Talk:Cardiovascular System - Lymphatic Development
Rondon-Galeano M, Skoczylas R, Bower NI, Simons C, Gordon E, Francois M, Koltowska K & Hogan BM. (2020). MAFB modulates the maturation of lymphatic vascular networks in mice. Dev. Dyn. , , . PMID: 32525258 DOI.
MAFB Modulates the Maturation of Lymphatic Vascular Networks in Mice
DOI: 10.1002/dvdy.209 Abstract
Background: Lymphatic vessels play key roles in tissue fluid homeostasis, immune cell trafficking and in diverse disease settings. Lymphangiogenesis requires lymphatic endothelial cell (LEC) differentiation, proliferation, migration and co-ordinated network formation, yet the transcriptional regulators underpinning these processes remain to be fully understood. The transcription factor MAFB was recently identified as essential for lymphangiogenesis in zebrafish and in cultured human LECs. MAFB is activated in response to VEGFC-VEGFR3 signalling and acts as a downstream effector. However, it remains unclear if the role of MAFB in lymphatic development is conserved in the mammalian embryo.
Results: We generated a Mafb loss-of-function mouse using CRISPR/Cas9 gene editing. Mafb mutant mice presented with perinatal lethality associated with cyanosis. Mafb mutant lymphatics were normal during initial LEC differentiation and sprouting. However, we identify a role for MAFB in modifying lymphatic network morphogenesis in the developing dermis, as well as developing and postnatal diaphragm. Furthermore, mutant vessels displayed excessive smooth muscle cell coverage, suggestive of a defect in the maturation of lymphatic networks.
Conclusions: This work confirms a conserved role for MAFB in murine lymphatics that is subtle and modulatory and may suggest redundancy in MAF family transcription factors during lymphangiogenesis. This article is protected by copyright. All rights reserved.
Keywords: MAFB; development; lymphangiogenesis; lymphatic; vascular.
Paraxial Mesoderm Is the Major Source of Lymphatic Endothelium
Dev Cell. 2019 May 15. pii: S1534-5807(19)30331-4. doi: 10.1016/j.devcel.2019.04.034. [Epub ahead of print]
Stone OA1, Stainier DYR2.
Endothelial cells (ECs), which line blood and lymphatic vessels, are generally described to come from the lateral plate mesoderm despite experimental evidence for a broader source of origin, including the paraxial mesoderm (PXM). Current dogma suggests that following specification from mesoderm, local environmental cues establish the distinct molecular and functional characteristics of ECs in different vascular beds. Here we present evidence to challenge this view, showing that lymphatic EC fate is imprinted during transition through the PXM lineage. We show that PXM-derived cells form the lymphatic endothelium of multiple organs and tissues, with a more restricted contribution to blood vessel endothelium. By deleting Prox1 specifically in PXM-derived cells, we show that this lineage is indispensable for lymphatic vessel development. Collectively, our data establish lineage history as a critical determinant of EC specialization, a finding with broad implications for our understanding of vascular development and heterogeneity.
Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.
KEYWORDS: cell lineage; endothelial differentiation; endothelial heterogeneity; lineage tracing; lymphangiogenesis; lymphatic; mesoderm; paraxial mesoderm; vasculogenesis PMID: 31130354 DOI: 10.1016/j.devcel.2019.04.034
Key molecules in lymphatic development, function, and identification
Ann Anat. 2018 Sep;219:25-34. doi: 10.1016/j.aanat.2018.05.003. Epub 2018 May 26.
Jha SK1, Rauniyar K1, Jeltsch M2.
While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.
Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.
KEYWORDS: Cell surface receptors; Growth factors; Lymphangiogenesis; Lymphatic marker; Transcription factors; VEGF-C/VEGFR-3 signaling; Vascular biology PMID: 29842991 DOI: 10.1016/j.aanat.2018.05.003
Development of the mammalian lymphatic vasculature
J Clin Invest. 2014 Mar;124(3):888-97. doi: 10.1172/JCI71609. Epub 2014 Mar 3.
Yang Y, Oliver G.
The two vascular systems of our body are the blood and lymphatic vasculature. Our understanding of the cellular and molecular processes controlling the development of the lymphatic vasculature has progressed significantly in the last decade. In mammals, this is a stepwise process that starts in the embryonic veins, where lymphatic EC (LEC) progenitors are initially specified. The differentiation and maturation of these progenitors continues as they bud from the veins to produce scattered primitive lymph sacs, from which most of the lymphatic vasculature is derived. Here, we summarize our current understanding of the key steps leading to the formation of a functional lymphatic vasculature.
Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature
Development. 2013 May;140(9):1857-70. doi: 10.1242/dev.089565.
Koltowska K1, Betterman KL, Harvey NL, Hogan BM.
The lymphatic vascular system develops from the pre-existing blood vasculature of the vertebrate embryo. New insights into lymphatic vascular development have recently been achieved with the use of alternative model systems, new molecular tools, novel imaging technologies and growing interest in the role of lymphatic vessels in human disorders. The signals and cellular mechanisms that facilitate the emergence of lymphatic endothelial cells from veins, guide migration through the embryonic environment, mediate interactions with neighbouring tissues and control vessel maturation are beginning to emerge. Here, we review the most recent advances in lymphatic vascular development, with a major focus on mouse and zebrafish model systems.
Comparative and developmental anatomy of cardiac lymphatics
ScientificWorldJournal. 2014 Jan 27;2014:183170. doi: 10.1155/2014/183170. eCollection 2014.
Ratajska A1, Gula G2, Flaht-Zabost A1, Czarnowska E3, Ciszek B4, Jankowska-Steifer E5, Niderla-Bielinska J5, Radomska-Lesniewska D5.
The role of the cardiac lymphatic system has been recently appreciated since lymphatic disturbances take part in various heart pathologies. This review presents the current knowledge about normal anatomy and structure of lymphatics and their prenatal development for a better understanding of the proper functioning of this system in relation to coronary circulation. Lymphatics of the heart consist of terminal capillaries of various diameters, capillary plexuses that drain continuously subendocardial, myocardial, and subepicardial areas, and draining (collecting) vessels that lead the lymph out of the heart. There are interspecies differences in the distribution of lymphatic capillaries, especially near the valves, as well as differences in the routes and number of draining vessels. In some species, subendocardial areas contain fewer lymphatic capillaries as compared to subepicardial parts of the heart. In all species there is at least one collector vessel draining lymph from the subepicardial plexuses and running along the anterior interventricular septum under the left auricle and further along the pulmonary trunk outside the heart and terminating in the right venous angle. The second collector assumes a different route in various species. In most mammalian species the collectors run along major branches of coronary arteries, have valves and a discontinuous layer of smooth muscle cells.
Major lymphatic precollectors/collectors accompany
- coronary artery branches - human, pig, and dog heart
- branches of cardiac veins (close proximity to major cardiac veins) - mouse and rat hearts.
Lymphatic vessel formation proceeds from the base to the apex (similar to mouse and other mammal hearts)
Mouse heart lymphatics are restricted to the outer zone of the myocardial wall (contrary to those in other mammalian species)
Digging deeper into lymphatic vessel formation in vitro and in vivo
BMC Cell Biol. 2011 Jun 24;12:29.
Detry B, Bruyère F, Erpicum C, Paupert J, Lamaye F, Maillard C, Lenoir B, Foidart JM, Thiry M, Noël A. Source Laboratory of Tumor and Development Biology, Groupe Interdisciplinaire de Génoprotéomique appliqué-Recherche (GIGA-Cancer), University of Liège, B-4000 Liège, Belgium.
BACKGROUND: Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures. RESULTS: In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation. CONCLUSIONS: In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.
The embryonic origins of lymphatic vessels: an historical review
Ribatti D, Crivellato E. Br J Haematol. 2010 Jan 13. [Epub ahead of print]
- "Summary Work on the lymphatic system began in the 17th century, and by the beginning of the 19th century the anatomy of most of the lymphatic system had been described. One of the most important questions in this field has been the determination of the embryological origin of the lymphatic endothelium. Two theories were proposed. The first suggested that lymphatic endothelium derived by sprouting from venous endothelium, the so-called centrifugal theory. The second, the so-called centripetal theory, suggested that lymphatic endothelium differentiates in situ from primitive mesenchyme, and secondarily acquires connection with the vascular system. More recent evidence has provided support for both hypotheses."
Embryonic vascular endothelial cells are malleable to reprogramming via Prox1 to a lymphatic gene signature
BMC Dev Biol. 2010 Jun 28;10:72.
