J Appl Physiol 115: 297–308, 2013.
First published May 2, 2013; doi:10.1152/japplphysiol.00201.2013.
Synthesis
Lymphatic regulation in nonmammalian vertebrates
Michael S. Hedrick,1 Stanley S. Hillman,2 Robert C. Drewes,3 and Philip C. Withers4
1
Developmental Integrative Biology Cluster, Department of Biological Sciences, University of North Texas, Denton, Texas;
Department of Biology, Portland State University, Portland, Oregon; 3Department of Herpetology, California Academy
of Sciences, San Francisco, California; and 4School of Animal Biology, University of Western Australia, Crawley,
Western Australia
2
Submitted 13 February 2013; accepted in final form 26 April 2013
birds; comparative physiology; ectothermic vertebrates; lymph; plasma volume
THE LYMPHATIC SYSTEM OF VERTEBRATES is generally defined from
a mammalian perspective as a vessel system draining the
interstitial space into the venous circuit but with no connection
to arterial circuit of the cardiovascular system. It transports a
fraction of the interstitial fluid, known as lymph, that originates
from the net filtration of plasma across the capillaries of the
cardiovascular system. This definition incorporates both anatomical and functional features of the mammalian lymphatic
system. In nonmammalian vertebrates, however, there is considerable variation in lymphatic structures, thus lymphatics is
best defined from a functional perspective. The lymphatic
vasculature of mammals has several essential physiological
functions, including the maintenance of fluid balance between
the plasma and interstitial compartments of the extracellular
space by returning protein and fluid filtered out of the capillaries to the vascular system, involvement in immune defense,
and the distribution of diet-derived fat (see Refs. 2, 3, 35, 62,
69). However, the lymphatics have historically been given
short shrift as an organ system. In the general medical school
Address for reprint requests and other correspondence: M. S. Hedrick, Dept.
of Biological Sciences, Univ. of North Texas, Denton, TX 76203 (e-mail:
[email protected]).
http://www.jappl.org
curriculum, it is the system that frequently is ignored or
perhaps mentioned only in passing (20, 96, 115). Why this
should be the case is unclear given the extremely important
role that the lymphatic system plays in fluid volume homeostasis. This attitude has changed recently, with an ever-increasing number of publications and journals devoted to lymphatics,
reflecting the obvious importance of this system when something goes wrong. Genetically aberrant or damaged lymphatic
vessels lead to lymphedema (excess swelling of the extremities). Lymphatic damage is a common postsurgical problem
with limited therapeutic solutions. Approximately 120 million
people worldwide currently suffer from lymphatic filariasis
(extreme lymphedema, commonly known as “elephantiasis”)
caused by a filarial worm that inhabits the lymphatic vessels
and prevents lymph return to the circulation by narrowing the
diameter of lymph vessels (53, 90). In addition, lymphatic
vessels promote metastatic spread of cancer cells to distant
organs—a leading cause of death in patients with cancer and a
major obstacle in the design of effective therapies (20).
The field of mammalian lymphatics has recently seen tremendous strides in our understanding of the molecular control
of lymph development, modeling of lymph function, regulation
of lymphatic pumping, immune function, and metastasis (2, 3,
8750-7587/13 Copyright © 2013 the American Physiological Society
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Hedrick MS, Hillman SS, Drewes RC, Withers PC. Lymphatic regulation in
nonmammalian vertebrates. J Appl Physiol 115: 297–308, 2013. First published
May 2, 2013; doi:10.1152/japplphysiol.00201.2013.—All vertebrate animals share
in common the production of lymph through net capillary filtration from their
closed circulatory system into their tissues. The balance of forces responsible for
net capillary filtration and lymph formation is described by the Starling equation,
but additional factors such as vascular and interstitial compliance, which vary
markedly among vertebrates, also have a significant impact on rates of lymph
formation. Why vertebrates show extreme variability in rates of lymph formation
and how nonmammalian vertebrates maintain plasma volume homeostasis is
unclear. This gap hampers our understanding of the evolution of the lymphatic
system and its interaction with the cardiovascular system. The evolutionary origin
of the vertebrate lymphatic system is not clear, but recent advances suggest
common developmental factors for lymphangiogenesis in teleost fishes, amphibians, and mammals with some significant changes in the water-land transition. The
lymphatic system of anuran amphibians is characterized by large lymphatic sacs
and two pairs of lymph hearts that return lymph into the venous circulation but no
lymph vessels per se. The lymphatic systems of reptiles and some birds have lymph
hearts, and both groups have extensive lymph vessels, but their functional role in
both lymph movement and plasma volume homeostasis is almost completely
unknown. The purpose of this review is to present an evolutionary perspective in
how different vertebrates have solved the common problem of the inevitable
formation of lymph from their closed circulatory systems and to point out the many
gaps in our knowledge of this evolutionary progression.
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THE PHYSIOLOGICAL ROLE OF THE LYMPHATIC SYSTEM
IN PLASMA VOLUME REGULATION
The lymphatic system has many different functions, but
paramount among these is its role in removing accumulating
protein and fluid from the interstitial space and returning it to
the vascular space. The net rate of filtration of fluid (plasma)
from the vascular to the interstitial space, the transvascular
fluid flux that occurs across the capillary wall is described by
the well-known Starling fluid flux equation
J ⫽ Fcap [(Pcap ⫺ Pist) ⫺ ␴ (⌸cap ⫺ ⌸ist)]
(1)
where J is net transvascular fluid flux (ml·min⫺1·kg⫺1), Fcap is
filtration coefficient of the capillaries (ml·min⫺1·kg⫺1·kPa⫺1),
Extracellular Fluid Volume Regulation
Interstitial Volume
Interstitial Fluid
Lymphatic Fluid
Transcapillary
Uptake
Drinking
Lymphatic
Return
Plasma Volume
Interstitial Volume
Hemorrhage
Fig. 1. Pathways for interstitial fluid addition (black arrows) and loss from
(gray arrows) the plasma volume; drinking additions to and hemorrhagic loss
from the plasma volume are also included.
Hedrick MS et al.
Pcap is mean capillary hydrostatic pressure (kPa), Pist is interstitial
hydrostatic pressure (kPa), ␴ is reflection coefficient for plasma
proteins, ⌸cap is colloid osmotic pressure of the plasma (kPa), and
⌸ist is colloid osmotic pressure of the interstitial fluid (kPa).
