Mary E. Dickinson

Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a-/-). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.

Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation

The planar cell polarity (PCP) pathway is conserved throughout evolution, but it mediates distinct developmental processes. In Drosophila, members of the PCP pathway localize in a polarized fashion to specify the cellular polarity within the plane of the epithelium, perpendicular to the apicobasal axis of the cell. In Xenopus and zebrafish, several homologs of the components of the fly PCP pathway control convergent extension. We have shown previously that mammalian PCP homologs regulate both cell polarity and polarized extension in the cochlea in the mouse. Here we show, using mice with null mutations in two mammalian Dishevelled homologs, Dvl1 and Dvl2, that during neurulation a homologous mammalian PCP pathway regulates concomitant lengthening and narrowing of the neural plate, a morphogenetic process defined as convergent extension. Dvl2 genetically interacts with Loop-tail, a point mutation in the mammalian PCP gene Vangl2, during neurulation. By generating Dvl2 BAC (bacterial artificial chromosome) transgenes and introducing different domain deletions and a point mutation identical to the dsh1 allele in fly, we further demonstrated a high degree of conservation between Dvl function in mammalian convergent extension and the PCP pathway in fly. In the neuroepithelium of neurulating embryos, Dvl2 shows DEP domain-dependent membrane localization, a pre-requisite for its involvement in convergent extension. Intriguing, the Loop-tail mutation that disrupts both convergent extension in the neuroepithelium and PCP in the cochlea does not disrupt Dvl2 membrane distribution in the neuroepithelium, in contrast to its drastic effect on Dvl2 localization in the cochlea. These results are discussed in light of recent models on PCP and convergent extension.

TECHNICOLOUR TRANSGENICS: IMAGING TOOLS FOR FUNCTIONAL GENOMICS IN THE MOUSE

Over the past decade, a battery of powerful tools that encompass forward and reverse genetic approaches have been developed to dissect the molecular and cellular processes that regulate development and disease. The advent of genetically-encoded fluorescent proteins that are expressed in wild type and mutant mice, together with advances in imaging technology, make it possible to study these biological processes in many dimensions. Importantly, these technologies allow direct visual access to complex events as they happen in their native environment, which provides greater insights into mammalian biology than ever before.

Four-dimensional cardiac imaging in living embryos via postacquisition synchronization of nongated slice sequences

Being able to acquire, visualize, and analyze 3D time series (4D data) from living embryos makes it possible to understand complex dynamic movements at early stages of embryonic development. Despite recent technological breakthroughs in 2D dynamic imaging, confocal microscopes remain quite slow at capturing optical sections at successive depths. However, when the studied motion is periodic--such as for a beating heart--a way to circumvent this problem is to acquire, successively, sets of 2D+time slice sequences at increasing depths over at least one time period and later rearrange them to recover a 3D+time sequence. In other imaging modalities at macroscopic scales, external gating signals, e.g., an electro-cardiogram, have been used to achieve proper synchronization. Since gating signals are either unavailable or cumbersome to acquire in microscopic organisms, we have developed a procedure to reconstruct volumes based solely on the information contained in the image sequences. The central part of the algorithm is a least-squares minimization of an objective criterion that depends on the similarity between the data from neighboring depths. Owing to a wavelet-based multiresolution approach, our method is robust to common confocal microscopy artifacts. We validate the procedure on both simulated data and in vivo measurements from living zebrafish embryos.

Rapid three-dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development.

