signature of various ocular surface cell populations scRNA-Seq of human developing cornea identifies stage-specific definitions of corneal epithelial, stromal and endothelial layers scRNA-Seq analysis results in identification of novel markers for qLSCs and TA cells Combined scRNA- and ATAC-Seq analysis reveals key transcriptional networks in qLSCs and TA cells and close interactions with immune cells Expansion of limbal epithelium results in downregulation of qLSCs and acquisition of proliferative limbal epithelial progenitor markers scRNA-Seq of keratoconus corneas reveals activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium Graphical abstract Schematic presentation of main techniques and findings presented in this manuscript.

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell (DC) subsets emerging for the first time. B-lymphocyte expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.