Normally, the balance of the transcapillary hydrostatic
forces favors efflux of fluid from the capillary, whereas balance
of the colloid forces favors influx. On average, the net hydrostatic force dominates and there is a consequential net efflux of
fluid from the vasculature into the mammalian interstitium;
⬃15% filtered out of the capillaries remains in the interstitial
space, and 85% is returned to the capillaries (38). This interstitial fluid, which is filtered from the capillaries, is returned as
lymph to the circulation via the lymphatic system (Fig. 1). It is
important to recognize that the lymph and interstitial fluid are
chemically identical, and it is essentially impossible to quantify
the relative volume fraction of the interstitial volume that
represents lymph volume. The fluid that comprises lymph is
present in both the interstitial space and lymph vessels, and
some fluid volume is always present. The fraction of this fluid
volume returned is quantified as the rate of fluid added to the
plasma volume from the lymphatic system. Consequently,
lymphatic return is generally characterized as a fluid volume
flux rather than a volume. If fluid is added to the plasma space,
then Pcap will increase and ⌸cap will decrease; both changes
lead to greater efflux and consequently a return toward the
original plasma volume. If fluid is lost from the plasma space,
then Pcap will decrease and ⌸cap will increase, both changes
favoring increased absorption of fluid from the interstitium and
a return toward the original plasma volume. Consequently,
there is an inherent mechanism in Starling’s equilibrium that
promotes homeostatic redistribution of blood and interstitial
fluid independent of a regulated negative feedback loop. Because Fcap and ␴ vary in different organs (108), the use of a
capillary-focused equation to describe blood volume regulation
would require an integration of both the blood flow and
Starling variables for every organ in the body, which is an
impractical approach. Furthermore, the relationship between
the plasma filtration and both Pcap and Pist is also determined
by both the vascular compliance and the interstitial compliance
of different organs (107). For instance, organs encased by
inelastic connective tissue or bone show large increases in Pist
in response to any filtration (108), which would immediately
prevent further hydrostatic filtration. A low Cist and high
reflection coefficient may explain why lymphatic vessels are
absent in specific tissue compartments of mammals, including
the brain, spinal cord, retina, cartilage, and bone (20). Consequently, the Starling fluid flux equation is an excellent model
for describing fluid fluxes across an individual capillary in a
particular organ, but it has limited capacity for interpreting
transcapillary fluid redistribution at an organismal level.
A more useful model for understanding the short-term effects of volume loading and hemorrhage on plasma volume in
mammals is a derivation of the Starling fluid flux equation that
emphasizes whole body compliance and its interaction with
fluid volume and pressure (107). The pressure in a fluid space
is determined by the volume of fluid and the elastic compliance
(capacitance) of the space (i.e., P ⫽ V/C). We can incorporate
the compliances of the vascular and interstitial spaces and a
whole body filtration coefficient to examine the kinetics of
fluid flux (J) between the vascular and interstitial spaces, by the
following equation:
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78, 86, 106). Although the importance of the lymphatic system
in mammals for maintenance of cardiovascular homeostasis in
health and disease is clear, much less is known about the
comparative biology of the lymphatic system. Because the
lymphatic system of mammals evolved from that of nonmammalian vertebrates, it is instructive to trace the evolutionary
history of this system with respect to its functional role in
plasma volume homeostasis.
The goal of this review is to introduce readers to lymphatic
function in nonmammalian vertebrates, especially the role that
the lymphatic system plays in the maintenance of plasma
volume (Fig. 1). We suggest that studying lymphatic function in
nonmammalian vertebrates will not only provide insight into the
evolution of the lymphatic system but may also provide alternative models for studying plasma volume regulation. Although
there are some very different types of lymphatic regulatory mechanisms in nonmammalian vertebrates, we believe that the general
concepts of plasma volume homeostasis, transvascular fluid flux,
and vascular and interstitial compliance are common to all vertebrates, and thus a discussion of the various modes of lymph flux
regulation is important for understanding the diversity of vascular
fluid regulatory capacities in vertebrates. Burggren et al. (14) have
reviewed the structure, function, and evolution of the vertebrate
circulatory system. The reader is also referred to Kampmeier’s
(67) excellent monograph on the evolution and morphology of the
lymphatic system.
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J ⫽ FC (Pvas ⫺ Pist) ⫽ FC [(Vvas ⁄ Cvas) ⫺ (Vist ⁄ Cist)] (2)
PLASMA TURNOVER AND LYMPH FORMATION
Measurement of the turnover rate of labeled plasma proteins
(Fig. 2) indicate that the net rates of fluid exchange between the
circulation and interstitium vary dramatically among vertebrates. Plasma turnover values are very high for anurans, 3–5%
of plasma volume per minute in bullfrogs (Lithobates catesbeianus ⫽ Rana catesbeiana) and cane toads (Rhinella marina ⫽
Bufo marinus) (52), compared with 0.9% per minute for fish
(82.101) and 0.03– 0.1% per minute for mammals (5, 8).
Fig. 2. Comparison of plasma turnover in a variety of vertebrate species.
Human data are from Bianchi et al. (8), fish (trout) data are from Nichols (82),
and bullfrog (R. catesbeiana) and cane toad (B. marinus) data are from
Hillman et al. (52). Note that anuran plasma turnover is significantly faster than
trout despite similar capillary filtration coefficients (see text). [Figure reproduced from Hillman et al. (47) with permission from the University of Chicago
Press.]
Hedrick MS et al.
299
There are two principal reasons for the higher plasma turnover of anurans compared with fishes and mammals: 1) a much
higher interstitial compliance (higher Cist) and 2) a “leakier”
vasculature (lower ␴). The whole body FC determined for
amphibian vascular systems (25 ml·min⫺1·kg⫺1·kPa⫺1; 41, 46)
is ⬃3- to 6-fold higher than for mammals (5, 107) but is lower
than for trout (88). Interstitial compliance is more than an order
of magnitude higher for anurans (1.5–3 ml·kg ⫺1·Pa⫺1; 52)
than for either the fish (88) or mammals (107), reflecting the
low resistance pathway for fluid flux into an anuran’s interstitial space. Fish data (Fig. 2) present an interesting example of
the interplay between FC and Cist. Trout have a high capillary
permeability (42) and a higher FC (37 ml·min⫺1·kg⫺1·kPa⫺1;
88) than anurans that would predict a higher plasma turnover
than anurans, but trout plasma turnover is actually lower. This
is because trout have a much lower Cist (0.09 ml·kg⫺1·Pa⫺1;
88) than anurans, which prevents fluid loss from the circulatory
system because Pist increases dramatically with any filtration,
even with a higher FC. The high Cist of anurans favors storage
of extracellular fluid volume in the interstitium without increasing Pist or Pcap, but entails the penalties of high filtrational
losses and plasma turnover and requires a substantial nonStarling mechanism (lymphatic return) for fluid uptake. It is
important to recognize that anurans are unique relative to the
few other vertebrate groups that have been studied in having
such a high Cist, and storage of lymph may be a specialized
adaptation of this highly derived vertebrate lineage for handling the blood volume stress of dehydration in a terrestrial
environment.