We report an accurate method for studying the functional dynamics of the beating embryonic zebrafish heart. The fast cardiac contraction rate and the high velocity of blood cells have made it difficult to study cellular and subcellular events relating to heart function in vivo. We have devised a dynamic three‐dimensional acquisition, reconstruction, and analysis procedure by combining (1) a newly developed confocal slit‐scanning microscope, (2) novel strategies for collecting and synchronizing cyclic image sequences to build volumes with high temporal and spatial resolution over the entire depth of the beating heart, and (3) data analysis and reduction protocols for the systematic extraction of quantitative information to describe phenotype and function. We have used this approach to characterize blood flow and heart efficiency by imaging fluorescent protein‐expressing blood and endocardial cells as the heart develops from a tube to a multichambered organ. The methods are sufficiently robust to image tissues within the heart at cellular resolution over a wide range of ages, even when motion patterns are only quasiperiodic. These tools are generalizable to imaging and analyzing other cyclically moving structures at microscopic scales. Developmental Dynamics 235:2940–2948, 2006.

Cell death in the CNS of the Wnt-1 mutant mouse

Using the benzothiazolium-4-quinolium dye, TO-PRO-1, to detect cell death in live embryos, we labeled a developmental series of Wnt-1 null mutant and wild type embryos to determine if cell death contributed to the absence of the midbrain and rostral metencephalon observed in Wnt-1 mutant embryos. We found that there is no detectable cell death at early somite stages in Wnt-1 mutant embryos. However, we detected a significant, but transient, population of dying cells within the anterior dorsal metencephalon in 20-29 somite stage embryos. These cells located in the anterior dorsal metencephalon also stain positive using the TUNEL technique that utilizes terminal transferase to label DNA fragments that are typical in the nuclei of apoptotic cells. Thus, programmed cell death plays a role in the loss of the metencephalon, but apparently does not contribute to the earliest aspect of the mutant phenotype, namely the loss of the midbrain.

Sensitive imaging of spectrally overlapping flourochromes using the LSM 510 META

Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.

Dynamic In Vivo Imaging of Mammalian Hematovascular Development Using Whole Embryo Culture

The yolk sac is the initial site of hematopoiesis in the mammalian embryo. As the embryo develops, blood vessels form around primitive erythroblasts to connect the yolk sac to the embryo, delivering newly formed blood cells to the embryonic circulation. The limited accessibility of the mammalian embryo has made it difficult to study the dynamic changes in cellular development during the formation of the early hematovascular system. Therefore, we have developed a culture system for studying early hematopoiesis, vasculogenesis. and angiogenesis in the mouse embryo. Early embryos (E7.5-E9.5) can be grown on the microscope stage to study the dynamics as vessels form and circulation begins. In addition, this mouse embryo culture system provides an excellent model for understanding the interplay between flow dynamics and cellular development.

10 – FRET Measurements Using Multispectral Imaging

This chapter discusses the use of multispectral imaging in Forster resonance energy transfer (FRET) measurements. Multispectral imaging, also known as hyperspectral imaging, is an important tool for confocal microscopy as it uses the entire spectral response from cells. By acquiring data from multiple wavelengths, the relative contribution of individual molecular components is determined through mathematical analysis. The variations in spectral information are determined by measuring intensity differences at different wavelengths from the same field of view, much like using a spectrometer to measure fluorescence from a cuvette at different wavelengths. Image data sets are analyzed for individual spectral frequencies and pixels with matching spectral information, often showing similar structures, are classified to produce an image. Spectral variations can be used to resolve details in the field of view that cannot be seen in single images taken when all the light from the visible spectrum is used or with single filters.

Wavelet-based synchronization of nongated confocal microscopy data for 4D imaging of the embryonic heart

With the availability of new confocal laser scanning microscopes, fast biological processes, such as the blood flow in living organisms at early stages of the embryonic development, can be observed with unprecedented time resolution. When the object under study has a periodic motion, e.g. a beating embryonic heart, the imaging capabilities can be extended to retrieve 4D data. We acquire nongated slice-sequences at increasing depth and retrospectively synchronize them to build dynamic 3D volumes. Here, we present a synchronization procedure based on the temporal correlation of wavelet features. The method is designed to handle large data sets and to minimize the influence of artifacts that are frequent in fluorescence imaging techniques such as bleaching, nonuniform contrast, and photon-related noise.