Molecular Basis of Vertebrate Embryonic Migration

Embryology aims at understanding how a single fertilized cell develops into a complex multicellular organism. Initially the embryo is no more than a ball of cells where the three primordial layers, the ectoderm, mesoderm and the endoderm are one on top of the other. The three germ layers will go on to form all the tissues and organs of the embryo. For example, the ectoderm will give rise to epidermis and the nervous system; the mesoderm to muscles, the skeletal system, the dermis or inner layer of the skin, the circulatory, excretory, and reproductive systems; and, finally, the endoderm will give rise to the inner lining of the alimentary canal and the structures derived from it, such as lungs, the liver, pancreas, and the bladder. Correct positioning of the germ layers paves the way for the inductive interactions that are the hallmark of both axis determination and organogenesis. Therefore, the formation of the body plan requires highly integrated and regulated cell movements. The study of these movements is central to the field of embryology.Historically, the amphibian gastrula became one of the predominant models for experimental embryologists. This was partially due to the major influence of studies that lead to the eventual discovery of the organizer by Spemann and Mangold in 1924. After decades of research there is an imposing literature on the subject of inductive interactions in the amphibian and other embryos but the investigation of the movements leading from the relatively simple architecture of a blastula to the advanced and highly complex architecture of the late gastrula has been lacking. Perhaps it is not surprising, given the difficulties of studying these movements, that after almost a century of research fundamental questions still have not been answered. Haeckel first proposed the name gastrula in 1872, and although there was a long debate concerning the movements leading to the formation of the gastrula structures, the first experimental evidence for epibolic and inward morphogenetic movements was provided by Kopsch in 1895. Epiboly refers to the intercalation of cells in the animal cap (Figure IA, B and D ) while inward movements are the movements that lead to the internalization of the mesoderm, which is now referred to as involution (Figure 1 compares the location of the orange colored mesoderm at F stage 9 with that of G stage 10). The morphogenetic movements involved in gastrulation were later described by Vogt (1925, 1929) and then studied by Holtfreter (1943, 1944) and, more recently, by Keller(Gerhart and Keller 1986; Keller 1991; Weliky, Minsuk et al. 1991; Wilson and Keller 1991; Keller, Shih et al. 1992). The vast majority of this work was descriptive and only recently have we begun to gain some molecular insight regarding the pathways that are involved in specific morphogenetic movements

Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (IN). The C-terminus half (IC) of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes

Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo

<p>Abstract</p> <p>Background</p> <p>Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or <it>in vivo</it>. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins <it>in vivo</it>. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions.</p> <p>Results</p> <p>Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, <it>in vivo</it>, without any cross talk between the two intein systems.</p> <p>Conclusions</p> <p>Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, <it>in vivo</it>, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.</p

High-Resolution Whole-Mount In Situ Hybridization Using Quantum Dot Nanocrystals

The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mount in situ hybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D

Spatiotemporal Identification of Cell Divisions Using Symmetry Properties in Time-Lapse Phase Contrast Microscopy

A variety of biological and pharmaceutical studies, such as for anti-cancer drugs, require the quantification of cell responses over long periods of time. This is performed with time-lapse video microscopy that gives a long sequence of frames. For this purpose, phase contrast imaging is commonly used since it is minimally invasive. The cell responses of interest in this study are the mitotic cell divisions. Their manual measurements are tedious, subjective, and restrictive. This study introduces an automated method for these measurements. The method starts with preprocessing for restoration and reconstruction of the phase contrast time-lapse sequences. The data are first restored from intensity non-uniformities. Subsequently, the circular symmetry of the contour of the mitotic cells in phase contrast images is used by applying a Circle Hough Transform (CHT) to reconstruct the entire cells. The CHT is also enhanced with the ability to “vote” exclusively towards the center of curvature. The CHT image sequence is then registered for misplacements between successive frames. The sequence is subsequently processed to detect cell centroids in individual frames and use them as starting points to form spatiotemporal trajectories of cells along the positive as well as along the negative time directions, that is, anti-causally. The connectivities of different trajectories enhanced by the symmetry of the trajectories of the daughter cells provide as topological by-products the events of cell divisions together with the corresponding entries into mitoses as well as exits from cytokineses. The experiments use several experimental video sequences from three different cell lines with many cells undergoing mitoses and divisions. The quantitative validations of the results of the processing demonstrate the high performance and efficiency of the method