One way to assess how variation in the factors involved in
transvascular flux and lymph formation affect transvascular
fluid flux is to perform a sensitivity analysis based on Tanaka’s
(107) equation (Eq. 2) to determine which factors have the
greatest contribution to filtration. This analysis (Fig. 3) reveals
a very different pattern for anurans compared with either fish or
mammals, largely as a consequence of their high Cist. Anurans
have a net efflux from the vascular to the interstitial space
under all modeled conditions, regardless of how each variable
changes (Fig. 3A). This indicates that transcapillary uptake of
interstitial fluid based on Starling balance is impossible and
points out the enormous importance of their lymphatic system
in returning interstitial fluid to the circulatory system. This
conclusion is consistent with the finding that lymph heart
ablation in toads causes hemoconcentration and death within a
few days (124) and an inability to compensate for hemorrhage
(7). Reducing Cvas and Vb have the biggest effects (increase
and decrease, respectively) on fluid loss from capillaries.
Changes in Vist and Cist have relatively small effects, reflecting
their initially high values. The pattern for trout under normal
conditions indicates zero flux with no net driving force (100%;
Fig. 3B). Thus any variation in FC will have no effect on flux
as there is no net driving force, but changing the other parameters in this analysis increases or decreases flux rates between
the vasculature and intersititium. Reductions in Cvas or Vist
increase flux to the interstitium whereas reductions in Vb or Cist
increase flux from the interstitium to the vasculature. For
example, during exercise in fish, increased arterial pressure
(hence Vb) drives fluid out of the capillaries, owing to the high
FC, resulting in hemoconcentration and increased hematocrit
(70, 104, 105). The direction of fluid flux is determined by the
relative difference in pressure between the vasculature and
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where FC is the whole body filtration coefficient (ml·kg⫺1·
kPa⫺1·min⫺1), Cvas is the vascular compliance (ml·kg⫺1·kPa⫺1),
Cist is the whole body interstitial compliance (ml·kg⫺1·kPa⫺1),
Vvas is the vascular blood volume (ml/kg), and Vist is the
interstitial fluid volume (ml/kg).
Starling models emphasizing a transcapillary mechanism of
fluid flux are well appreciated and appear to adequately explain
plasma volume maintenance in mammals (Fig. 1; Ref. 95).
Mammals, when dehydrationally or hyperthermally challenged,
decrease the efflux of plasma protein by increasing lymphatic flow
(resulting in increased plasma protein concentration and decreased
interstitial protein concentration) and, in some species by decreasing the filtration coefficient (11, 55– 60, 98, 99). This
combination of effects increases the driving force for transcapillary uptake. Compliance models are generally not necessary
to invoke for mammals because plasma turns over slowly,
hence lymphatic flux is a relatively small fraction of the total
plasma volume (see below) and can be ignored in normal
physiological states. This partially explains why the lymphatic
system is poorly popularized as a mechanism for plasma
volume maintenance in mammals. However, pathological situations where lymphatic flux is compromised, or compliance
changes, are a different story. As we point out below, such
circumstances of high capillary filtration rates and overall
plasma protein turnover are in the normal circumstance for
nonmammalian vertebrates, and application of the modified
compliance form of the Starling equation as developed by
Tanaka (107) is usually more appropriate.
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10.0
5.0
2.5
Vist
Cist
Fc
Vb
LYMPHATIC FUNCTION IN FISHES
0.0
50
75
gain
loss
50
Vist
0
Fc
-25
Vb
-50
Cist
150
trout
Cvas
25
100
-75
50
150
dog
C
20
loss
Edema
0
Control
gain
Transcapillary Flux
(ml kg -1 min-1)
100
-20
-40
Cvas
Fc
Vist
Vb
Cist
-60
50
100
150
% of Mean Value
Fig. 3. Sensitivity analysis of the effect of individually increasing and decreasing each variable in Tanaka’s (107) analysis by 50% (Eq. 2 in text) on plasma
flux. Each point is a mean value calculated from Eq. 2. Positive flux values
indicate loss of plasma from the vascular space to the interstitium, and negative
values indicate gain of fluid from the intersitium to the vascular space.
Cardiovascular variables are the whole body filtration coefficient (Fc), blood
volume (Vb), interstitial volume (Vist), vascular compliance (Cvas) and interstitial compliance (Cist). [Figure modified from Hillman et al. (52) with
permission from the University of Chicago Press.]
interstitial spaces, and the fluxes in either direction are very
high owing to the very high FC. Therefore, just as for anurans,
Starling equilibrium driving transcapillary uptake appears to
play little role in determining transvascular fluid flux in trout
(15, 88). The normal mammalian pattern (Fig. 3C) is a net fluid
uptake with a driving force of about ⫺3.2 kPa, which is
approximately the value predicted from vascular and interstitial
colloid osmotic differences. Lowering Cist has the greatest
effect on net fluid uptake, illustrating its importance in determining transvascular flux. Decreases in Vist, FC, and Cvas
reduce the inward flux of fluid. Increased Cist and decreased
Vist with continuous angiotensin II infusion in dogs (110) shift
transvascular fluid flux to an outward flux, which is very
similar to the normal situation for the toad. Interstitial fluid
pressure in mammals is normally negative (37, 39, 40) and
represents a significant “safety factor” for preventing edema
(37) in conjunction with the variable capacity of the lymphatic
system to return fluid. However, once fluid loss from the vascular
The roles of, and indeed the very existence of, a lymphatic
system in fishes are controversial (87, 111–113). Until about
1980, it was thought that the lymphatic system of fish was
similar in structure and function to that of mammals, but early
descriptions of “lymphatic” vessels in fish may have been, in
fact, descriptions of a secondary circulatory system (67, 103,
111, 117). Vogel (111, 113) first described the secondary
circulatory system of teleost fishes and argued that this secondary circulation is not a lymphatic system owing to a direct
connection (i.e., interarterial anastomoses) between the primary and secondary vasculature (112). This mammalian definition of lymphatics has confused the issue of lymphatics in
teleost fishes that we describe below.
There are two fundamental questions to be addressed with
regard to lymphatic regulation in fishes: 1) do fishes have a
lymphatic system? and 2) is their secondary circulatory system
part of the lymphatic system? In our view, the answer to the
first question, based upon several recent studies with zebrafish
outlined below, is that teleost fishes do have a lymphatic
system that shares many molecular and morphological features
with the mammalian lymphatic system. With regard to the
second question, it is still unclear whether the secondary
circulation is connected to, or a component of, the lymphatic
system. More studies are needed to resolve this question.
