BackgroundHuman inner ear contains macrophages whose functional role in early development is yet unclear. Recent studies describe inner ear macrophages act as effector cells of the innate immune system and are often activated following acoustic trauma or exposure to ototoxic drugs. Few or limited literature describing the role of macrophages during inner ear development and organogenesis.Material and MethodsWe performed a study combining immunohistochemistry and immunofluorescence using antibodies against IBA1, CX3CL1, CD168, CD68, CD45 and CollagenIV. Immune staining and quantification was performed on human embryonic inner ear sections from gestational week 09 to 17.ResultsThe study showed IBA1 and CD45 positive cells in the mesenchymal tissue at GW 09 to GW17. No IBA1 positive macrophages were detected in the sensory epithelium of the cochlea and vestibulum. Fractalkine (CX3CL1) signalling was initiated GW10 and parallel chemotactic attraction and migration of macrophages into the inner ear. Macrophages also migrated into the spiral ganglion, cochlear nerve, and peripheral nerve fibers and tissue-expressing CX3CL1. The mesenchymal tissue at all gestational weeks expressed CD163 and CD68.ConclusionExpressions of markers for resident and non-resident macrophages (IBA1, CD45, CD68, and CD163) were identified in the human fetal inner ear. We speculate that these cells play a role for the development of human inner ear tissue including shaping of the gracile structures.

As the human heart ages, the myocardium undergoes fibrotic remodelling, there is declining cardiovascular performance and eventual heart failure. It is suggested that exercise is an important intervention to ameliorate these changes. In this study we establish zebrafish as a laboratory model to understand how aging and exercise affect cardiomyocyte turnover. We show the zebrafish heart does not exhibit indeterminate growth but follows the pattern seen in human aging. In zebrafish, cardiomyocyte proliferation remains constant, but a late increase cell death underlies the pattern of initial cardiac growth and later fibrosis. These anatomical findings are corelated with the human like decline in cardiovascular performance reflected in voluntary swimming activity, critical swimming speed (Ucrit) and biomarkers of cardiac insufficiency. Whilst the vertebrate heart can respond to injury through cardiomyocyte proliferation, it is not known if a proliferative response occurs when the cardiovascular system is exposed to prolonged severe physiological stress, or if this changes with age. To investigate this, young and old adult zebrafish were challenged by 72 hours of enforced swimming in a purpose-built flume at levels close to maximal Ucrit. Whilst young adult fish produced a significant proliferative response older fish had a dramatically impaired response, provided by a smaller proliferative cardiomyocyte population. Finally, we asked if these aging responses could be improved by increased activity throughout adulthood. Whilst there was some improvement in the aged proliferative response the size of the reduced proliferative cardiomyocyte pool remained unchanged and importantly, there was increased myocardial fibrosis. The zebrafish heart thus provides a laboratory model to study cardiomyocyte turnover during aging and physiological stresses, revealing the important trade-off between preserving cardiovascular fitness through exercise and accelerated fibrotic change, whilst the available proliferative pool of cardiomyocytes continues to diminish.

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.

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-Seq and spatial transcriptomics we present for the first-time a single cell spatio-temporal transcriptome of retinal organoid development. Our data demonstrate that retinal organoids recapitulate key events of retinogenesis including optic vesicle/cup formation, formation of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-Seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.