Dorsalization of the neural tube by the non-neural ectoderm

The patterning of cell types along the dorsoventral axis of the spinal cord requires a complex set of inductive signals. While the chordamesoderm is a well-known source of ventralizing signals, relatively little is known about the cues that induce dorsal cell types, including neural crest. Here, we demonstrate that juxtaposition of the non-neural and neural ectoderm is sufficient to induce the expression of dorsal markers, Wnt-1, Wnt-3a and Slug, as well as the formation of neural crest cells. In addition, the competence of neural plate to express Wnt-1 and Wnt-3a appears to be stage dependent, occurring only when neural tissue is taken from stage 8–10 embryos but not from stage 4 embryos, regardless of the age of the non-neural ectoderm. In contrast to the induction of Wnt gene expression, neural crest cell formation and Slug expression can be induced when either stage 4 or stage 8–10 neural plates are placed in contact with the non-neural ectoderm. These data suggest that the non-neural ectoderm provides a signal (or signals) that specifies dorsal cell types within the neural tube, and that the response is dependent on the competence of the neural tissue

Analysis of somitogenesis using multiphoton laser scanning microscopy (MPLSM)

In order to study complex cellular interactions in the developing somite and nervous system, we have been refining techniques for labeling and imaging individual cells within the living vertebrate embryo. Most recently, we have been using MPLSM to analyze cellular behaviors, such as cell migration, filopodial extension, cell process collapse, and neuron pathfinding using time-lapse microscopy in 3-dimensions (3-d). To enhance the efficiency of two-photon excitation in these samples, we have been using a Zeiss LSM 510 NLO fiber delivery system with a Grating Dispersion Compensator (GDC). This system not only offers the convenience of fiber delivery for coupling our Ti:Sapphire laser to the microscope, but also affords us precise control over the pulsewidth of the mode- locked beam. In addition, we have developed a novel peptide/non-cationic lipid gene delivery system to introduce GFP plasmid into somite cells. This approach has allowed us to generate detailed 3-d images of somite cell morphologies at various stages of somite development in a way that best preserves the vitality of the cells being imaged

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 sa

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

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

Nonuniform temporal alignment of slice sequences for four-dimensional imaging of cyclically deforming embryonic structures

The temporal alignment of nongated slice-sequences acquired at different axial positions in the living embryonic zebrafish heart permits the reconstruction of dynamic, three-dimensional data. This approach overcomes the current acquisition- speed limitation of confocal microscopes for real-time three-dimensional imaging of fast processes. Current synchronization methods align and uniformly scale the data in time, but do not compensate for slight variations in the heart rhythm that occur within a heartbeat. Therefore, they impose constraints on the admissible data quality. Here, we derive a nonuniform registration procedure based on the minimization of the absolute value of the intensity difference between adjacent slice-sequence pairs. The method compensates for temporal intra-sample variations and allows the processing of a wider range of data to build functional, dynamic models of the beating embryonic heart. We show reconstructions from data acquired in living, fluorescent zebrafish embryos

Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons

Fura-2 recording of Ca^(2+) influx was used to show that incubation in 1 μM nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHβE-sensitive component that presumably corresponds to α4β2 receptors. To study changes in α4β2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the α4 or β2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca^(2+) influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent α4 and β2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of α4β2 receptors, coexpression of the α4-YFP and β2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic α4β2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 μMnicotine, there was increased FRET in the cell body, denoting increased assembly of α4β2 receptors. Thus, changes in α4β2 receptor assembly play a role in the regulation of α4β2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca^(2+)-stimulated pathways

Analysis of somitogenesis using multiphoton laser scanning microscopy (MPLSM)

In order to study complex cellular interactions in the developing somite and nervous system, we have been refining techniques for labeling and imaging individual cells within the living vertebrate embryo. Most recently, we have been using MPLSM to analyze cellular behaviors, such as cell migration, filopodial extension, cell process collapse, and neuron pathfinding using time-lapse microscopy in 3-dimensions (3-d). To enhance the efficiency of two-photon excitation in these samples, we have been using a Zeiss LSM 510 NLO fiber delivery system with a Grating Dispersion Compensator (GDC). This system not only offers the convenience of fiber delivery for coupling our Ti:Sapphire laser to the microscope, but also affords us precise control over the pulsewidth of the mode- locked beam. In addition, we have developed a novel peptide/non-cationic lipid gene delivery system to introduce GFP plasmid into somite cells. This approach has allowed us to generate detailed 3-d images of somite cell morphologies at various stages of somite development in a way that best preserves the vitality of the cells being imaged

Rotational imaging optical coherence tomography for full-body mouse embryonic imaging

Optical coherence tomography (OCT) has been widely used to study mammalian embryonic development with the advantages of high spatial and temporal resolutions and without the need for any contrast enhancement probes. However, the limited imaging depth of traditional OCT might prohibit visualization of the full embryonic body. To overcome this limitation, we have developed a new methodology to enhance the imaging range of OCT in embryonic day (E) 9.5 and 10.5 mouse embryos using rotational imaging. Rotational imaging OCT (RI-OCT) enables full-body imaging of mouse embryos by performing multiangle imaging. A series of postprocessing procedures was performed on each cross-section image, resulting in the final composited image. The results demonstrate that RI-OCT is able to improve the visualization of internal mouse embryo structures as compared to conventional OCT

Runners’ Engagement and Social Support Practices: Exploring the Uses and Role of Online Activities

Tracking technologies that monitor exercise and health have grown in popularity, while online communities have emerged to encourage healthier lifestyles. Runners display high engagement levels with these technologies, however, studies are inconclusive about the effectiveness of these tools, and half of tracking device owners stop use within three months. This exploratory study analyses the engagement practices of UK based long distance runners with tracking technologies and online communities in order to explore how these provide social support for runners. Data were derived from nine interviews. Findings identify distinct use practices associated with social networking sites, running club online communities and tracking applications. Social support is not as prominent as social comparison, identity formation, motivation and information gathering for runners. The nature of the community, co-present connections between individuals and running experience have implications for engagement and support practices, while emotional attachment to data represents an opportunity for development