There is growing use and acceptance of zebrafish as a model
for lymphatic system development in vertebrates, stressing the
potential importance of comparative vertebrate model systems
for studying lymphatic function. This is based on several
studies showing that highly specific molecular markers for
lymphangiogensis in mammals are also expressed in zebrafish
vessels that are clearly not primary circulatory vessels (20, 61).
These vessels have been interpreted as lymphatic in nature,
based on molecular expression data, but whether they are also
part of the secondary circulation is unclear (see below). The
thoracic duct was the first lymphatic vessel identified in zebrafish (71, 122), and several other lymphatic vessels have
been subsequently identified in various regions, including the
head and gastrointestinal tract (21). During development, a
population of lymphatic endothelial cell (LEC) precursors
sprout from the cardinal veins and form embryonic lymph sacs,
as occurs in mammals. These LEC precursors are marked by
the expression of key transcriptional regulators that specify
LEC identity (35). The transcription factor Prox1, a mammalian homolog of the Drosophila protein Prospero, is confined to
LEC nuclei in both zebrafish and mammals (100). Targeted
inactivation of Prox1 completely arrests lymphatic vasculature
development without affecting angiogenesis (118), and prox1b
knockdown inhibits formation of the thoracic duct in zebrafish
(29). Knockdown of delta-like-4 (DII4) or its receptors
Notch-1b or Notch-6 in zebrafish also impair lymphangiogenesis, including formation of the thoracic duct (36). Vascular
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Transcapillary Flux
(ml kg -1 min-1)
B
Hedrick MS et al.
to the interstitial space reaches or exceeds atmospheric pressure,
interstitial compliance increases dramatically (39). Changing vascular and interstitial variables do not markedly affect the net
outward flux during this edematous state (Fig. 2), which is consistent with the difficulty in correcting excess interstitial fluid retention during situations that produce edema, such as lymphedema or
congestive heart failure (37, 39).
cane toad
Cvas
7.5
loss
Transcapillary Flux
(ml kg-1 min-1)
A
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has direct connections with both the arterial and venous vasculature of the cardiovascular system. It has been argued that
the secondary circulation cannot be lymphatic because it does
not fit the mammalian definition of a lymphatic system (112).
Cyclostome and elasmobranch fishes do not appear to have a
secondary circulation, but they do have some vessels that have
been described as “venolymphatic,” with no clear distinction
between the lymphatic and venous system (103). The fluid
contained within the secondary circulation of teleosts appears
to be composed of plasma skimmed from the primary circulation and contains very few red blood cells (RBCs) (103, 111,
113). The function of the secondary circulation is unknown,
but it has been suggested to be a primitive lymphatic system
(67) or have nutritive or immune sensing functions (87, 94).
Given the location of the secondary circulation in regions
where injuries might occur, an immune function for the secondary circulation seems a likely possibility (94) but this
hypothesis has not been tested. The near absence of RBCs in
the fluid of the secondary circulation would rule out a significant role in oxygen transport; however, a study with zebrafish
and glass catfish found that hypoxia promoted nitric oxideregulated perfusion of the interarterial anastomoses (63). These
anastomoses were termed “arterial-lymphatic conduits” because the hypoxia-induced perfusion of interarterial conduits
allowed RBCs to enter the lymphatic vessels (which expressed
Prox-1), indicating there are direct connections between the
primary arterial system and the lymphatic system of zebrafish
(63) if the secondary circulation is considered lymphatic in
function. This experimental evidence is important because it
establishes a direct connection between the primary circulation
and the presumptive lymphatic system (Fig. 4). The direct
connection may also be important as a potential driving force
to propel lymph through the lymphatic system and back to the
primary circulation. The small anastomoses would siphon
some of the pressure and use the energy generated from cardiac
contraction to create a pressure head to propel the lymph. In the
absence of lymphatic pumps (see below) this arrangement may
have been necessary and possibly represents the original mechanism for lymphatic flow and its return to the circulatory
system. Given the current evidence, our view is that the
secondary circulation in teleost fish likely represents a “primitive” lymphatic system. Whether these secondary vessels are
identical to the lymphatic vessels described for zebrafish is
unclear, and more studies are needed to ascertain the connection, if any, between the lymphatic system and the secondary
circulation in teleosts.
LYMPHATIC FUNCTION IN LUNGFISHES AND THE
WATER-LAND TRANSITION
Lungfishes (Dipnoi) occupy an important evolutionary position as the sister group of modern tetrapods. This group
appears to have the first recognizable tetrapod-like lymphatic
system among the vertebrates. The lymphatic system of Dipnoi
is characterized by the presence of lymphatic capillaries and a
large number of lymphatic micropumps located throughout the
body, except for the central nervous system (114). There are
high concentrations of lymphatic micropumps in the fin regions, up to 100 mm⫺3 in the South American lungfish (Lepidosiren paradoxa). The lymphatic micropumps are fed by
thin-walled afferent lymphatic vessels that have no direct
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endothelial growth factor C (VEGFC), which is critical for
lymphangiogenesis in mammals (68), is also necessary for
development of the lymphatic system in zebrafish (71). The
expression of Prox1 and neuropilin-2 (Nrp-2), which play a
role in the development of small lymphatic vessels in mammals
(123), were found in presumptive lymphatic vessels in zebrafish (122). However, a more recent study with transgenic
zebrafish expressing the lymphatic endothelial hyaluronan receptor 1 (LYVE1) promotor, identified previously uncharacterized lymphatic vessels in the head, intestine, and superficial
areas of the lateral trunk (85). LYVE1 is one of the most
specific and widely used mammalian lymphatic endothelial
markers (106). The general consensus from these studies is that
molecular pathways controlling lymphangiogenesis are highly
conserved from zebrafish to mammals.
In addition to molecular evidence for lymphatic vessel
structure, functional studies also point to these zebrafish vessels as being lymphatic. For example, subcutaneous injection
of fluorescin dextran or rhodamine dextran into the trunk
muscle of zebrafish resulted in dye taken up by the putative
lymphatic vessels, which accumulated in the venous circulation as would be expected of lymphatic vessels (71). A genetic
screen identified an edematous zebrafish mutant, full of fluid,
which lacked previously identified lymphatic vessels. including the thoracic duct, intersegmental lymphatic vessels. and
dorsal longitudinal lymphatic vessels, but retained normal
vasculature (54). Mutants developed severe edema in the lower
intestine and around the eyes, suggesting the presence of
lymphatic vessels in these regions. The mutation was localized
to a single gene, ccbe1 (collagen and calcium binding EGF
domain 1), which involved a single amino acid substitution.
