A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy

Abstract

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic-specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination-based ultramicroscopy to entire wholemount-immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF-C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.

Figure 1

Emergence of the initial lymphatic progenitor cells from the cardinal vein. (A–D) Wholemount immunostaining of the vasculature in mouse embryos at 9.5/9.75 (A, C) and 10.5 (B, D) days post fertilization. PECAM-1 preferentially stained arterial, Endomucin mostly venous vessels. Prox1 identified lymphatic endothelial cells (LECs). The box in (A) delineates the jugular-thoracic region, in which LECs emerged from the cardinal vein (CV). DA, dorsal aorta; ISA, intersegmental artery; PAAs, pharyngeal arch arteries. Scale bar=100 μm. (E) Schematic sagittal section through one of the paired CVs. Venous ECs, blue; developing heart, dark green. The relative position of the superficial venous plexus (sVP) is indicated. CCV, common cardinal vein; SV, sinus venosus; H, heart; ISV, intersegmental vein. (F) 3D representation of the paired CCVs and SV draining into the heart. Depicted on the averted half of the symmetrical CVs are the ISVs and myotomes (M). Blue arrows indicate the flow of venous blood. (G) Schematic transverse section at the jugular-thoracic region. DA, ISA and arterial plexus in red; CV, ISV and sVP in blue. NT, neural tube; DRG, dorsal root ganglion; iLECs, initial lymphatic endothelial cells. (H–K) Volume reconstruction of optical sections of embryos wholemount immunostained for the proteins noted on the left side of each panel. E, developmental stages in days post fertilization. (H, I, K transverse sections; J sagittal sections). White arrows, emerging iLECs; dotted line, dorsal roof of the CV. Scale bar=100 μm. (L–O) Schematic depiction of the emergence of the first iLECs between E10.0 and E10.25. Prox1+ cells, green with yellow nuclei. The Prox1 expression domain (P1ED) in the averted branch of the CCV is indicated as a green surface.

Figure 2

Budding of lymphatic endothelial cells from the CV is associated with a change in cell and nuclear shape, but also with a switch in protein marker expression. (A, B) Sagittal view of the CCV of embryos wholemount immunostained for the proteins indicated on the left side of each panel. Developmental stages in days post fertilization (E); iLECs, initial lymphatic endothelial cells; cranial, left; caudal, right. Scale bar=100 μm. Upon egress from the CV, LEC shape changed from squamous (arrows indicate Prox1+ ECs in the roof of the CV) to spindle shape. White arrowheads, extremely thin connections between iLECs; red arrowheads, erythrocytes frequently found in luminized venous vessels (but never in iLECs). (B) Also see corresponding scheme Figure 1O. (C) At E10.5, VEGFR-3 and its co-receptor Nrp2 were upregulated in emerging iLECs, while Lyve-1 levels remained unchanged in the CV and iLECs. ***P≤0.001, NS, non-significant. (D, E) 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–H) Surface rendering of Prox1+ nuclei inside and outside the CCV of wholemount immunostained embryo in sagittal (F) and transversal view (G, H). (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. (H) Anatomical location of cells is depicted by superposition. Software package: Imaris Vantage, Scale bar=100 μm.

Figure 3

iLECs condense at the level of the major lateral branch of the intersegmental vessels to form a lumenized peripheral longitudinal lymphatic vessel (PLLV). (A–D) Sagittal view reconstructed from optical sections of embryos wholemount immunostained for the proteins indicated above each panel. E, developmental stages in days post fertilization; cranial, left; caudal, right. (A) During the first stages of iLECs emergence, iLECs appeared in a fan-shaped pattern, which extended caudally and cranially from the CCV. Dotted line, ventral border of iLEC detection. (A–D) iLECs immediately condensed at the level of the first lateral branch of the intersegmental vessels to form the PLLV. Long-hatched lines denote the position of the CCV and SV; short-hatched line, area of iLECs condensation and PLLV formation. (E–H) Schematic depiction of the position of iLECs appearing dorsal to the CV between E10.5 and E10.75. Prox1+ iLECs outside the CCV are depicted in light green, Prox1+ cells within the CV and heart muscle in dark green. The Prox1 expression domain (P1ED) in the averted branch of the CCV is indicated as a light green surface. Position of the superficial venous plexus, as a possible alternative source of iLECs, is indicated in blue (G, H). Prox1+ endothelial cells within the sVP are highlighted in red. sVP, superficial venous plexus; scale bars=100 μm.

Figure 4

LECs between the CV and PLLV aggregate and form increasingly larger lumenized structures finally giving rise to the primordial thoracic duct. (A–C) Sagittal and (D) transversal views reconstructed from optical section of embryos wholemount immunostained for the proteins indicated above each panel. (A) Arrowheads indicate rapid and progressive aggregation of LECs located between CV and PLLV, which resulted in the formation of a large lumenized structure, the pTD (B–D). (C, D) Superficial lymphatics (sLECs) appeared to sprout from the PLLV dorsally and the pTD laterally. PLLV and pTD were connected at the cranial end of the pTD. (F–H) Schematic depiction of the cellular aggregation/condensation events that result in pTD formation. (I) At E11.5, VEGFR-3 and its co-receptor Nrp2 were upregulated in sLECs, while Lyve-1 levels were strongly reduced as compared to the CV and iLECs. ***P≤0.001. Developmental stages in days post fertilization (E); cranial, left; caudal, right. ACV, anterior cardinal vein; CCV, common cardinal vein; PCV, posterior cardinal vein; ISV, intersegmental vein; PLLV, peripheral longitudinal lymphatic vessel; pTD, primordial thoracic duct; sLECs, superficial lymphatics. Scale bars=100 μm.

