Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment‐based computational modelling and analysis of early‐stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV‐OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four‐dimensional SV‐OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle‐based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture : Four‐dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.     

Optical coherence tomography for dynamic investigation of mammalian reproductive processes

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 investigations of the oocyte/embryo transport through the oviduct. We describe how the functional OCT approach can be applied to the analysis of cilia dynamics within the male and female reproductive systems. We also discuss the areas of research, where OCT could find potential applications to progress our understanding of normal reproductive physiology and reproductive disorders.     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Inheritance of OCT4 predetermines fate choice in human embryonic stem cells

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single‐cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.     

Measuring hemodynamic changes during mammalian development

The pathogenesis of many congenital cardiovascular diseases involves abnormal flow within the embryonic vasculature that results either from malformations of the heart or defects in the vasculature itself. Extensive genetic and genomic analysis in mice has led to the identification of an array of mutations that result in cardiovascular defects during embryogenesis. Many of these mutations cause secondary effects within the vasculature that are thought to arise because of altered fluid dynamics. Presumably, cardiac defects disturb or reduce flow and thereby lead to the disruption of the mechanical signals necessary for proper vascular development. Unfortunately, a precise understanding of how flow disruptions lead to secondary vasculature defects has been hampered by the inadequacy of existing analytical tools. Here, we used a fast line-scanning technique for the quantitative analysis of hemodynamics during early organogenesis in mouse embryos, and we present a model system for studying cellular responses during the formation and remodeling of the mammalian cardiovascular system. Flow velocity profiles can be measured as soon as a heart begins to beat even in newly formed vessels. These studies establish a link between the pattern of blood flow within the vasculature and the stage of heart development and also enable analysis of the influence of mechanical forces during development.     

Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells

Nanog, Oct4, and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated, the mechanistic functions are not well defined. Here, we identify general and cell-line-specific requirements for NANOG, OCT4, and SOX2 in hESCs. We show that OCT4 regulates, and interacts with, the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages, whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus, instead of being panrepressors of differentiation, each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs.     

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

Noninvasive visualization of embryos at different development stages is crucial for the understanding of the basic developmental biology. It is therefore desirable to have an imaging tool capable of rapidly evaluating the effects of gene manipulation or genome editing in developing embryos for the studies of gene functions and genetic engineering. Here, we propose and demonstrate a novel use of optical coherence tomography (OCT) to noninvasively exam the embryonic development of the migratory locusts in real time with 3‐dimensional (3D) view capability. In particular, we obtain the sufficiently high spatial resolution tomographic 2D and 3D images of live locust embryos throughout their development processes. We show that not only we are able to noninvasively observe all previously known forms of blastokinesis as an embryo develops, such as anatrepsis, katatrepsis, revolution, rotation and diapauses, and determine their precise occurring time or duration, but also discover an unreported rotation form we named “twist.” In addition, with the OCT images we determined the exact occurring time of diapauses of the locusts from Tibetan plateau for the first time. Finally, we demonstrate that OCT systems can be used to rapidly capture the development defects of genetically modified embryos in which certain genes essential for embryonic development were suppressed by RNA interference. Our work shows that OCT is an enabling imaging tool with sufficient spatial resolution for the rapid evaluation of embryonic variations of small animals.     

Defining the molecular pathologies in cloaca malformation: similarities between mouse and human