Interestingly, this finding in zebrafish led to the discovery of a
link between the ccbe1 mutation and lymphatic hypoplasia in
a human disorder (1). Knockdown of prox1b, which prevents
thoracic duct formation, also results in an edematous phenotype (29). Morpholino-mediated knockdown of Prox1 and
VEGF-C in Xenopus tadpoles also produces lymph vessel
defects and lymphedema (83), indicating a similar genetic
program for lymphangiogenesis in zebrafish and anurans. Finally, rapamycin, a specific inhibitor of the mammalian target
of rapamycin (mTOR) that exhibits antilymphangiogenic properties in mammals, suppresses rostral and trunk lymphangiogenesis without affecting blood vessels in zebrafish (34).
Taken together, the morphological and functional studies
strongly suggest that zebrafish, and presumably other teleosts,
have a lymphatic system that presumably functions similarly to
that of other vertebrates.
The “secondary vascular system” of teleost fishes is in
parallel with, and distinct from, the primary vasculature. This
secondary vascular system (also known as the secondary circulation) arises from a very large number of interarterial
anastomoses from the primary arterial vessels, particularly in
regions with external contact with the environment, e.g., skin,
gills, fins (14, 103, 111). An alternative view of the interarterial
anastomoses that emerge from the primary arteries is that they
are artifacts of the casting methods used to fill the vascular
system (61), although this seems unlikely. These secondary
arteries, similar in size to arterioles, coalesce to form secondary
arteries that supply a capillary network of their own that drains
into secondary veins and ultimately empty into the primary
veins. The secondary vascular system is thus defined because it
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LYMPHATIC FUNCTION IN AMPHIBIANS
Fig. 4. Hypoxia-induced linearization of arterial-lymphatic conduits (ALCs), lymphatic dilation, and blood perfusion in zebrafish and glass catfish. A: ALCs appear to
be secondary circulation vessels that create anastomoses between the primary circulation (segmental artery, SA) and the segmental lymphatic (SL) vessels. B: ALCs in glass
catfish (outlined in black boxes) are linearized during hypoxia. C: ALCs in transgenic
zebrafish tail region close to the SA and lymphatic vessel (LV) as shown by enhanced
green fluorescent protein (EGFP)-positive structures that include blood and endothelial
cells and lymphatic endothelial cells that express EGFP. D: hypoxia-induced dilation
and blood perfusion in a collecting duct lymphatic vessel (CLV). Dashed lines indicate
the border of the CLV. E: dilation of the lymphatic thoracic duct (TD) under hypoxic
conditions. Dashed lines indicate the border of the TD. F: summary of vessel diameters
for ALC, lymphatic vessel, and segmental artery under normoxia (open bars) and
hypoxia (solid bars). Note that hypoxia increases diameter of ALCs and lymphatic
vessels but not segmental arteries. G: linearization of ALCs under normoxia and
hypoxia. [Figure reproduced from Jensen et al. (63) with permission from the Proceedings of the National Academy of Sciences.]
The lymphatic system and lymph hearts of caecilians (order
Gymnophiona) and salamanders (order Urodela) are poorly
known; much more is known about lymphatics in frogs and
toads (order Anura). Caecilians are legless, burrowing amphibians with a “wormlike” appearance (49). They are highly
segmented, with a bilateral pair of lymph hearts between each
segment; thus more than 200 pairs of lymph hearts have been
found (67). Salamanders also have numerous lymph hearts,
with anywhere from 8 to 23 pairs depending on the species
(67). An unusual and characteristic feature of the salamanders
is the presence of what has been termed the “cardial lymph
propulsor” (67). This lymphatic propulsor lies tightly against
the truncus arteriosus, the major outflow tract of the amphibian
heart. It is in direct communication with the venous circulation,
but it is not clear if it is distinct from typical lymph hearts.
Anatomically, anurans are a highly derived and specialized
group of amphibians, as is their lymphatic system. The anuran
lymphatic system consists of extensive subcutaneous and intrapleuroperitoneal sacs separated by connective tissue walls
and interconnected by one-way valves. These valves appear to
be controllable rather than being simply passive flaps (64). The
various lymph sacs have been generally described for a variety
of anurans, but they do vary interspecifically (18). These large
subcutaneous lymph sacs account for the high Cist of anurans
compared with either fishes or mammals. Owing to their very
high Cist, the addition of even a volume equivalent to 4% of a
toad’s plasma volume (their minute plasma turnover) will only
generate an increased interstitial pressure of 1–2 Pa. This low
interstitial pressure means that lymph will preferentially move
gravitationally and pool in the ventral lymph sacs. The problem
for anurans, then, is that this lymph must be moved from
ventral regions to the dorsally located lymph hearts that pump
the lymph into the venous circulation. To describe this, we
have used the analogy of a “sump pump” that is placed in an
attic instead of a basement to move ventrally pooled lymph
(47). The pooled lymph is at ⬃200 Pa below the location of the
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connection with the primary circulation and contain valves at
the inlet and outlet that are adjacent to a blood capillary,
presumably to collect filtrate from the adjacent interstitial
space. There was no evidence for the “secondary circulation”
vessels characteristic of teleosts (114). No studies have examined lymphatic development, the rate of volume flux, nor
measured the pressures within the lymphatic system in this key
group of vertebrates.
The recently sequenced genome of the African coelacanth
(Latimeria chalumnae) provides some additional insight into
the evolution of the lymphatic system in the water-land transition of vertebrates (4). The data indicate that tetrapods have
expansion in at least four conserved regions of the genome
involved in lymphatic development and function compared
with teleosts. This suggests a more elaborate lymphatic system
and provides evidence for a potential elaboration of lymphatics
in the water-land transition. This raises questions about the
adequacy of teleost models, but also suggests that comparisons
among tetrapods could be useful for correlates of lymphatic
structure and function. The sequencing of the coelacanth genome opens up a myriad of potential studies to examine the
evolution of the tetrapod lymphatic system.
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Comparative Vertebrate Lymphatics
Hedrick MS et al.
303
Fig. 5. A series of pressure-volume loops of a posterior lymph heart (LH) of
the cane toad with saline infusion into the lymphatic sac surrounding the LH.
