Analyzing The Developing Lymphatic System With Imaris | Imaris

Analyzing the developing lymphatic system


Dr. Friedemann Kiefer and colleagues, Max Planck Institute for Molecular Biomedicine

Circulatory blood vessels transport cells, nutrients, gases and waste products to and from tissues. In contrast, the tree-shaped lymphatic vessel system collects fluid, proteins, dietary fats and cells from the interstitial space and returns these into the venous circulation. Although they closely cooperate functionally, blood and lymph vessels form two physically separate vessel systems and are connected in only two places at the subclavian veins. Segregation of established blood and lymphatic vessel is actively maintained throughout adult life, however, details of the process are poorly understood.

Researchers led by Dr. Friedemann Kiefer from the Max Planck Institute for Molecular Biomedicine in Germany used ultramicroscopy and Imaris software to analyze the morphogenetic mechanisms that lead to the first partition of lymphatic endothelial cells from venous endothelium during mammalian fetal development.

These images are 3D reconstructions of single optical slices (2000-3000 slices for each channel), created using Surpass Mode in Imaris. They show a wildtype embryo 10 days post fertilization (left) and a CCBE-1 knockout embryo at 10.5 days post fertilization (right). CCBE-1 is a matrix component that the researchers found was necessary for the migration of lymphatic endothelial cells. Pecam-1 was stained in red (stains strongly arterial vasculature), Prox1 as a lymphatic endothelial cell marker in green (green nuclei), and VEGFR-3 in blue (stains venous and lymphatic endothelial cells). The threshold levels are adjusted for proper visualization of each channel and the clipping planes were used to focus on the region of interest. Images courtesy of René Hägerling, Max Planck Institute for Molecular Biomedicine.

The researchers used selective-plane based ultramicroscopy to observe the entire developing vascular system of whole mount mouse embryos at cellular resolution. They captured image stacks at 1-micron steps under various magnifications and then used Imaris for 3D reconstruction and morphometric analysis. The volume reconstructions made from the image stacks and surface rendering allowed the researchers to perform a comprehensive analysis. “At the time we initiated the study, Imaris provided a user friendly solution for the 3D rendering of very large stacks of optical sections,” Dr. Kiefer says.

During the first steps of lymph vessel formation, a group of endothelial cells in the cardinal vein develop into the first lymphatic structures. As the lymphatic endothelial cells emerge from the cardinal vein they change in cell and nuclear shape. To analyze change nuclear shape, the research team made use of the ImarisVantage module. ImarisVantage, statistical data-visualization module, enables the creation of fully customizable multiple dimensions colourful plots using actual segmented objects. These plots help to reveal relationships between cellular shape change and segmented nuclei sphericity and ellipticity. The analysis revealed that as the lymphatic endothelial cells left the cardinal vein, their nuclear shape changed from round to oblong as the cells underwent a pronounced change from squamous to spindle shape.

Nuclear shape change from spherical to elliptical associated with the emergence of iLECs. Nuclei of Prox1+ cells outside (red) and within (green) the CCV were depicted by nuclear surface rendering (D) and plotted against sphericity and ellipticity (prolate) (E). Scale bar=100 μm. (F–G) Surface rendering of Prox1+ nuclei inside and outside the CCV of wholemount immunostained embryo in sagittal (F) and transversal view (G). (F, G) Prox1 expression strength is indicated by pseudo-colouring using a heat map, i.e. highest Prox1 expression is indicated in red, low expression in blue. Images courtesy of EMBO J. 2013 Feb 12; 32(5): 629-44 Hägerling R et al. A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy

Dr. Kiefer explains that their study showed that lymphatic endothelial cells emerge from the dorsal roof of the cardinal vein as streams of cells, rather than by sprouting or pinching off of luminal structures. They also found that the cardinal vein is not the only source of lymphatic endothelium in the mouse embryo, but that the lower edge of the superficial venous plexus is also a source. Next, the researchers will work to better understand the molecular mechanisms that govern the migratory process during the emergence of the first lymphatic endothelial cells.

Research Paper: Hägerling R, Pollmann C, Andreas M, Schmidt C, Nurmi H, Adams RH, Alitalo K, Andresen V, Schulte-Merker S, Kiefer F. A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J. 2013 Feb 12;32(5):629-44. doi: 10.1038/emboj.2012.340.

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