Kim H, Nguyen VP, Petrova TV, Cruz M, Alitalo K, Dumont DJ. Sunnybrook Research Institute University of Toronto 2075 Bayview Avenue Toronto, Ontario M4N 3M5, Canada. email@example.com
BACKGROUND: In vivo studies demonstrate that the Prox1 transcription factor plays a critical role in the development of the early lymphatic system. Upon Prox1 expression, early lymphatic endothelial cells differentiate from the cardinal vein and begin to express lymphatic markers such as VEGFR-3, LYVE-1 and Podoplanin. Subsequent in vitro studies have found that differentiated vascular endothelial cells can be reprogrammed by Prox1 to express a lymphatic gene profile, suggesting that Prox1 can initiate the expression of a unique gene signature during lymphangiogenesis. While the in vitro data suggest that gene reprogramming occurs upon Prox1 expression, it is not clear if this is a direct result of Prox1 in vascular endothelial cells in vivo.
RESULTS: Overexpression of Prox1 in vascular endothelial cells during embryonic development results in the reprogramming of genes to that of a more lymphatic signature. Consequent to this overexpression, embryos suffer from gross edema that results in embryonic lethality at E13.5. Furthermore, hemorrhaging and anemia is apparent along with clear defects in lymph sac development. Alterations in junctional proteins resulting in an increase in vascular permeability upon Prox1 overexpression may contribute to the complications found during embryonic development.
CONCLUSION: We present a novel mouse model that addresses the importance of Prox1 in early embryonic lymphangiogenesis. It is clear that there needs to be a measured pattern of expression of Prox1 during embryonic development. Furthermore, Prox1 reprograms vascular endothelial cells in vivo by creating a molecular signature to that of a lymphatic endothelial cell.
PMID 20584329 http://www.biomedcentral.com/1471-213X/10/72
Tbx1 regulates Vegfr3 and is required for lymphatic vessel development
J Cell Biol. 2010 May 3;189(3):417-24.
Chen L, Mupo A, Huynh T, Cioffi S, Woods M, Jin C, McKeehan W, Thompson-Snipes L, Baldini A, Illingworth E. Program of Cardiovascular Sciences, Baylor College of Medicine, Houston, TX 77030, USA.
Lymphatic dysfunction causes several human diseases, and tumor lymphangiogenesis is implicated in cancer spreading. TBX1 is the major gene for DiGeorge syndrome, which is associated with multiple congenital anomalies. Mutation of Tbx1 in mice recapitulates the human disease phenotype. In this study, we use molecular, cellular, and genetic approaches to show, unexpectedly, that Tbx1 plays a critical role in lymphatic vessel development and regulates the expression of Vegfr3, a gene that is essential for lymphangiogenesis. Tbx1 activates Vegfr3 transcription in endothelial cells (ECs) by binding to an enhancer element in the Vegfr3 gene. Conditional deletion of Tbx1 in ECs causes widespread lymphangiogenesis defects in mouse embryos and perinatal death. Using the mesentery as a model tissue, we show that Tbx1 is not required for lymphatic EC differentiation; rather, it is required for the growth and maintenance of lymphatic vessels. Our findings reveal a novel pathway for the development of the lymphatic vessel network.
Int J Dev Biol. 2010;54(2-3):421-30. doi: 10.1387/ijdb.082800et.
Taglauer ES, Adams Waldorf KM, Petroff MG. Source Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA.
The genetic disparity between the mother and fetus has long enticed immunologists to search for mechanisms of maternal tolerance to fetal antigens. The study of antigen-specific tolerance in murine and human pregnancy has gained new momentum in recent years through the focus on antigen-presenting cells, uterine lymphatics and fetal antigen-specific maternal T cell responses. In mice, we now know that these responses occur within the secondary lymphoid structures as they can be conveniently tracked through the use of defined, often transgenic fetal antigens and maternal T cell receptors. Although the secondary lymphoid organs are sites of both immunization and tolerization to antigens, the immunological processes that occur in response to fetal antigens during the healthy pregnancy must invariably lead to tolerance. The molecular properties of these maternal-fetal tolerogenic interactions are still being unraveled, and are likely to be greatly influenced by tissue-specific microenvironments and the hormonal milieu of pregnancy. In this article, we discuss the events leading to antigen-specific maternal tolerance, including the trafficking of fetal antigens to secondary lymphoid organs, the properties of the antigen-presenting cells that display them to maternal T lymphocytes, and the nature of the ensuing tolerogenic response. Experimental data generated from human biological specimens as well as murine transgenic models are considered. PMID 19876825
- lymphoid tissue inducer cells - (LTi cells) are the first hematopoietic cells to enter the primordial secondary lymphoid and induce lymphoid tissue neogenesis. PMID 19403040
Butler MG, Isogai S, Weinstein BM. Birth Defects Res C Embryo Today. 2009 Sep;87(3):222-31. Review.