Note that end-diastolic pressure increased the end systolic pressure of the LH,
but stroke volume was independent of end diastolic volume and pressure,
illustrating that LH output does not behave in a manner consistent with
Starling’s law of the heart with increased preload volume. [Figure reproduced
from Crossley and Hillman (24) with permission from the Journal of Experimental Biology.]
hearts pump (24). Interestingly, anuran lymph hearts share
some common characteristics of isolated mammalian lymphangions. Both lymph hearts and lymphangions increase contractility in response to increased input pressure, and stroke volume is limited by increased afterload (24, 26, 97). Both
produce similar systolic pressures (⬃1 kPa), but lymphangions
produce greater stroke volumes in response to increased preload (i.e., Starling mechanism) whereas lymph hearts do not.
Given that lymph heart output is primarily determined by
afterload, the main problem facing anurans is how lymph can
be moved from ventral regions to the dorsally located lymph
hearts. We found that anuran amphibians have three mechanisms to move lymph from ventral lymph sacs to the dorsally
located lymph hearts. First, lymph is moved along the hindlimb
in a distal to proximal direction owing to differential compliance of the lymph sacs, a mechanism that we have termed a
“compliance pump” (48). As lymph enters the various
hindlimb sacs (foot, calf, thigh), the lower compliance of the
distal (foot) sac creates a higher pressure than the more
proximal (calf, thigh) lymph sacs, thus lymph moves in the
distal to proximal direction because of the pressure gradient
created by the differential lymph sac compliance. However, the
pressures generated are small (⬍20 Pa) and although able to
move lymph horizontally along the hindlimb, are not sufficient
to move lymph vertically; other mechanisms are necessary to
move lymph from the ventral portion of the animal to the
dorsally located lymph hearts, a vertical distance equivalent to
⬃200 Pa (see Fig. 6). This compliance pump mechanism
appears to be important only in terrestrial/semiterrestrial species (e.g., cane toads and bullfrogs) because aquatic species
(e.g., African clawed frogs) do not have differential hindlimb
lymph sac compliance (48). This might be expected given there
is no gravitational influence on lymph movement for aquatic
species (see below).
The second mechanism that promotes the vertical movement
of lymph is the contraction of various skeletal muscles, many
of which insert on the skin (31, 32). Contraction of these
muscles, which are also closely associated with lymph sacs,
alter lymph sac compliance and volume and, therefore, cause
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lymph hearts (sump pumps) and must be moved this vertical
distance against gravity to be pumped back into the vascular
circulation by the lymph hearts. Salamanders and caecilians do
not have a high interstitial compliance and are more “fishlike”
with tight skin. Thus the anatomical arrangement found of the
lymphatic system of anurans is unique among both amphibians
and vertebrates.
Anurans generally have two pairs of dorsal lymph hearts
(67, 81). Both pairs are innervated by spinal nerves (28, 84, 92,
93) and are under feedback control of the arterial baroreceptors
(23, 121) as well as hormonal control by angiotensin II and
arginine vasotocin (27). Lymph hearts generate pressures of
⬃1.0 –1.5 kPa at rates of ⬃40 – 60 beats/min (23, 27, 65, 66,
119). Lymph hearts are critical to vascular homeostasis in
anurans: their destruction results in hemoconcentration, interstitial edema, and death within a few days (124). Anurans with
cauterized lymph hearts are unable to replace fluids lost
through hemorrhage (7). Clearly, lymph heart transport is
critically important in maintaining blood volume and cardiovascular homeostasis, and anurans provide the clearest case for
the necessity of negative feedback control of lymphatic flux to
maintain plasma volume.
Lymph hearts consist of three tissue layers, a tunica interna
intima consisting of an endothelial cell lining, a middle tunica
media that contains the musculature of the lymph heart, and an
outer tunica externa made up of fibroelastic tissue (89). In
Xenopus, as in mammals, the endothelial tissue arises from the
blood vasculature and requires the homeobox transcription
factor prox1 (83). The lymph heart musculature, however, is
under different developmental control and expresses the skeletal muscle marker myoD, rather than cardiac markers (89). In
the absence of Hedgehog signaling and Engrailed-1 knockdown, lymph heart muscle fails to develop despite normal
development of the endothelium, and embryos develop edema
(89). The separate developmental pathways for endothelial and
lymph muscle may provide the basis for the origin of the
jugular (endothelial) lymph sac in mammals, which has the
same anatomical location as the anterior lymph hearts of
amphibians (89).
Lymph heart output can be rapidly modulated by both rate
and stroke volume changes (65, 66). Lymph heart stroke
volume is ⬃20% of end-diastolic volume, which is ⬃10 ␮l/kg
measured by conductance manometry and up to 29 ␮l/kg
measured by ultrasound (24). Lymph heart stroke volume
varies widely (66), but little is known of the effect of physical
factors, such as interstitial fluid pressure or venous pressure, on
regulation of lymph heart stroke volume. Nevertheless, lymph
heart stroke volume is clearly determined, in part, by lymph
input (like cardiac heart stroke volume). This is apparent from
the following three findings: 1) lymph heart stroke volume
declines with dehydrational fluid loss, despite no change in
lymph heart systolic pressure (66); 2) postural shifts in anesthetized animals that favor pooling of lymph around the posterior hearts cause a dramatic rise in stroke volume (66); and
3) lymph heart output in general has been reported to correlate
with the rate of lymph formation (6). However, direct measurements of lymph heart pressure-volume (P-V) indicate that
stroke volume is not correlated with preload variables, as
predicted by a Frank-Starling mechanism (Fig. 5; 24). Rather,
lymph heart stroke volume appears to be determined by venous
(afterload) pressure, the pressure against which the lymph
•
Synthesis
Comparative Vertebrate Lymphatics
0.8
0.6
0.4
0.2
ub
v
lS
oi
C
A
bl
at
e
A
er
ll
t
0.0
Fig. 7. Lymph flux rates measured from the femoral lymph sac after tendon
ablation of skeletal lymph muscles or placing a plastic coil into the subvertebral lymph sac to prevent complete filling of the lung in cane toads. Ablation
of the m. sphincter ani cloacalis (SAC) and m. abdominal crenators (Ab Cr) or
the m. piriformis (Piri) is required to cause a significant reduction in lymph
flux. Lymph flux also decreases significantly when the lungs are prevented
from fully expanding. [Figure adapted from Hillman et al. (50) with permission
from the Journal of Experimental Biology.]
