Lavision Light Film Illumination (SPIM) Microscope - Lymphatic Formation Mechanism

Lavision Light Slice Lighting (SPIM) Microscope Ultramicroscope Application

Multi-step mechanism of initial lymphatic vessel formation in mouse embryos

Rene ́ Ha ̈ gerling1,7, Cathrin Pollmann1,7,Martin Andreas1, Christian Schmidt1,Harri Nurmi2, Ralf H Adams3, Kari Alitalo2,Volker Andresen4, Stefan Schulte-Merker5,6and Friedemann Kiefer1,* The EMBO Journal (2013), 1 –16

Abstract: During mammalian development, an internal cell subpopulation in the main venous blood vessels begins to express lymphatic-specific genes, which in turn develop primary lymphoid structures, which are collectively named lymphatic vesicles. The budding, expansion, and swelling of lymphatic endothelial cells are thought to be the basis for the production of lymphatic endothelial cells from the main vein, but the exact mechanism of lymphangiogenesis is still unknown. Using a selective light illumination microscope Ultramicroscope to observe mouse embryos for overall immunostaining, we observed a complete developing vascular system with cell resolution. In this paper, we report that the earliest lymphatic endothelial cells that can be detected are loosely connected to the main and superficial vascular bundles. The next step in lymphatic endothelial cell aggregation results in two distinct, unconfirmed lymphoid structures, the dorsal peripheral longitudinal lymphatic vessels and the ventral primary thoracic ducts, which form a direct connection to the main vein at a later stage. We found that vascular endothelial growth factor C and the matrix component CCBE1 are essential for lymphocyte endothelial cell sprouting and migration. In summary, we provide a significantly more detailed perspective and a novel model of early lymphatic development.

Main results:

Figure 1. Initial lymphoid progenitor cells are produced from the main vein.
(AD) Overall staining of mouse embryonic vasculature at 9.5/9.75 (A, C) and 10.5 (B, D) days after fertilization. PECAM-1 preferentially stains endogenous mucin in arterial and venous blood vessels. Lymphocytes recognized by Prox1. (A) The thoracic jugular vein area and lymphatic endothelial cells are framed. DA, dorsal aorta; ISA, intersegmental artery; PAAs, pharyngeal arch artery. The ruler is 100um. E The icon arrow traverses one of a pair of main veins. Venous endothelial cells, blue; developing heart, dark green; the location of superficial venous plexus is marked. CCV, general main vein; SV, sinus; H, heart; ISV, interstitial blood vessels. (F) Three-dimensional reconstruction of the paired CCV and SV leading into the heart. Remove the ISVs and the myogenic muscle knife (M) after half of the symmetrical main vein. The blue arrow indicates the flow of venous blood. (G) Cross section of the thoracic jugular vein area. DA, ISA and arterial plexus marked red; CV, ISV and sVP marked blue. NT, neural tube; DRG, dorsal root ganglion; iLECs, initial lymphatic endothelial cells. (HK) 3D reconstruction of the optical section of the protein distribution labeled on the left side of the overall immunostained embryo. E, the developmental stage of a few days after fertilization (H, I, K cross section; J sagittal section). White arrows, emerging iLECs; dotted lines, the back roots of CV. The ruler is 100um. (LO) An illustration of the earliest iLECs that occurred during E10.0 and E10.25. Prox1+ cells, green and yellow are the nucleus. The green surface indicates the Prox1 expression region in the CCV removal branch.

