Developmental Biology

Post-GWAS screening of candidate genes for refractive error in mutant zebrafish models

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Genome-wide association studies (GWAS) have dissected numerous genetic factors underlying refractive errors (RE) such as myopia. Despite significant insights into understanding the genetic architecture of RE, few studies have validated and explored the functional role of candidate genes within these loci. To functionally follow-up on GWAS and characterize the potential role of candidate genes on the development of RE, we prioritized nine genes (TJP2, PDE11A, SHISA6, LAMA2, LRRC4C, KCNQ5, GNB3, RBFOX1, and GRIA4) based on biological and statistical evidence; and used CRISPR/cas9 to generate knock-out zebrafish mutants. These mutant fish were screened for abnormalities in axial length by spectral-domain optical coherence tomography and refractive status by eccentric photorefraction at the juvenile (2 months) and adult (4 months) developmental stage. We found a significantly increased axial length and myopic shift in refractive status in three of our studied mutants, indicating a pot...

Open-source, highly efficient, post-acquisition synchronization for 4D dual-contrast imaging of the mouse embryonic heart over development with optical coherence tomography

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Dynamic imaging of the beating embryonic heart in 3D is critical for understanding cardiac development and defects. Optical coherence tomography (OCT) plays an important role in embryonic heart imaging with its unique imaging scale and label-free contrasts. In particular, 4D (3D + time) OCT imaging enabled biomechanical analysis of the developing heart in various animal models. While ultrafast OCT systems allow for direct volumetric imaging of the beating heart, the imaging speed remains limited, leading to an image quality inferior to that produced by post-acquisition synchronization. As OCT systems become increasingly available to a wide range of biomedical researchers, a more accessible 4D reconstruction method is required to enable the broader application of OCT in the dynamic, volumetric assessment of embryonic heartbeat. Here, we report an open-source, highly efficient, post-acquisition synchronization method for 4D cardiodynamic and hemodynamic imaging of the mouse embryonic ...

Optical Coherence Tomography Is a Promising Tool for Zebrafish-Based Research—A Review

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The zebrafish is an established vertebrae model in the field of biomedical research. With its small size, rapid maturation time and semi-transparency at early development stages, it has proven to be an important animal model, especially for high-throughput studies. Three-dimensional, high-resolution, non-destructive and label-free imaging techniques are perfectly suited to investigate these animals over various development stages. Optical coherence tomography (OCT) is an interferometric-based optical imaging technique that has revolutionized the diagnostic possibilities in the field of ophthalmology and has proven to be a powerful tool for many microscopic applications. Recently, OCT found its way into state-of-the-art zebrafish-based research. This review article gives an overview and a discussion of the relevant literature and an outlook for this emerging field.

Washington University Receives NIH Grant for High-throughput integrated live imaging and optogenetic pacing platform to assess hypoxia responsiveness in the fly heart

Washington University received a 2023 NIH Grant for $432,968 for High-throughput integrated live imaging and optogenetic pacing platform to assess hypoxia responsiveness in the fly heart.  The principal investigator is Chao Zhou.  Below is a summary of the proposed study. Ischemic preconditioning is a well-established phenomenon, in which a brief episode(s) of controlled ischemia and reperfusion renders cardioprotection from a subsequent sustained episode of ischemia. An emerging body of evidence demonstrated that neural regulated heart rate modulation confers cardiac preconditioning responses. Understanding the mechanism through model systems of preconditioning would help us identify the genes and proteins when designing future drug targets for the prevention of ischemic cardiac injury. As a promising alternative to electrical pacing to modulate heart rate, optogenetic pacing does not require physical contact, has high spatial and temporal precision, offers more specific excitat...

Optical coherence tomography for dynamic investigation of mammalian reproductive processes

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The biological events associated with mammalian reproductive processes are highly dynamic and tightly regulated by molecular, genetic, and biomechanical factors. Implementation of live imaging in reproductive research is vital for the advancement of our understanding of normal reproductive physiology and for improving the management of reproductive disorders. Optical coherence tomography (OCT) is emerging as a promising tool for dynamic volumetric imaging of various reproductive processes in mice and other animal models. In this review, we summarize recent studies employing OCT-based approaches toward the investigation of reproductive processes in both, males and females. We describe how OCT can be applied to study structural features of the male reproductive system and sperm transport through the male reproductive tract. We review OCT applications for in vitro and dynamic in vivo imaging of the female reproductive system, staging and tracking of oocytes and embryos, and investigati...

