Non-invasive imaging of blood cell concentration and blood distribution in zebrafish Danio rerio incubated in hypoxic conditions in vivo
Institute for Zoology and Limnology, University of Innsbruck, Austria
Author for correspondence (e-mail: thorsten.schwerte{at}uibk.ac.at)
Accepted 16 January 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: ontogeny, erythropoiesis, angiogenesis, hypoxia, digital video imaging, zebrafish, Danio rerio
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypoxic conditions are observed in the flowing and stagnant waters that are
the natural environment of the tropical zebrafish. The coupling of convective
oxygen transport and metabolic activity ensures sufficient oxygen supply to
the cells and prevents oxygen shortages at the organ level. Accordingly, in
adult animals hypoxia itself acts a stimulus and induces profound changes in
cardiac activity and peripheral resistance, and even stimulates
erythropoiesis. If this coupling is not yet established in early developmental
stages, it could mean that hypoxia does not act as a stimulus in early
developmental stages. In a recent study in zebrafish we were able to
demonstrate that long before coupling between metabolic requirements and blood
flow is established, environmental hypoxia can be sensed and induces
stimulation of cardiac activity (Jacob et
al., 2002). A reduction in the oxygen-carrying capacity of the
blood, however, had no effect on cardiac activity. Thus, hypoxia does exert a
signaling effect, even in early larval stages. The aim of the present study
was to investigate whether hypoxia, in addition to the modification of cardiac
activity shown in an earlier study, would also induce a redistribution of
blood and/or stimulate the production of red blood cells in the zebrafish. In
addition, the vascular bed of various organs was compared in animals raised
under normoxic and hypoxic conditions in order to test the hypothesis that
hypoxia stimulates the formation of blood vessels even at early developmental
stages. To answer these questions for millimetre-sized zebrafish, we used the
recently developed method of digital motion analysis
(Schwerte and Pelster, 2000
)
and extended it so that we could determine the concentration of red bloods
cells in a defined volume of blood and also visualize blood distribution
within the animal.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The imaging system
An inverted microscope (Zeiss Axiovert 25 CF) was placed on a solid,
heavy-weight steel plate to reduce vibration and the illumination set to
infrared light (913 nm wavelength) to prevent any light-induced stress
reactions in the animals. The microscope was equipped with a 2/3'' CCD
camera (Hamamatsu C-2400 without infrared cut-off filter) which, in turn, was
connected to the luminance input of a SVHS video recorder (Sony S-9500),
remote-controlled via the RS232 serial communication port. Recorded
images were digitized by a monochrome frame-grabber card (Imagenation PX-610)
with a personal computer (PIII 450 MHz). The depth of view was adjusted to
provide images that visualized most of the erythrocytes in one field.
Visualization of the vascular bed
A cast of the vascular bed was obtained by accumulation of the shifting
vectors of moving erythrocytes from a number of subsequent difference
pictures, as described in a previous study
(Schwerte and Pelster, 2000).
Briefly, by subtracting the two fields of a video frame, any movement that
occurred within the 20 ms necessary for the acquisition of one field was
visualized. The length of the shifting vectors, generated by this subtraction,
represent a direct measurement of the velocity of a moving particle, i.e. an
erythrocyte in the vascular system. By accumulation of shifting vectors
generated from several consecutive video frames, a complete trace of the
routes moved by the erythrocytes was obtained
(Fig. 1). Vascular beds of the
entire animal can be visualized non-invasively using this method
(Schwerte and Pelster, 2000
).
Typically the difference pictures of about 30 consecutive images were
accumulated to obtain a complete cast of the vascular bed.
|
As shown in Fig. 1, an image showing a complete cast of the vasculature of a body section or an organ permits measurement of the total area covered by these vessels relative to the size of the total area covered by the organ. This value can be used as an indicator for the vascularization of a tissue. Values for the whole tail and the gut were calculated.
Blood distribution using digital motion analysis
Developing this method further, it is also possible to obtain data about
the concentration of erythrocytes in a given section of a blood vessel. The
grey-scale value of any given pixel, or of a defined number of pixels, in the
image generated by digital motion analysis increases linearly from 0 to 255,
depending on the number of erythrocytes passing it. Although the depth of the
grey scale display on the screen is limited to 8 bit, the actual range for the
calculations was extended to 24 bit. Thus the erythrocyte distribution could
automatically be recorded in defined blood vessels or in the whole animal.
Calibration of the signal was done by correlating the grey scale values of a
defined area with the number of erythrocytes passing this area, as counted by
the conventional frame-to-frame technique
(Schwerte and Pelster, 2000).
These recordings were made using a 20x objective.
To evaluate blood redirection, mean values for the number of blood cells passing a specific tissue were compared and taken as an indicator of relative blood flow. A comparison of the concentration of red cells in various sections of the vascular system did not reveal the existence of any significant organ-specific differences in the hematocrit in zebrafish larvae, and our studies were focused on the brain, gut and tail musculature. The mean grey levels of the specific tissues obtained by this method were used to determine relative changes in tissue blood flow.
Red blood cell count
Using a 40x objective, 4-8 image frames had to be patched in order to
obtain a picture from the whole animal. After accumulating sufficient
difference pictures in order to obtain a cast of the vascular bed, the blood
vessel diameter of defined sections of the vessels was measured and the volume
of a defined blood vessel section calculated, assuming a circular
cross-section of the vessels.