Figure 2. The budding of lymphatic endothelial cells from CV is accompanied by changes in the shape of the cells and nucleus, as well as the expression of a protein-labeled switch.
(A, B) Sagittal view of the labeled protein on the left side of the CCV of the overall immunostained embryo. Developmental stage after fertilization (E); initial lymphatic endothelial cells of iLECs; at the head, left; tail, right. The ruler is 100um. The upper exit of the CV changes from a scaly to a spindle-shaped LEC shape (arrows indicate Prox1+ ECs in the CV root). White arrows, extremely thin connections between iLECs; red arrows, frequent red blood cells found in venous vessels (but never in iLECs). (B) You can also see the corresponding diagram 1O. (C) At the E10.5 stage, the levels of VEGFR-3 and its co-receptor Nrp2 in iLECs appear to be up-regulated, while the levels of Lyve-1 in CV and iLECs remain unchanged. ***P<0.001, NS, not significant. (D, E) The shape of the nucleus changes from a circular shape to an elliptical shape as the iLECs appear. The Prox1+ nuclei inside and outside the CCV are described by nuclear surface reconstruction and a scatter plot (E) is made for the sphericity and ellipsometry. The ruler is 100um. (FH) Prox1+ nuclear surface remodeling of CCV internal and external CCV of whole immunostained mouse embryos in the sagittal (F) and cross-sectional (G, H) views. (F, G) Prox1 expression intensity maps with pseudo-color markers by thermal imaging, for example, the highest intensity expression marker is red and the low intensity expression marker is blue. (H) Anatomy positioning of the cell by superposition of images: Imaris Vantage, ruler 100um

Figure 3. iLECs are concentrated at the level of the main branch of the interstitial vessel to form an illuminated peripheral longitudinal lymphatic vessel (PLLV).
(AD) Sagittal reconstruction of the whole immunostained embryonic optical section of each of the displayed proteins. E, the number of days after fertilization; head cover, left; tail end, right. (A) In the early stages of the emergence of iLECs, iLECs are distributed in a fan-shaped pattern, extending from the CCV to the head and tail. The dotted line, the boundary detected by iLECs. (AD) iLECs are immediately concentrated at the level of the first side branch of the interstitial vessel to form a PLLV. Long hatching indicates the location of CCV and SV; short hatching, iLECs concentration and areas formed by PLLV. (EH) illustrates the location of the iLECs, which appear on the back of the CV during the E10.5 and E10.7 phases. Prox1+iLECs outside CCV are marked in light green, and Prox1+ cells and myocardium in CV are marked in dark green. The Prox1 expression domain (P1ED) in the branch of the CCV removal is shown in a pale green surface. Superficial venous plexus is a possible alternative source of iLECs and its location is marked in blue (G, H). Prox1+ endothelial cells within sVP are marked in red. sVP, superficial venous plexus; scale 100um

Figure 4. LECs between CV and PLLV aggregate and form a growing larger illuminated structure and eventually form the original thoracic duct. The (AC) sagittal and (D) cross-sectional views of the labeled proteins are taken from the images of the mouse embryonic optical sections of the overall immunostaining.
The (A) arrow indicates the rapid and ongoing aggregation of LECs located between the CV and the PLLV, which results in the formation of a larger illumination structure pTD (BD). (C, D) Superficial lymphatic sLECs begin to extend from the dorsal side of the PLLV and next to the pTD. PLLV and pTD are connected together at the pTD cap end. (FH) illustrates the cell aggregation and concentration events that result in pTD formation. (I) At the E11.5 stage, VEGFR-3 and its co-receptor Nrp2 levels were up-regulated in sLECs, whereas Lyve-1 levels were strongly down-regulated compared to CV and iLECs. ***P<0.001. Developmental stage (E); head cover, left, tail, right. ACV, anterior main vein; CCV, general main vein; PCV, posterior main vein; ISV, intersegmental vein; PLLV, peripheral longitudinal lymphatic vessels; pTD, original thoracic duct; sLECs, superficial lymph nodes. Ruler 100um

Figure 5. Newly formed paired contact points between pTD and CV characterized by the highest level of expression of Prox1.
(AC) Sagittal view of the overall immunostained embryo. The newly formed pTD is quickly consolidated into a huge illumination structure with the skull connected to the PLLV in a U shape (left A, B). The two connections between CV and pTD express the highest level of Prox1 (arrow). (BE) A short-lived lateral branch that is always located between the pTD and CV connections as the subclavian artery is marked with an asterisk. (C) Red arrow: Red blood cells accumulated in pTD. The arrow marks the Prox1+ cells opposite the pTD junction. (D, E) A single plane (optical slice) of the connection region through pTD and CV. (FH) illustrates the development of indirect contacts of pTD and CV, and Prox1+ cells with high expression at the contact points are labeled as dark green and red nuclei. Ruler 100um