Anorectal malformations are congenital anomalies that form a spectrum of disorders, from the most benign type with excellent functional prognosis, to very complex, such as cloaca malformation in females in which the rectum, vagina and urethra fail to develop separately and instead drain via a single common channel into the perineum. The severity of this phenotype suggests that the defect occurs in the early stages of embryonic development of the organs derived from the cloaca. Owing to the inability to directly investigate human embryonic cloaca development, current research has relied on the use of mouse models of anorectal malformations. However, even studies of mouse embryos lack analysis of the earliest stages of cloaca patterning and morphogenesis. Here we compared human and mouse cloaca development and retrospectively identified that early mis-patterning of the embryonic cloaca might underlie the most severe forms of anorectal malformation in humans. In mouse, we identified that defective sonic hedgehog (Shh) signaling results in early dorsal-ventral epithelial abnormalities prior to the reported defects in septation. This is manifested by the absence of Sox2 and aberrant expression of keratins in the embryonic cloaca of Shh knockout mice. Shh knockout embryos additionally develop a hypervascular stroma, which is defective in BMP signaling. These epithelial and stromal defects persist later, creating an indeterminate epithelium with molecular alterations in the common channel. We then used these animals to perform a broad comparison with patients with mild-to-severe forms of anorectal malformations including cloaca malformation. We found striking parallels with the Shh mouse model, including nearly identical defective molecular identity of the epithelium and surrounding stroma. Our work strongly suggests that early embryonic cloacal epithelial differentiation defects might be the underlying cause of severe forms of anorectal malformations in humans. Moreover, deranged Shh and BMP signaling is correlated with severe anorectal malformations in both mouse and humans.KEY WORDS: Anorectal malformation, Cloaca, Patterning, Epithelial differentiation, Sonic hedgehog     

Embryonic Carcinoma Cells Show Specific Dielectric Resistance Profiles during Induced Differentiation

Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2′-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2). Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS). We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating cultures and real-time label-free monitoring of differentiation processes. This work might provide a basis for further analyses of drug candidates for differentiation therapy of cancers.     

High-resolution optical coherence tomographic imaging of osteoarthritic cartilage during open knee surgery

This study demonstrates the first real-time imaging in vivo of human cartilage in normal and osteoarthritic knee joints at a resolution of micrometers, using optical coherence tomography (OCT). This recently developed high-resolution imaging technology is analogous to B-mode ultrasound except that it uses infrared light rather than sound. Real-time imaging with 11-μm resolution at four frames per second was performed on six patients using a portable OCT system with a handheld imaging probe during open knee surgery. Tissue registration was achieved by marking sites before imaging, and then histologic processing was performed. Structural changes including cartilage thinning, fissures, and fibrillations were observed at a resolution substantially higher than is achieved with any current clinical imaging technology. The structural features detected with OCT were evident in the corresponding histology. In addition to changes in architectural morphology, changes in the birefringent or the polarization properties of the articular cartilage were observed with OCT, suggesting collagen disorganization, an early indicator of osteoarthritis. Furthermore, this study supports the hypothesis that polarization-sensitive OCT may allow osteoarthritis to be diagnosed before cartilage thinning. This study illustrates that OCT, which can eventually be developed for use in offices or through an arthroscope, has considerable potential for assessing early osteoarthritic cartilage and monitoring therapeutic effects for cartilage repair with resolution in real time on a scale of micrometers.     

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around gastrulation, days 8-17 postinsemination, introducing a stereomicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers was observed at defined stages, suggesting correlations to lineage specification. In the endoderm, clearance of OCT4 was apparent from early during its formation at the primitive streak stage. The endoderm harbored progenitors of the "fourth germ layer," the primordial germ cells (PGCs), the only cells maintaining expression of OCT4 at the end of gastrulation. In the ectodermal and mesodermal cell lineages, OCT4 became undetectable at the neural groove and somite stage, respectively. As in the mouse, PGCs showed onset of c-kit expression when located in extraembryonal compartments. They appeared to follow the endoderm during extraembryonal allocation and the mesoderm on return to the genital ridge.     

Genetic Control of Spermatogenesis and Sex Determination in Mammals

The material was analyzed on the main problems of genetics of mammalian spermatogenesis, sex determination, its reversion and other defects from the standpoint of current cytological and molecular-genetic concepts of functional activity of the parental genomes after fertilization and behavior of their chromosomes at the early embryonic stages. On the basis of this analysis, a hypothesis has been proposed, which explains a high percentage (50% or more) of early embryonic mortality in placental mammals under the conditions of natural and extracorporeal fertilization, as well as regular appearance of defects in the course of natural sex determination, including the appearance of representatives of both sex minorities. We do not make pretense to comprehensive and deep analysis of male gametogenesis and sex determination in mammals.