OCT based four-dimensional cardiac imaging of a living chick embryo using an impedance signal as a gating for post-acquisition synchronization

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Optical coherence tomography (OCT) is a non-invasive imaging modality with high spatial resolution suitable for early embryonic heart imaging. However, the most commonly used OCT systems cannot provide direct 4-D imaging due to acquisition speed limitations. We proposed a retrospective gating 4-D reconstruction method based on spectral domain OCT. A special circuit was designed to measure the impedance change of chick embryos in response to the heart beating. The impedance signal was acquired simultaneously with the OCT B-scan image sequence at several different locations along the heart. The impedance signal was used as a gating for 4-D reconstruction. The reconstruction algorithm includes cardiac period calculation, interpolation from multi-cardiac cycle image sequence into one cardiac cycle, and cardiac phase synchronization among the different locations of the heart. The synchronism of the impedance signal change with the heartbeat was verified. Using the proposed method, we rec...

Optical coherence tomography for dynamic investigation of mammalian reproductive processes

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The biological events associated with mammalian reproductive processes are highly dynamic and tightly regulated by molecular, genetic, and biomechanical factors. Implementation of live imaging in reproductive research is vital for the advancement of our understanding of normal reproductive physiology and for improving the management of reproductive disorders. Optical coherence tomography (OCT) is emerging as a promising tool for dynamic volumetric imaging of various reproductive processes in mice and other animal models. In this review, we summarize recent studies employing OCT-based approaches toward the investigation of reproductive processes in both, males and females. We describe how OCT can be applied to study structural features of the male reproductive system and sperm transport through the male reproductive tract. We review OCT applications for in vitro and dynamic in vivo imaging of the female reproductive system, staging and tracking of oocytes and embryos, and investigati...

A novel uveitis model induced by lipopolysaccharide in zebrafish

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Objective: Endotoxin-induced uveitis (EIU) is an important tool for human uveitis study. This study was designed to develop a novel EIU model in zebrafish. Methods: An EIU model in zebrafish was induced by intravitreal lipopolysaccharide (LPS) injection and was assessed dynamically. Optical coherence tomography (OCT) was used to assess infiltrating cells in the vitreous body. The histological changes wereevaluated using HE staining and immune cells were measured by immunofluorescence. The retinal RNA Sequencing (RNA-Seq) was used to explore the transcriptional changes during inflammation. RNA-Seq data were analyzed using time-course sequencing data analysis (TCseq), ClueGO plugin in Cytoscape, and Gene Set Enrichment Analysis (GSEA) software. Flow cytometry and retinal flat mounts were used to dynamically quantify the immune cells. Results: EIU was successfully induced in zebrafish following intravitreal LPS injection. Inflammation appeared at 4 hours post injection (hpi), reached i...

Embryonic aortic arch material properties obtained by optical coherence tomography-guided micropipette aspiration

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It is challenging to determine the in vivo material properties of a very soft, mesoscale arterial vesselsof size ∼ 80 to 120 μm diameter. This information is essential to understand the early embryonic cardiovascular development featuring rapidly evolving dynamic microstructure. Previous research efforts to describe the properties of the embryonic great vessels are very limited. Our objective is to measure the local material properties of pharyngeal aortic arch tissue of the chick-embryo during the early Hamburger-Hamilton (HH) stages, HH18 and HH24. Integrating the micropipette aspiration technique with optical coherence tomography (OCT) imaging, a clear vision of the aspirated arch geometry is achieved for an inner pipette radius of Rp = 25 μm. The aspiration of this region is performed through a calibrated negatively pressurized micro-pipette. A computational finite element model is developed to model the nonlinear behaviour of the arch structure by considering the geom...