Figure 6. Different lymphatic endothelial cell populations express different marker protein sets.
Transverse cryosection of immunostained embryos at the developmental stage indicated by (AG). Visible antigens are labeled with the corresponding colors labeled on each map. Panels of typical exemplary marker expressions are summarized in (I). (A) No expression of mucin in LECs cells at stage E10.0, first detected in the E11.0 phase and enriched in LE1s of E12.0. Note that Prox1+ cells in CV are negative at all stages. In the E11.5 phase, Nrp2 is moderately expressed in CV and pTD, while iLECs outside CV are strongly positive. (C) Endothelin has only a short-lived retention in iLECs. (D) Lyve-1 is strongly expressed in Prox1+ ECs of CV and pTD, while only residual expression (arrow) is present in the displayed sLECs. (E) Integrin α6 is moderately expressed in all vascular structures. (F) In the E11.5 phase, the nerve growth factor Netrin-4 is strongly expressed in BECs, is weakly expressed in CV, is moderately expressed in pTD, but is not detected in iLECs (arrow). (G, H) Unc5B is strongly expressed in iLECs (G, arrow) and sLECs (H, arrow), while expression is weak in pTD. (H) Sagittal remodeling of Prox1 (green) and Unc5B (blue) optical sections from whole immunostained mouse embryos. (I) Expression of labeled proteins in different LEC populations during the second trimester. Data were obtained from immunostained frozen sections or whole immunostaining. Structure and cell population expressed: CV, main vein; iLECs, initial LECs (first-round spindle-like LE from CV, loosely connected cells); sLECS, superficial LECs (extended from PLLV (back side) LECs); pTD, initial thoracic duct. CV*, expression restriction on CV dorsal Prox1+ cells. Ruler 100um

Figure 7. Failure of Prox1+ cells to separate from CV due to CCBE1 deficiency and leading to rapid loss of initial lymphoid structure.

Figure 8

Prox1+ endothelial cells in VEGF-C (vascular endothelial factor C)-deficient mouse embryos are labeled with a venous source of LECs because they cannot leave the blood vessels at their origin. Sagittal 3D reconstruction of E10.75 stage wild type (A, B) and Vegfc_/_ type (C–F) embryos, and overall immunostaining of labeled proteins. In VEGF-C-deficient embryos, Prox1+ endothelial cells cannot leave the venous blood vessels resulting in the absence of developing lymphoid structures. (E, F) In addition to the Prox1+ cells in the CV (arrow), a second group of Prox1t lymphoid initial tissues was captured in a larger venous vessel at the ventral sVP (arrow). (G, H) Prox1+ cells in wild type (G) and VEGF-C deficient (H) embryos are shown. NE, Prox1+ expression stripes of neurons. sVP, superficial venous plexus. Ruler 100 um

Figure 9. Synergistic interaction of CCBE1 and VEGF-C during iLECs outing and lymphangiogenesis.
Wild type (A–C), Vegfct/_ (D–F), Ccbe1t/_ (G–I) and Vegfct/_/Ccbe1t/_ (J–L) for the overall immunostaining of the proteins noted in the E10.5 phase 3-dimensional reconstruction of the embryonic sagittal map. The roots of CCV and ISVs are marked with dashed lines and Prox1+ cells are marked with arrows. Vegfct/_embryo (A–C) showed a decrease in the migration of iLECs from CCV (D, E) compared to wild-type litter. In contrast, in the Ccbe1t/_ embryo, damaged ISVs were detected. Moreover, atypical, illuminated branches appear in the main vein root (G–I) of Prox1+ and high levels of VEGFR-3 expression. (J–L) In complex heterozygous embryos, this phenotype is very exaggerated. The synergistic effect of VEGF-C and CCBE1 in lymphatic vessel formation was demonstrated. Ruler 100um