Functional characterization of developing heart in embryos using Electric Potential Sensors

The characterization of the electrocardiographic activity of the living zebrafish heart during early developmental stages is a challenging task. Most of the available techniques are limited to heartbeat rate quantification being this inaccurate. Other invasive methodologies require the insertion of electrodes noise isolated environments and advanced amplification stages making these techniques very expensive. In this paper, we present a novel and non-invasive sensor development to characterize the functional activity of the developing heart of in vivo zebrafish embryos. The design is based on the Electric Potential Sensing technology patented at Sussex which has been developed to achieve reproducibility and continuous detection. We present preliminary functional characterization data of the developing zebrafish heart starting at 3 days-post-fertilization. Results show that using the proposed system for mapping the electrocardiographic activity of the zebrafish heart at early developmental stages is successfully accomplished. This is the first time that such a sensitive sensor has been developed for measuring the electrical changes occurring on micron sized (< 100 µm) living samples such as the zebrafish heart

New Tools for Real-Time Study of Embryonic Development

Embryonic development represents one of the most complex and dynamic cellular processes in biology, and plays vital roles in understanding of functions of embryonic stem cells (ESCs) and design of ESC-based therapy. Conventional assays and fluorescence-based imaging methods have been widely used for the study of embryonic development. These conventional methods cannot effectively provide spatial and temporal resolutions with sufficient sensitivity and selectivity that are required to depict embryonic development in vivo in real-time at single-cell and single-molecule resolutions. In this dissertation, we have developed a wide range of innovative tools for real-time study of embryonic development. These new tools include biocompatible and photostable plasmonic gold (Au) and silver (Ag) nanoparticle (NP) imaging probes, dark-field optical microscopy and spectroscopy (DFOMS), and ultrashort electric pulses. We have designed and synthesized a mini-library of Au and Ag NPs with different sizes and chemical properties. We have used developing zebrafish embryos as in vivomodel organisms to study embryonic development and as in vivo assays to study size- and chemical-dependent nanotoxicity. We found that these multicolored imaging probes can passively diffuse into embryos and enter into embryos non-invasively. These NPs exhibit superior photostability and enable us to study embryonic environments for a desired period of time. They can be illuminated under a standard microscope halogen lamp and characterized simultaneously using DFOMS equipped with a multi-spectral imaging system to achieve real-time multiplexing imaging. Our studies show that Au NPs are much more biocompatible than Ag NPs, while Ag NPs are much more sensitive and colorful than Au NPs. Notably, we can make Ag NPs nearly as biocompatible as Au NPs by functionalizing their surfaces with biocompatible peptides. Furthermore, Ag NPs can incite stage-specific embryonic phenotypes, and enable us to generate distinctive mutants for further identification of biomarkers for better understanding of embryonic development and for potential diagnosis of birth defects. We have developed new methods to effectively culture and sustain ESCs of zebrafish, mouse and human, laying down the foundation for real-time study of differentiation of ESCs both in vitro and in vivo for a wide variety of biomedical applications

Neo-SENSE: a non-invasive smart sensing mattress for cardiac monitoring of babies

Within the first minute of life a newborn must take its first breath to make the transition from life inside the womb to the outside world. If a baby does not start breathing, its heart rate will drop and the circulation of blood carrying oxygen to the organs will be seriously affected. The damage done to a newborn who is deprived of oxygen happens so quickly that rapid response is imperative. During birth, the attending neonatal staff manually listen to the baby´s heart and count the heart rate; however, this has proven inaccurate and inefficient. Nowadays, there is not a reliable method to monitor newborn heart rate promptly throughout birth. In this paper, we report the design and development of a novel smart mattress device to measure the babies’ electrocardiogram and respiration non-invasively. The device is based on electrometer-based amplifier sensors combined with novel screen-printing techniques. Proof of concept tests are carried out to demonstrate the suitability of the smart-mattress for new born ECG monitoring. We perform tests with a young infant and demonstrate the potential of this sensing technology to provide a quick and reliable application as ECG readings were displayed within a time < 30 seconds. This will aid the neonatal staff to assess the success of the resuscitation technology aiming to lower the incidence of long-term consequences of poor adaptation to life outside the womb

Biomaterials and Stem Cells: Promising Tools in Tissue Engineering and Biomedical Applications

Biomaterial sciences and tissue engineering approaches are currently fundamental strategies for the development of regenerative medicine. Stem cells (SCs) are a unique cell type capable of self‐renewal and reconstructing damaged tissues. At the present time, adult SCs isolated from postnatal tissues are widely used in clinical applications. Their characteristics such as a multipotent differentiation capacity and immunomodulatory activity make them a promising tool to use in patients. Modern material technologies allow for the development of innovative biomaterials that closely correspond to requirements of the current biomedical application. Biomaterials, such as ceramics and metals, are already used as implants to replace or improve the functionality of the damaged tissue or organ. However, the continuous development of modern technology opens new insights of polymeric and smart material applications. Moreover, biomaterials may enhance the SCs biological activity and their implementation by establishing a specific microenvironment mimicking natural cell niche. Thus, the synergistic advancement in the fields of biomaterial and medical sciences constitutes a challenge for the development of effective therapies in humans including combined applications of novel biomaterials and SCs populations

The 1988-1989 NASA Space/Gravitational Biology Accomplishments

This report consists of individual technical summaries of research projects of NASA's space/gravitational biology program, for research conducted during the period May 1988 to April 1989. This program is concerned with using the unique characteristics of the space environment, particularly microgravity, as a tool to advance knowledge in the biological sciences; understanding how gravity has shaped and affected life on Earth; and understanding how the space environment affects both plant and animal species. The summaries for each project include a description of the research, a list of the accomplishments, an explanation of the significance of the accomplishments, and a list of publications