A new mechanochemical model for apical constriction: coupling calcium signalling and viscoelasticity

Embryonic epithelial cells exhibit strong coupling of mechanical responses to chemical signals and most notably to calcium. Recent experiments have shown that the disruption of calcium signals during neurulation strongly correlates with the appearance of neural tube defects. We, thus, develop a multi-dimensional mechanochemical model and use it to reproduce important experimental findings that describe anterior neural plate morphogenetic behaviour during neural tube closure. The governing equations consist of an advection-diffusion-reaction system for calcium concentration which is coupled to a force balance equation for the tissue. The tissue is modelled as a linear viscoelastic material that includes a calcium-dependent contraction stress. We implement a random distribution of calcium sparks that is compatible with experimental findings. A finite element method is employed to generate numerical solutions of the model for an appropriately chosen range of parameter values. We analyse the behaviour of the model as three parameters vary: the level of IP3 concentration, the strength of the stretch-sensitive activation and the maximum magnitude of the calcium-dependent contraction stress. Importantly, the simulations reproduce important experimental features, such as the spatio-temporal correlation between calcium transients and tissue deformation, the monotonic reduction of the apical surface area and the constant constriction rate, as time progresses. The model could also be employed to gain insights into other biological processes where the coupling of calcium signalling and mechanics is important, such as carcinogenesis and wound healing

Cell-Autonomous Ca2+ Flashes Elicit Pulsed Contractions of an Apical Actin Network to Drive Apical Constriction during Neural Tube Closure

Neurulation is a critical period in all vertebrates and results in the formation of the neural tube, which gives rise to the CNS. Apical constriction is one of the fundamental morphogenetic movements that drives neural tube closure. Using live imaging, we show that apical constriction during the neurulation is a stepwise process driven by cell-autonomous and asynchronous contraction pulses followed by stabilization steps. Our data suggest that contraction events are triggered by cell-autonomous Ca2+ flashes and are driven by a transient contractile apical pool of actin. In addition, we provide evidence that the cell autonomy and asynchrony of contraction are required for the correct spatial distribution of constriction and, as a result, are critical for tissue morphogenesis. Finally, we identify Calpain2 as a regulator of apical constriction and show that it is required for the stabilization step, but is dispensable during contraction

Spatiotemporal Identification of Cell Divisions Using Symmetry Properties in Time-Lapse Phase Contrast Microscopy

A variety of biological and pharmaceutical studies, such as for anti-cancer drugs, require the quantification of cell responses over long periods of time. This is performed with time-lapse video microscopy that gives a long sequence of frames. For this purpose, phase contrast imaging is commonly used since it is minimally invasive. The cell responses of interest in this study are the mitotic cell divisions. Their manual measurements are tedious, subjective, and restrictive. This study introduces an automated method for these measurements. The method starts with preprocessing for restoration and reconstruction of the phase contrast time-lapse sequences. The data are first restored from intensity non-uniformities. Subsequently, the circular symmetry of the contour of the mitotic cells in phase contrast images is used by applying a Circle Hough Transform (CHT) to reconstruct the entire cells. The CHT is also enhanced with the ability to &ldquo;vote&rdquo; exclusively towards the center of curvature. The CHT image sequence is then registered for misplacements between successive frames. The sequence is subsequently processed to detect cell centroids in individual frames and use them as starting points to form spatiotemporal trajectories of cells along the positive as well as along the negative time directions, that is, anti-causally. The connectivities of different trajectories enhanced by the symmetry of the trajectories of the daughter cells provide as topological by-products the events of cell divisions together with the corresponding entries into mitoses as well as exits from cytokineses. The experiments use several experimental video sequences from three different cell lines with many cells undergoing mitoses and divisions. The quantitative validations of the results of the processing demonstrate the high performance and efficiency of the method