1 Lymphatic System

1.1 Embryology of the Lymphatic System

Florian Früh, Patrick A. Will, and Epameinondas Gousopoulos

The lymphatic vascular system has been substantially neglected in the history of vascular biology. Due to the challenge of its visualization and the absence of specific markers, the lymphatic system has been ignored and its anatomy was specified only at the beginning of the 19th century. The identification of specific lymphatic markers in the late 1990s as well as subsequent molecular genetic studies revolutionized our understanding of the mechanisms involved in specification, expansion, and maturation of the lymphatic system. ▶ [1]

From a morphological point of view, the murine lymphatic development starts at embryonic day E10.5, corresponding to week 6.5 to 7 in human embryos. ▶ [2],​ ▶ [3] Endothelial cells of the anterior cardinal vein give rise to the lymphatic primordia, which subsequently form the first primitive lymphatic structures termed lymph sacs. Despite interspecies variabilities of these primordia, the jugular region is generally accepted to be the site of lymphatic induction, ▶ [4] and mammalian embryos exhibit eight lymph sacs ( ▶ Fig. 1.1a,b). ▶ [4],​ ▶ [5] By the end of the 9th development week in humans, lymphatic vessels connect the lymph sacs. In the fetal period, the lymphatic system already exhibits the asymmetrical condition characteristic of the adult lymphatic system ( ▶ Fig. 1.1c).

More than 100 years ago, the American anatomist Florence Sabin introduced the now widely accepted centrifugal theory of lymphatic development. ▶ [6] She based her theory upon ink-injection experiments in pig embryos and suggested that the peripheral lymphatic system arises from the primary lymph sacs. Then it would spread to the surrounding tissues and organs by endothelial sprouting, where local capillaries are formed. However, in 1910, Huntington and McClure suggested a contradictory centripetal theory, stating that lymphatic vessels arise from peripheral mesenchymal lymphatic endothelial cell (LEC) progenitors, called lymphangioblasts. ▶ [7]

Expression studies of the lymphatic-specific marker vascular endothelial growth factor receptor 3 (VEGFR-3) and experiments in mice lacking the homeobox gene PROX1 (prospero homeobox protein 1) confirmed the venous origin of lymphatic vessels. ▶ [8],​ ▶ [9] Importantly, in PROX1 −/− knockout mice, budding and sprouting of LEC progenitors is arrested without affecting the vasculature of blood vessel development. ▶ [9] These landmark studies led to the proposal of a stepwise model for the development of the lymphatic vasculature in mammals. ▶ [2],​ ▶ [10],​ ▶ [11] LEC competence of endothelial progenitor cells in the cardinal veins is achieved around E9.0 by PROX1 expression under the control of the transcription factor gene Sox18 (sex determining region Y box 18). ▶ [12] Under the influence of the lymphatic master-regulator gene PROX1, the endothelial progenitors undergo LEC commitment and start budding from the cardinal veins at approximately E10.5. ▶ [11] After commitment, cells may give rise to a particular cell type or structure, depending on the surrounding tissue. ▶ [2] Lymphatic vessel sprouting from the embryonic veins is guided by a graded mesenchymal expression of the VEGFR-3 ligand vascular endothelial growth factor C (VEGF-C). Paracrine VEGF-C signaling is crucial for the migration and survival of PROX1-expressing cells from the cardinal veins and for the subsequent formation of lymph sacs. ▶ [13] Furthermore, as LEC progenitor cells bud from the embryonic veins into the surrounding mesenchymal tissue, they begin expressing the lymphatic marker podoplanin, indicating lymphatic differentiation and maturation. ▶ [14] At around E11.5, PROX1+/VEGFR-3+/podoplanin+ differentiating LECs migrate in interconnected cell groups into the surrounding mesenchyme to form large lymph sacs. ▶ [11]

Even though a major part of the lymphatic vasculature develops in a centrifugal manner through lymphangiogenesis (i.e., the growth of new lymphatic vessels from pre-existing capillaries), a dual origin of the lymphatic vasculature has been demonstrated in grafting experiments. ▶ [15] This study revealed that the avian lymphatic vasculature possesses both venous and mesenchymal cell contributions. The unequivocal proof of this dual origin came with recent genetic tracing studies in mice, demonstrating that both venous and nonvenous LECs participate in the development of lymphatic vessels. ▶ [16],​ ▶ [17] LECs were initially observed as isolated cell clusters separate from the sprouting lymphovascular front which subsequently coalesced to form vessels through the process of lymphovasculogenesis. ▶ [17],​ ▶ [18]

After the establishment of the primitive lymphatic network from E14.5 onward, the lymphatic vessels undergo maturation and remodeling to form a hierarchical tree, characterized by lymphatic capillaries, precollectors, and collecting vessels. ▶ [18] Finally, the collecting vessels form lymphatic valves, recruit smooth muscle cells (SMCs), and deposit basement membrane. ▶ [11],​ ▶ [18]

1.2 Anatomy of the Lymphatic System

Hiroo Suami

Italian anatomist Gasparo Aselli is credited with the discovery of the lymphatic system in 1622. ▶ [19] While dissecting live canines, he chanced upon white cords in the mesentery and concluded that these structures were a new anatomic feature related to the absorption of nutrients. To further investigate that new structure, the mercury injection method developed by Anton Nuck was used as the standard technique in the study of cadaver models between the 17th and 20th centuries. ▶ [20] Our current anatomical knowledge about the lymphatic system is largely based on the findings that are generated by using the mercury method. However, this method fell out of favor because of the toxicity of mercury and anatomical studies using adult cadavers eventually ceased. A new, safer technique to identify the lymphatics in a cadaver was developed by Hiroo Suami, using hydrogen peroxide to inflate the lymphatic vessel, after which microscope manipulation was used to inject a contrast medium directly into the vessel via needle to allow radiographic demonstration. ▶ [21] A further modification was the application of indocyanine green (ICG) lymphangiography to map the superficial lymphatic collectors prior to dissection. ▶ [22] These new techniques have allowed us to conduct further anatomical studies of the lymphatics. Precise anatomical knowledge about the normal lymphatics is crucially important because it provides the baseline information required to identify the anatomical changes...