The role of ryanodine receptors in development

PhDCalcium ions (Ca2+) are fundamental to the regulation of many cellular processes; however, the coordination of these signals during embryogenesis is not well understood. Ryanodine receptors (RyR) are a family of important intracellular ion channels that are responsible for the release of Ca2+ and they regulate the cytosolic Ca2+ concentration. Humans have three differentially expressed ryr genes (ryr1, ryr2 and ryr3) and mutations can cause both skeletal and cardiac diseases. Although the primary function of RyR is to mediate excitation-contraction coupling in muscle, they may also regulate Ca2+ signalling during developmental processes. The project has addressed the role of RyR during embryonic development, using the zebrafish as an in vivo vertebrate model. Five zebrafish RyR genes (ryr1a, ryr1b, ryr2a, ryr2b and ryr3) were characterised and a comprehensive overview of their spatial and temporal expression in the embryo was determined. At 24 hours post-fertilisation (hpf), ryr1a, ryr1b and ryr3 are expressed in the skeletal muscle, ryr2a in specific neuronal populations and ryr2b in the cardiac muscle. Semi-quantitative PCR data and wholemount in situ hybridisation revealed strong maternal expression of ryr3 during the cleavage and blastula periods and into adulthood. The early expression of the ryr3 gene suggests that this receptor functions during the initial stages of development; a role that has not been described previously. The functional significance of RyR3 during early embryogenesis was investigated in a loss-of- 3 function model using antisense morpholino oligonucleotides. The ryr3 specific knockdown experiments appeared to affect the establishment of embryonic axis prior to the segmentation periods (before 10 hpf). In addition, by 19 to 20 hpf ryr3 morphants failed to exhibit spontaneous muscle contractions and displayed a defect in neuromuscular development. In conclusion, this study has characterised the ryr genes and provided an overview on their temporal and spatial expression. The work provides evidence that ryr3 expression provides the Ca2+ vital for myofibrils organisation and that is required for the spontaneous movements during zebrafish embryonic development. The knowledge of RyR tissue distribution in zebrafish has provided a strong foundation for loss-of-function studies aimed at addressing their role in development. In the long term, the work will also facilitate more focused studies on disease.School of Biological and Chemical Sciences Queen Mary University of London. Central Research Fund and Physiological Society Travel Grant

Comparative Embryonic Spatio-Temporal Expression Profile Map of the Xenopus P2X Receptor Family

International audienceP2X receptors are ATP-gated cations channels formed by the homo or hetero-trimeric association from the seven cloned subunits (P2X1-7). P2X receptors are widely distributed in different organs and cell types throughout the body including the nervous system and are involved in a large variety of physiological but also pathological processes in adult mammals. However, their expression and function during embryogenesis remain poorly understood. Here, we report the cloning and the comparative expression map establishment of the entire P2X subunit family in the clawed frog Xenopus. Orthologous sequences for 6 mammalian P2X subunits were identified in both X. laevis and X. tropicalis, but not for P2X3 subunit, suggesting a potential loss of this subunit in the Pipidae family. Three of these genes (p2rx1, p2rx2, and p2rx5) exist as homeologs in the pseudoallotetraploid X. laevis, making a total of 9 subunits in this species. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. RT-PCR revealed that all subunits are expressed during the development although zygotic p2rx6 and p2rx7 transcripts are mainly detected at late organogenesis stages. Whole mount in situ hybridization shows that each subunit displays a specific spatio-temporal expression profile and that these subunits can therefore be grouped into two groups, based on their expression or not in the developing nervous system. Overlapping expression in the central and peripheral nervous system and in the sensory organs suggests potential heteromerization and/or redundant functions of P2X subunits in Xenopus embryos. The developmental expression of the p2rx subunit family during early phases of embryogenesis indicates that these subunits may have distinct roles during vertebrate development, especially embryonic neurogenesis

What makes cilia beat and how do different laterality mutations impact on heart morphology and physiology?

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, 2023, Universidade de Lisboa, Faculdade de CiênciasDespite their external bilateral symmetry, vertebrates are not as symmetrical as we think. Under their skin, several organs and corresponding vasculature display an asymmetric arrangement in the body cavity. This archetypal visceral patterning is the outcome of the left-right (LR) axis formation during embryonic development and is critical to ensure optimal organ functioning. For example, patients with laterality disorders tend to develop congenital cardiovascular malformations. In several species, in a transient organ known as LR organizer (LRO), directional fluid flow yielded by motile cilia beating has been reported to trigger asymmetric Nodal signaling expansion, which is critical for differentiating the left and right sides of the embryo. Zebrafish LRO comprises motile and immotile cilia and their balance is critical to develop an efficient fluid flow that promotes the correct positioning of visceral organs and, subsequently, healthy vertebrate development. Intriguingly, both types of cilia express cilia motilityrelated genes and are equipped with similar motility apparatus, and yet some beat and others do not. A previous study determined that the transcription repressor Her12 was a potential candidate to mediate this motility fate decision, and this hypothesis underpinned the beginning of this master thesis. We first aimed to ascertain the correlation between Her12 and cilia behavior by assessing her12 expression in the cilia population and identifying her12 targets. However, many pitfalls were encountered, and no conclusions could be drawn. Experiments must be repeated to solve this outstanding question. We also investigated how error-prone execution of asymmetric LR patterning in motile cilia and non-cilia mutants, ccdc40-/- and dand5-/- mutants, respectively, affected heart situs and morphogenesis. We characterized, for the first time, similar cardiovascular defects in both mutants. However, how the disrupted LR spatial-temporal signals impaired coordination between the heart situs and development remains to be enlightened and further studies should be conducted