Cardiac development demystified by use of the HDBR atlas.

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.

Abundance and diversity of fungal endophytes isolated from monk fruit (Siraitia grosvenorii) grown in a Canadian research greenhouse.

Monk fruit (Siraitia grosvenorii) is an herbaceous perennial vine of the Cucurbitaceae family cultivated commercially mainly in southern China. There is very little information available about the fungal endophytes in monk fruit. In this study, monk fruit plants were grown from seeds in a research greenhouse at Kwantlen Polytechnic University in British Columbia, Canada to explore the abundance and diversity of their fungal endophytes. Fungal endophytes were isolated from seeds, seedlings, mature monk fruit plants, and fruits, and cultured on potato dextrose agar and water agar media. Isolates were identified by microscopic examination and BLAST comparison of ITS sequences to published sequences in GenBank. At least 150 species of fungal endophytes representing 60 genera and 20 orders were recovered from monk fruit tissues. Non-metric multidimensional scaling (NMDS) was carried out to explore the similarity of fungal communities among roots, stems, leaves, flowers, fruits, and seeds based on fungal orders. Our study showed that monk fruit plants are a rich source of fungal endophytes with the greatest abundance and diversity in leaves. This work has deepened our understanding of the intricate interactions between plants and fungi that sustain ecosystems and underpin plant health and resilience.

Development of the arterial roots and ventricular outflow tracts.

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.

Functional analysis of germline VANGL2 variants using rescue assays of vangl2 knockout zebrafish.

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 plays essential roles in the developing heart and aorta linked to disruption of Notch signalling.

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.

Spatial transcriptomics reveals novel genes during the remodelling of the embryonic human arterial valves.

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.

Yolk sac cell atlas reveals multiorgan functions during human early 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.

The Left Ventricular Myocardium in Hypoplastic Left Heart Syndrome.

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.

Sequential action of JNK genes establishes the embryonic left-right axis.

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.

Submission or subversion: survival and resilience of Chinese international research students in neoliberalised Australian universities.

Although scholars have noted the detrimental nature of the various changes in higher education prompted by neoliberalism, its impact on the experiences of international Higher Degree by Research (HDR) students has yet to be adequately studied. Informed by Bourdieu's concepts of doxa, field, habitus, and capital, this paper examines the ways in which neoliberalism as doxa in the Australian higher education field has colonised the perception and practice of Chinese international HDR students whilst some students were able to demonstrate resilience to the pervasive neoliberal practices. The paper draws on a larger qualitative research project including interviews with 18 Chinese HDR students from four Australian universities. Data suggest that Chinese HDR research students gradually developed intensified dispositions of self-reliance and self-exploitation in response to neoliberal academic practices whilst others were enculturated into a floating habitus (or vulnerable position) in relation to academic publishing as they attempted to negotiate the tensions across fields and over time. Data further reveal that some participants demonstrated resilience to neoliberalism when empowered by their supervisors with less utilitarian and more critically reflexive supervisory practices. The paper argues that the embrace of neoliberalism in the Australian higher education field has become widespread yet controversial, and that thinking and enacting resilience sociologically may de-neoliberalise the higher education field in Australia and beyond.

Blood and immune development in human fetal bone marrow and Down syndrome.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

Exercise, programmed cell death and exhaustion of cardiomyocyte proliferation in aging zebrafish.

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.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC 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 limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated 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 LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

The Editors' Personal Biography of Professor Robert Anderson.

Robert (Bob) Henry Anderson was born in Wellington, Shropshire, UK, in 1942 and he completed his medical training in Manchester (UK) in 1966 [...].

Development of the Human Arterial Valves: Understanding Bicuspid Aortic Valve.

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.

New Concepts in the Development and Malformation of the Arterial Valves.

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.

Early Embryonic Expression of AP-2α Is Critical for Cardiovascular Development.

Congenital cardiovascular malformation is a common birth defect incorporating abnormalities of the outflow tract and aortic arch arteries, and mice deficient in the transcription factor AP-2α (Tcfap2a) present with complex defects affecting these structures. AP-2α is expressed in the pharyngeal surface ectoderm and neural crest at mid-embryogenesis in the mouse, but the precise tissue compartment in which AP-2α is required for cardiovascular development has not been identified. In this study we describe the fully penetrant AP-2α deficient cardiovascular phenotype on a C57Bl/6J genetic background and show that this is associated with increased apoptosis in the pharyngeal ectoderm. Neural crest cell migration into the pharyngeal arches was not affected. Cre-expressing transgenic mice were used in conjunction with an AP-2α conditional allele to examine the effect of deleting AP-2α from the pharyngeal surface ectoderm and the neural crest, either individually or in combination, as well as the second heart field. This, surprisingly, was unable to fully recapitulate the global AP-2α deficient cardiovascular phenotype. The outflow tract and arch artery phenotype was, however, recapitulated through early embryonic Cre-mediated recombination. These findings indicate that AP-2α has a complex influence on cardiovascular development either being required very early in embryogenesis and/or having a redundant function in many tissue layers.