- "The lymphatic system is essential for fluid homeostasis, immune responses, and fat absorption, and is involved in many pathological processes, including tumor metastasis and lymphedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by lack of defining molecular markers and difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. Recent identification of new molecular markers, new genes with important functional roles in lymphatic development, and new experimental models for studying lymphangiogenesis has begun to yield important insights into the emergence and assembly of this important tissue. This review focuses on the mechanisms regulating development of the lymphatic vasculature during embryogenesis."
Lymph node lymphangiogenesis: a new concept for modulating tumor metastasis and inflammatory process
Ji RC. Histol Histopathol. 2009 Mar;24(3):377-84. Review. PMID 19130407
Organization and developmental aspects of lymphatic vessels
Arch Histol Cytol. 2008 May;71(1):1-22.
Ohtani O, Ohtani Y. Source Department of Anatomy, Faculty of Medicine and Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. firstname.lastname@example.org Abstract The lymphatic system plays important roles in maintaining tissue fluid homeostasis, immune surveillance of the body, and the taking up dietary fat and fat-soluble vitamins A, D, E and K. The lymphatic system is involved in many pathological conditions, including lymphedema, inflammatory diseases, and tumor dissemination. A clear understanding of the organization of the lymphatic vessels in normal conditions would be critically important to develop new treatments for diseases involving the lymphatic vascular system. Therefore, the present paper reviews the organization of the lymphatic vascular system of a variety of organs, including the thyroid gland, lung and pleura, small intestine, cecum and colon in the rat, the diaphragm in the rat, monkey, and human, Peyer's patches and the appendix in the rabbit, and human tonsils. Methods employed include scanning electron microscopy of lymphatic corrosion casts and tissues with or without treatment of alkali maceration technique, transmission electron microscopy of intact tissues, confocal microscopy in conjunction with immunohistochemistry to some lymphatic-specific markers (i.e., LYVE-1 and VEGFR-3), and light microscopy in conjunction with enzyme-histochemistry to 5'-nucleotidase. Some developmental aspects of the lymphatic vessels and lymphedema are also discussed.
Observations on the prenatal development of human lymphatic vessels with focus on basic structural elements of lymph flow
Lymphat Res Biol. 2008;6(2):89-95.
Petrenko VM, Gashev AA. Department of Human Anatomy, St. Petersburg State Medical Academy, St. Petersburg, Russia.
BACKGROUND: The prenatal development of human lymphatic systems has not attracted enough attention by lymphatic researchers in the past. Yet clearly these critical, early events determine the fate and function of the human lymphatic system.
METHODS AND RESULTS: The main focus of these studies was to investigate the embryonic development of human lymphangions including lymphatic valves and muscle cells, to better understand the prenatal formation of basic structural elements of lymph flow. This review in most of its parts is a short summary of the findings. It provides important information necessary for understanding the development and functioning of the human lymphatic system.
CONCLUSIONS: The structural basis of the active lymph transport system--the lymphatic muscle cells and lymphatic valves--which is absolutely necessary for all functions of lymphatic system, is already formed during the first half of the prenatal development in humans. During the second half of this development maturation of this system is already underway. The enlargement of lymphatic muscle cells together with increases in their quantity leads to formation of the multi-layered lymphatic vessel wall, able to develop contractions strong enough to propel lymph downstream of the lymphatic channels against gravity in bipedal humans. The development of the competent valves in lymphatic vessels occurs at the same time creating the ground for effective net, unidirectional lymph flow. The data summarized here represents some of the first systematic studies of the prenatal development of lymphatic muscle cells and valves in humans.
Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature
Genes Dev. 2007 Oct 1;21(19):2422-32.
Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, Samokhvalov IM, Oliver G. Department of Genetics and Tumor Cell Biology, St. Jude Children's Hospital, Memphis, Tennessee 38105, USA.
Comment in: Lymphat Res Biol. 2007;5(4):275-6.