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movement of lymph into or out of lymph sacs. The actual
movement of lymph is difficult to quantify for individual sacs
because the initial volume of each sac determines its compliance and movement of lymph is dependent upon the compliance of neighboring sacs. The skeletal lymph muscles of the
posterior region of anurans were described nearly two centuries
years ago (cf. Ref. 33), but in many cases the function of these
muscles was unknown. Because many of these muscles insert
on the skin, their function remained elusive (47). We used
measurements of lymph sac pressures combined with electromyography of the putative lymph muscles to define their role
in lymph movement (31). Furthermore, there are both similarities and differences in the presence, absence, and degree of
development of some of these lymph muscles. This has allowed us to make phylogenetically independent determinations
of form and function in these muscle groups (32). Our analysis
of over 400 species of anurans, from a variety of habitats,
indicate there has been bidirectional selection within these
muscle groups with greater elaboration of these muscles in
more terrestrial species and reduction in more aquatic species
(32). The degree of development of these muscles is obviously
correlated with habitat; corresponding rates of lymph flux are
also correlated with both habitat and lymph muscle development in three species of anurans that span the environmental
gradient from aquatic to terrestrial habitats (51).
The third mechanism that is responsible for the vertical
movement of lymph is lung ventilation (Fig. 6; 43). Changes in
lung volume associated with ventilation facilitate lymph movement by changing the volume of the lymph sacs surrounding
the lungs, in particular the large subvertebral lymph sinus that
is located in the dorsal space between the lungs and vertebral
column. The subvertebral sinus has direct connections with the
anterior and posterior lymph hearts in anurans. During lung
inflation (inhalation), the increase in lung pressure is transmitted to the surrounding lymph sacs and this facilitates movement of lymph toward the lymph hearts by “squeezing” the
ri
4 Pa
Fig. 6. Schematic diagram of the anatomical arrangement of the hindlimb
lymph sacs for the cane toad. Arrows indicate pathways for lymph flux leading
to the posterior LH. Relevant pressures are indicated in hindlimb lymphatic
sacs and the gravitational potential energy (PE) that must be overcome to move
lymph from ventral regions to the dorsal LH. The 3 types of pumps (compliance, skeletal muscle, and respiratory) and where they act to facilitate lymph
movement are shown (see text). [Figure modified from Hillman et al. (47) with
permission from the University of Chicago Press.]
Pi
8 Pa
e
THIGH
16 Pa
at
CALF
bl
Interfemoral
FOOT
A
Crural
C
r
Femoral
Plantar
A
b
200 Pa
&
Intermuscular
C
on
tr
ol
s
Pubic
Iliac
Gravitational PE
Compliance Pumps
A
bl
at
e
LH
Lateral
Skeletal Muscle Pumps
Dorsal
Subvertebral
Hedrick MS et al.
lymph in the sacs. During lung deflation (exhalation), the
reduction in lung volume causes an increase in the volume of
the subvertebral sinus because the two structures are attached
through a thin pleural membrane. This causes pressure in the
subvertebral space to decrease, thus providing a “suction”
effect to pull lymph into the subvertebral sinus (43). Reductions in subvertebral sinus pressure during lung deflation are of
sufficient magnitude to move lymph from ventral to dorsal
locations (43). It is also clear that skeletal muscle contraction
and lung ventilation act in a coordinated fashion to move
lymph. Cutting the insertional tendons of the m. gracilis minor,
m. sphincter ani cloacalis, and m. piriformis significantly
reduced lymph flux rates in cane toads (50). The reduction of
lymph flux by tendon ablation required removal of all three
muscles, indicating there is redundancy in the role of the lymph
muscles for mobilizing lymph. In a separate experiment, inserting a plastic coil into the subvertebral sinus to prevent
ventilatory-induced changes in subvertebral volume also significantly reduced lymph flux rates to the same degree as
tendon ablation of the lymph muscles (50). These experiments
indicate that both lymph skeletal muscles and lung ventilation
have a significant impact on lymph flux rates in anurans (Fig. 7).
Given the importance of the lungs for determining lymph flux
rates in anurans, we hypothesized that lung morphological
properties such as lung volume and lung compliance might be
correlated with lymph flux rates in anurans. There are significant family level differences in lung volume and compliance,
with higher values in the more terrestrial Bufonidae (toads)
compared with intermediate values in semiaquatic Ranidae
(frogs) and the lowest values in the totally aquatic Pipidae
(clawed frogs) (44). Moreover, the values for lung volume and
compliance were correlated with those species for which
lymph flux rates have been measured (44, 50). These data
suggest that greater lung volumes and lung compliance values
Lymph Flux (ml/kg min)
Respiratory Pump
•
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Comparative Vertebrate Lymphatics
LYMPHATIC FUNCTION IN REPTILES AND BIRDS
Very little is known about the physiology of lymphatics in
reptiles and birds as it relates to plasma homeostasis. Reptiles
show large intraclass variation in lymphatic vessel and lymph
heart structure, but the basis for this variation is unknown (67).
On the other hand, birds show much less variation in the
lymphatic system except for the presence of posterior lymph
hearts in the large ratites (e.g., ostrich and cassowary; 67).
Indeed, basic cardiovascular variables such as vascular/interstitial volume and compliance have not been measured in these
groups, so there is a large gap in our knowledge of the
magnitude of lymphatic flux and its role in plasma volume
maintenance. Lymph hearts are widespread among the reptiles
and have been described in snakes, lizards, turtles, and crocodilians (19, 67, 79). In many snakes, the lymphatic system is
extensive and forms perivascular lymphatic networks associated with the major arterial and venous vessels (19). Posterior
lymph hearts in many reptiles are housed by modifications of
the posterior ribs and pelvis. For example, in lizards and snakes
the posterior vertebrae near the pelvis are distinguished from
more anterior vertebrae by the absence of ribs and enlargement
of the transverse processes, forming a “fork” in which the
lymph heart is contained (67). In some cases transverse pro-
Hedrick MS et al.
305
cesses have a small “cup” indentation into which the lymph
heart fits. Similar anatomical features have been found in large
dinosaurs and, based upon anatomical measurements, it has
been estimated that the posterior lymph heart of brontosaurus
(Apatosaurus excelsus) had a volume of ⬃8 liters (67). Lymph
hearts of the Eastern painted turtle (Chrysemys picta picta)
generate a pressure of ⬃0.32 kPa at a rate of ⬃38 beats/min
(116). These values are lower than in amphibians, suggesting
that lymph heart output in turtles may be lower than for
anurans; however, lymph stroke volume has not been measured
in turtles. Given the high rate of plasma turnover in amphibians
compared with other vertebrates, greater lymph heart outputs
would be expected in amphibians.