OCT Meets micro-CT: A Subject-Specific Correlative Multimodal Imaging Workflow for Early Chick Heart Development Modeling

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Structural and Doppler velocity data collected from optical coherence tomography have already provided crucial insights into cardiac morphogenesis. X-ray microtomography and other ex vivo methods have elucidated structural details of developing hearts. However, by itself, no single imaging modality can provide comprehensive information allowing to fully decipher the inner workings of an entire developing organ. Hence, we introduce a specimen-specific correlative multimodal imaging workflow combining OCT and micro-CT imaging which is applicable for modeling of early chick heart development-a valuable model organism in cardiovascular development research. The image acquisition and processing employ common reagents, lab-based micro-CT imaging, and software that is free for academic use. Our goal is to provide a step-by-step guide on how to implement this workflow and to demonstrate why those two modalities together have the potential to provide new insight into normal cardiac developme...

In vivo morphological study of obese development in mice model guided by swept-source optical coherence tomography (SSOCT)

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This article shows the adequacy of the custom-built optical imaging system in the advancement investigation of obese mice. Obesity is defined as increased adipose/fatty mass resulting from a chronic imbalance between energy intake and expenditure. The in vivo investigation was performed for the tissue characterization of obese mice utilizing swept-source optical coherence tomography (SSOCT) for in situ examination and histology of delicate tissues in mice skin. It provides a noninvasive, painless visualization of the subsurface in life systems. Our SSOCT system's data is comparable to the regular invasive histology. Cross-assessment is done in various skin layers in obese mice like epidermis, papillary dermis, dermis, and fat tissue, which are likewise separated from the nonobese mice group. Histopathology results were further assessed with the obtained SSOCT results. This high precision of characterizing tissues using SSOCT helps us perform in vivo imaging and can also be used for ...

An in-vivo depth-resolved imaging of developing zebrafish microstructure and microvasculature using swept-source optical coherence tomography angiography

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Zebrafish as vertebrate model has applications in the fields of embryology, oncology, toxicology, molecular genetics and drug discovery due to its high homology with the human genome. The zebrafish embryogenesis presents a propitious model for concurrent visualization of developmental stages in vivo owing to transparency and ex-utero development of the embryo. Developments in imaging tools and techniques focus on providing real-time quantitative imaging. The present work demonstrates depth-resolved imaging of developing zebrafish using swept-source optical coherence tomography (SSOCT). The SSOCT system provides an axial resolution of 4.5 µm and an imaging depth of 3.26 mm. The paper also presents imaging of the developing larval microvasculature using a 1 µm swept-source optical coherence tomography angiography (ssOCTA). A phase-contrast technique of optical coherence angiography calculates the phase differences between consecutive B-scans taken at single location. This ...

Automated endocardial cushion segmentation and cellularization quantification in developing hearts using optical coherence tomography

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Of all congenital heart defects (CHDs), anomalies in heart valves and septa are among the most common and contribute about fifty percent to the total burden of CHDs. Progenitors to heart valves and septa are endocardial cushions formed in looping hearts through a multi-step process that includes localized expansion of cardiac jelly, endothelial-to-mesenchymal transition, cell migration and proliferation. To characterize the development of endocardial cushions, previous studies manually measured cushion size or cushion cell density from images obtained using histology, immunohistochemistry, or optical coherence tomography (OCT). Manual methods are time-consuming and labor-intensive, impeding their applications in cohort studies that require large sample sizes. This study presents an automated strategy to rapidly characterize the anatomy of endocardial cushions from OCT images. A two-step deep learning technique was used to detect the location of the heart and segment endocardial cush...

Mouse embryo phenotyping with optical coherence tomography

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With the explosion of gene editing tools in recent years, there has been a much greater demand for mouse embryo phenotyping, and traditional methods such as histology and histochemistry experienced a methodological renaissance as they became the principal tools for phenotyping. However, it is important to explore alternative phenotyping options to maximize time and resources and implement volumetric structural analysis for enhanced investigation of phenotypes. Cardiovascular phenotyping, in particular, is important to perform in vivo due to the dramatic structural and functional changes that occur in heart development over relatively short periods of time. Optical coherence tomography (OCT) is one of the most exciting advanced imaging techniques emerging within the field of developmental biology, and this review provides a summary of how it is currently being implemented in mouse embryo investigations and phenotyping. This review aims to provide an understanding of the approaches us...

Regenerative hallmarks of aging: Insights through the lens of Pleurodeles waltl

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Background: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime. Results: Here, we used Optical Coherence Tomography (OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related decline in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms. Conclusions: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intri...

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