The origin of the mammalian lymphatic vasculature has been debated for more than 100 years. Whether lymphatic endothelial cells have a single or dual, venous or mesenchymal origin remains controversial. To resolve this debate, we performed Cre/loxP-based lineage-tracing studies using mouse strains expressing Cre recombinase under the control of the Tie2, Runx1, or Prox1 promoter elements. These studies, together with the analysis of Runx1-mutant embryos lacking definitive hematopoiesis, conclusively determined that from venous-derived lymph sacs, lymphatic endothelial cells sprouted, proliferated, and migrated to give rise to the entire lymphatic vasculature, and that hematopoietic cells did not contribute to the developing lymph sacs. We conclude that the mammalian lymphatic system has a solely venous origin. PMID 17908929
Keystones in lymph node development
J Anat. 2006 Nov;209(5):585-95.
Blum KS, Pabst R. Department of Functional and Applied Anatomy, Hannover Medical School, Germany. email@example.com
New molecular markers are constantly increasing our knowledge of developmental processes. In this review article we have attempted to summarize the keystones of lymphoid tissue development in embryonic and pathological conditions. During embryonic lymph node development in the mouse, cells from the anterior cardinal vein start to bud and sprout, forming a lymph sac at defined sites. The protrusion of mesenchymal tissue into the lymph sacs forms the environment, where so-called 'lymphoid tissue inducer cells' and 'mesenchymal organizer cells' meet and interact. Defects of molecules involved in the recruitment and signalling cascades of these cells lead to primary immunodeficiency diseases. A comparison of molecules involved in the development of secondary lymphoid organs and tertiary lymphoid organs, e.g. in autoimmune diseases, shows that the same molecules are involved in both processes. This has led to the hypothesis that the development of tertiary lymphoid organs is a recapitulation of embryonic lymphoid tissue development at ectopic sites.
Organogenesis of lymphoid tissues
Nat Rev Immunol. 2003 Apr;3(4):292-303.
Mebius RE. Source Department of Molecular Cell Biology, VU University Medical Center, v.d. Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands. firstname.lastname@example.org Erratum in Nat Rev Immunol. 2003 Jun;3(6):509. Abstract The development of lymphoid organs depends on the correct expression of several molecules within a defined timeframe during ontogeny. Although this is an extremely complex process, with each secondary lymphoid tissue requiring subtly different signals, a common framework for lymphoid development is beginning to emerge. Drawing on studies of lymph nodes, Peyer's patches and nasal-associated lymphoid tissue, an integrative model of lymphoid-tissue development, involving adhesion molecules, cytokines and chemokines, which emphasizes the role of interactions between CD3-CD4+CD45+ 'inducer' cells and VCAM1+ICAM1+ stromal 'organizer' cells is presented. PMID 12669020
The popliteal lymph node of the mouse: internal architecture, vascular distribution and lymphatic supply
J Anat. 1986 Oct;148:25-46.
Kowala MC, Schoefl GI. Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra, ACT.
The architecture of the mouse popliteal lymph nodes differs from that shown in conventional diagrams. The cortical lymphoid tissue, rather than forming a continuous outer layer, is organised into one or two hemispherical aggregates which project towards the hilus. These aggregates are surrounded by medullary tissue which thus extends to large areas of the surface of the node. The vascular distribution in the lymphoid aggregates is relatively sparse and contrasts with the dense meshwork of capillaries and venules around them. It also contrasts with the high vascularity of medullary tissue. Arterial vessels, especially those of larger calibre, are predominantly seen in the hilar area of the node suggesting that there is extensive branching as the artery enters the node. Capillaries associated with the lymphoid aggregates are usually lined by continuous endothelium, while those in the medulla are generally of the fenestrated type. The microcirculation has an extensive venous capacity and many venous segments are high endothelium venules whose walls are permeated by lymphocytes. Each node receives one or two afferent lymphatic vessels and is drained by up to four or five efferent lymphatic vessels. In approximately half the nodes examined, there were extranodal communications between afferent and efferent lymphatic vessels allowing some lymph to bypass the node. PMID 3693091
On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig
Sabin, F. R. (1902).
Am. J. Anat. 1, 367-389.
The lymphatic system in human embryos, with a consideration of the morphology of the system as a whole
Sabin, F. R. (1909).
Am. J. Anat. 9, 43-91.