Reptiles also have a remarkable capacity to maintain blood
volume when hemorrhagically stressed (73–77). A transcapillary (Starling) mechanism has been suggested for this regulation (77, 102), but this hypothesis has not been rigorously
tested for reptiles. Reptiles also have reflexive ventilatory and
muscular activity responses consistent with decreasing interstitial compliance during hemorrhage (73, 76), suggesting that
effectors similar to those in amphibians may be present to
facilitate lymph movement with hypovolemic stress. There is
also evidence for differences in tail interstitial compliance
consistent with interspecific tolerance of tilt-induced hypotension (74). Recent studies using lizard tail loss (autotomy, a
behavioral escape mechanism) as a model for lymphangiogenesis (10, 25) have shown for a gecko (Christinus marmoratus)
that, following voluntary tail loss, tail regeneration occurs
without any associated lymphedema. Tails developed lymphatic vessels early in the regeneration process and the lymphatic system was highly effective during tail regeneration at
removing fluid from the interstitial space. There are clear
similarities between reptiles and amphibians in variation in
interstitial compliance and ventilatory responses to hypovolemia, suggesting a role for lymphatics in plasma volume regulation that remains unexplored.
Similar to the situation in reptiles, lymph hearts and vessels
in birds are known primarily from anatomical descriptions, and
there are no studies that have characterized a role for the avian
lymphatic system for maintaining plasma volume. Avian embryos have a pair of lymph hearts that return lymph in ovo from
the extraembryonic membranes (120). These hearts partially
degenerate after hatching (9). Flightless ratite birds apparently
use a lymphatic pressure mechanism, rather than a blood
vascular mechanism, for penile erection (12), and lymph hearts
near the copulatory organ assist the return of lymph from the
penis to the venous system (13).
Birds also have a remarkable capacity to maintain blood
volume after hemorrhage or dehydration (16, 17, 30, 72,
91). They have high arterial pressures and presumably high
capillary pressures coupled to low colloid forces. Thus it is
difficult to imagine that a Starling-based mechanism can
account for their blood volume regulatory capabilities during dehydration and hemorrhage owing to the presumably
large efflux forces at the capillary level, although it has been
the mechanism invoked to explain mobilization of interstitial fluid to the plasma space (16). A clear unresolved issue
for both reptiles and birds is the role of lymphatic return in
the maintenance of plasma volume.
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allow greater lymph flux rates in more terrestrial species that
require greater lymph mobilization to maintain cardiovascular
homeostasis.
The regulation of lymph flux by the coordinated action of
lymph skeletal muscles and lung ventilation begs the question
as to how lymph mobilization is regulated. One hypothesis is
that central arterial pressure/volume homeostasis is the key to
lymph mobilization and that lung ventilation and lymph muscle
contraction are effectors of the baroreflex. Previous work has
shown that lymph heart frequency is inversely correlated with
peak arterial pressure, and this correlation is abolished by acute
transection of the recurrent laryngeal nerve, a primary baroreceptor nerve, in cane toads (23). Lung ventilation can be
altered by changes in mean arterial pressure, indicating that it
is linked to the baroreflex (45). There have been few studies in
ectothermic vertebrates of a link between blood pressure and
breathing, although there is a large literature on this topic for
mammals (see Ref. 80 for a recent review). The studies with
amphibians would indicate there is a significant influence of
blood pressure on breathing and that the proximate role is
regulation of lymph flux. The loose coupling of arterial blood
gases and ventilation in anurans (22) may be explained by the
finding that lung ventilation provides a significant cardiovascular function of lymph mobilization. There has been speculation about the origin of the link between blood pressure and
breathing in mammals (80); the amphibian studies suggest that
the origin of this reflex may lie in the role of lung ventilation
to mobilize lymph in anuran amphibians. Lymph hearts, lymph
skeletal muscles, and lung ventilation all appear to be under
control of negative feedback loops that may be linked to
pressure and/or volume sensing in the vascular space. Major
questions to be answered are how these various feedback loops
are integrated in the central nervous system, how the effectors
are coordinated to regulate lymph flux and plasma volume, and
whether these feedback loops also exist in the lungfish or
developed in the water-land vertebrate transition.
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Comparative Vertebrate Lymphatics
CONCLUSIONS AND PERSPECTIVES
GRANTS
Our work was generously supported by the National Science Foundation
(IBN-0110713 and IOS-0843082).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.S.H., S.S.H., and P.C.W. prepared figures; M.S.H.
drafted manuscript; M.S.H., S.S.H., R.C.D., and P.C.W. edited and revised
manuscript; M.S.H., S.S.H., R.C.D., and P.C.W. approved final version of
manuscript; S.S.H., R.C.D., and P.C.W. interpreted results of experiments.
Hedrick MS et al.
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We suggest that some important lymphatic-related variables
(lymphatic flux, vascular compliance, interstitial compliance,
whole body filtration coefficients) have remained in the recesses of comparative cardiovascular biology. However, their
measurement is central to our ability to understand the comparative role of the lymphatic system in plasma volume regulation and the evolution of the vertebrate cardiovascular system. The lymphatic system is poorly described for nonmammalian vertebrates, but developmental studies point to highly
conserved molecular pathways for lymph vessel formation in
zebrafish, anurans, and mammals. The evolution of the lymphatic system appears to have originated in teleost fishes,
perhaps with direct connections to the primary vasculature,
using the energy of the cardiac heart to propel lymph through
lymph vessels. This was followed by accessory lymph hearts,
originating in the Dipnoi and Amphibia to pump lymph into the
venous system. Most of our knowledge of lymphatic function
in nonmammalian vertebrates is from anuran amphibians,
which are highly derived and specialized. The anuran body
plan is also unique in having large subcutaneous lymph sacs
that confer a large interstitial compliance; this anatomical
feature appears to have evolved to maximize lymph storage
capabilities. Recent work has revealed a unique set of effectors
(specialized skeletal muscles and lung ventilation) that are
coordinated to mobilize lymph to the lymph hearts. Given that
anurans were the first vertebrates to successfully colonize a
large variety of terrestrial habitats, a large interstitial compliance appears to have been a successful evolutionary solution to
the problems associated with maintaining plasma volume in the
face of dehydration in the water-land transition. By contrast, a
large interstitial compliance in mammals is associated with
pathological conditions that cause edema and are difficult to
ameliorate owing to the imbalance of forces that maintain the
edematous state. Lymph hearts are retained in reptiles and
some birds, but in mammals they were replaced with contractile lymph vessels capable of intrinsic pumping of interstitial
fluid throughout the lymphatic system, and the skeletal muscle
pump, into the venous circulation. Perhaps a key adaptation in
mammals was the reduction in net transcapillary flux that
allowed lymphatic pumping confined to the lymphatic vessels.
The reduction in overall lymphatic return may have allowed
the lymph system of mammals to assume a greater role in
immune surveillance, including the proliferation of lymphoid
tissue that is far more developed in mammals compared with
other vertebrates.
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