Figure 5

Paired contact sites between the newly formed pTD and the CV are characterized by highest levels of Prox1 expression. (A–C) Sagittal views of wholemount immunostained embryos. The newly forming pTD rapidly consolidated into a massive lumenized structure, cranially connected in a U-shape to the PLLV (left side A, B). Two contact sites between CV and pTD expressed high levels of Prox1 (arrowheads). (B–E) An arterial vessel identified as a transient side branch of the subclavian artery and stereotypically located between the contacts of pTD and CV is marked by an asterisk. (C) Red arrowhead: packed erythrocytes within the pTD. Arrows denote Prox1+ cells in the CV opposing the pTD contact sites. (D, E) Individual planes (optical sections) through the contact area of pTD and CV. (F–H) Schematic representation of the development of the contact sites between pTD and CV with highly Prox1+ cells at the contact site depicted in dark green with red nuclei. Scale bars=100 μm.

Figure 6

Different lymphatic endothelial cell populations express distinct sets of marker proteins. (A–G) Immunostainings of transversal cryosections from embryos at the indicated developmental ages are shown. Visualized antigens are depicted in the corresponding colours indicated above each panel. Panels exemplify representatively the marker expression summarized in (I). (A) Podoplanin expression was absent on LECs at E10.0, was first detectable at E11.0 (arrowheads) and was abundant on LECs at E12.0. Note Prox1+ cells in the CV were negative at all stages. (B) At E11.5, Nrp2 was moderately expressed on the CV and the pTD, while iLECs emerging outside the CV were strongly positive (arrows). (C) Endomucin was only transiently retained on iLECs. (D) Lyve-1 was strongly expressed on Prox1+ ECs of the CV and the pTD, while sLECs showed only residual expression (arrowheads). (E) Integrin α6 was moderately expressed on all vascular structures. (F) At E11.5, Netrin-4 was expressed strongly on BECs (arrows), weakly on the CV and moderately on the pTD, but was undetectable on iLECs (arrowheads). (G, H) Unc5B was strongly expressed on iLECs (G, arrowheads) and sLECs (H, arrowheads), while it was weak on the pTD. (H) Sagittal projection reconstructed from optical sections of a wholemount immunostained embryo for Prox1 (green) and Unc5B (blue). (I) Expression of the indicated proteins in different LEC populations during midgestation. Data are based on either immunostained cryosections or wholemount immunostaining. Indicated structures and cell populations: CV, cardinal vein; iLECs, initial LECs (first wave of LECs emerging from the CV as spindle shaped, loosely connected cells); sLECS, superficial LECs (LECs extending from the PLLV (dorsally)); pTD, primordial thoracic duct. CV*, expression limited to Prox1+ cells on the dorsal side of the CV. Scale bars=100 μm.

Figure 7

CCBE1 deficiency results in a failure of Prox1+ cells to separate from the CV and in rapid loss of nascent lymphatic structures. (A, B, F, G) 3D reconstructions of wild-type (A) and Ccbe1^−/− (B, F, G) embryos, wholemount immunostained for the indicated proteins. (A, B) Sagittal view at E10.5. (B) In CCBE1-deficient embryos, abundant Prox1+ cells were detected in the CV and in a rudimentary PLLV, adjacent to the superficial venous plexus. In contrast to wild-type embryos (A), the meshwork of spindle shaped iLECs between CCV and PLLV was absent. (B, F) Prox1+ cells outline the CCV and SV, while atypical, large, lumenized sprouts emerged from the CV (arrowheads). (G) Highly VEGFR-3+, dysformed sprouts extended from the CV (arrowheads) and ISVs (arrow). (C–E) Schematic representation of Prox1+ cells in wild-type (C) and CCBE1-deficient (D, E) embryos. Strongly VEGFR-3+ venous endothelium is depicted in dark blue. sVP, superficial venous plexus; scale bars=100 μm.

Figure 8

Prox1+ endothelial cells in VEGF-C-deficient mouse embryos are unable to leave their vessels of origin and thereby mark the venous sources of LECs. 3D reconstruction of (A, B) wild-type and (C–F) Vegfc^−/− embryos, wholemount immunostained for the indicated proteins at E10.75, sagittal view. In VEGF-C-deficient embryos, Prox1+ endothelial cells were unable to leave venous vessels resulting in an absence of developing lymphatic structures. (E, F) Besides Prox1+ cells in the CV (arrow), a second population of Prox1+ lymphatic progenitors is trapped in a larger venous vessel at the ventral edge of the sVP (arrowhead). (G, H) Schematic representation of Prox1+ cells in wild-type (G) and VEGF-C-deficient (H) embryos NE, stripe of neuronal Prox1 expression; sVP, superficial venous plexus, scale bars=100 μm.

Figure 9

CCBE1 and VEGF-C interact synergistically during iLEC egression and lymph vessel formation. 3D reconstructions of wild-type (A–C), Vegfc^+/− (D–F), Ccbe1^+/− (G–I) and Vegfc^+/−/Ccbe1^+/− (J–L) embryos, wholemount immunostained for the indicated proteins at E10.5, sagittal view. CCV and roots of the ISVs are indicated by dashed lines, Prox1+ cells by arrows. Compared to wild-type littermates (A–C) Vegfc^+/− embryos showed a reduction of emigrating iLECs from the CCV (D, E). Contrary to this, in Ccbe1^+/− embryos an impaired formation of ISVs was detectable. In addition, atypical, lumenized sprouts appeared at the roof of the cardinal vein that were Prox1+ and expressed high levels of VEGFR-3 (G–I). (J–L) In compound heterozygous embryos, this phenotype was grossly exaggerated indicating a synergistic role of VEGF-C and CCBE1 during lymphatic vessel formation. Scale bars=100 μm.