Subsequently, erythrocytes within the defined section of the vessel were tracked on individual images and a series of images showing individual blood cells was stored to the computer hard drive. Blood cells were automatically detected by their characteristic grey scale value and motion, marked with a red cross and counted. The results were controlled by optical inspection of an image series and misinterpretations were eliminated. This determination was repeated five times for each animal to take possible clustering of blood cells into account. The process of counting red blood cells and the visualization of red blood cell distribution is described in Fig. 2.
|
Hypoxic incubation
Albino zebrafish eggs were incubated under hypoxic conditions
(PO2 8.7 kPa) at 28°C. Oxygen tension was
adjusted using a gas flow meter, which prepared a gas mixture of air and
nitrogen. The gas was infused into the water (28°C) of sealed 16 liter
aquaria through a fine-pored tube. Oxygen tension in the water was controlled
twice a day with a calibrated Clark oxygen electrode (Radiometer Copenhagen,
Willich, Germany) to ensure stable values.
Experimental protocol
Measurements were made at 3, 5, 7, 12 and 15 d.p.f. For measurement the
animals were anaesthetized with a neutralized tricaine solution (100 mg
l-1) at the adjusted PO2, and
embedded in low-melting-point agarose (containing 100 mg l-1
tricaine). For animals older than 11 d.p.f., the gills were sculpted free from
the agarose to allow gill ventilation
(Rombough, 2002). The animals
were covered with a thin layer of water (100-200 µm, containing 100 mg
l-1 tricaine). In the hypoxic groups all media were adjusted to the
desired PO2 and the animal chamber was sealed
tight. The sealed animal chamber had a reservoir of 0.7 ml gas with the
desired PO2, enough to maintain the
PO2 during the measurement. This was checked
using a miniature oxygen electrode. Oxygen deprivation has been shown to cause
general developmental retardation, so the staging was carefully done using the
following morphological criteria: yolk sac, animal length, diameter at the
position of the heart and vascular bed
(Isogai et al., 2001
)
(Fig. 1). The animals were not
fed prior to experiments. To avoid artefacts from differently fed animals
(older than 6 d.p.f.), the gut filling was microscopically inspected and found
to be similar in all animals prior to data acquisition. The experimental
groups were pooled from 2-3 clutches obtained from a group of 15 female with 5
male fish.
Statistics
For comparison of two means, statistical significance was evaluated by
unpaired Student's t-test. For multiple comparisons, one-way analysis
of variance (ANOVA) followed by StudentNewmanKeuls multiple
comparison test was used. Differences were considered significant at
P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Red blood cell count
Between 3 and 7 d.p.f. the concentration of red cells in the blood remained
quite stable and no differences between normoxic and hypoxic animals were
observed (Fig. 3). At 12 and 15
d.p.f., however, the concentration of red blood cells decreased significantly,
and at 15 d.p.f. it was reduced to approximately 30% of the value recorded at
7 d.p.f. in control animals. In hypoxic animals the concentration of red blood
cells also decreased at 12 and 15 d.p.f., but this decrease was significantly
smaller than in normoxic animals.
|
Vascularization
There were no significant changes in vascularization of the tail
musculature and the gut. The basic pattern of the vascular bed was similar in
normoxic and hypoxic animals (see Fig.
4). Nevertheless, in hypoxic animals the number of intersegmental
anastomosis was not significantly higher in hypoxic animals, and the number of
animals showing a caudal vascular tree was higher in hypoxic than in normoxic
animals. A statistical analysis of the morphometric data obtained from
sections through normoxic and hypoxic incubated animals
(Table 1), however, showed no
significant differences in the area covered by blood vessels.
|
|
Blood flow distribution
Fig. 4 shows typical
false-colour-coded images of the vascular cast obtained from 12 and 15 d.p.f.
animals raised under either normoxic or hypoxic conditions. It was obvious
that in 12 and in 15 d.p.f. animals, blood perfusion was higher in the muscle
tissue of hypoxic animals. By contrast, perfusion of the gut was significantly
lower only in 12 d.p.f. animals, but not in 15 d.p.f. animals. Brain perfusion
was not affected by hypoxia. Fig.
5 summarizes these differences as percentage of control value
(100%).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The quality of the automated red blood cell count was ensured by careful visual inspection of detected blood cells. Double labelling as well as underestimation because of blood cell clusters could easily be detected by visual inspection of the image series. Repeated measurements in the same animal were within an error range of 2%.
Visualization of blood vessels for morphometric analysis is based on the
movement of red blood cells, and therefore plasma skimming
(Schmid-Schönbein, 1988)
or layers of erythrocyte-free plasma may cause an underestimation of the
vessel diameter. On the basis of model calculations of red cell movements in a
capillary system, this plasma layer is estimated to have a thickness of
approximately 1 µm or less
(Schmid-Schönbein, 1988
).
To evaluate the accuracy of our method, we compared the blood vessel thickness
determined from the accumulated difference images with the diameter measured
from a complete video image of the same site assessed by microscopical
inspection. The results differed by no more than 2-3%. We therefore conclude
that our volumetric analysis based on the movement detection and the red blood
cell count provides very accurate and highly reproducible results.
The influence of hypoxia
In adult vertebrates, cardiac activity and blood flow to tissues are both
mainly determined by metabolic demand. Adult fish and amphibians are typically
oxy-regulators, but in the earliest embryonic and larval stages of these
vertebrates oxygen uptake appears to decrease with decreasing environmental
PO2, i.e. they are oxyconformers
(Hastings and Burggren, 1995).
In fact, the first embryonic stages of the zebrafish can survive complete
anoxia in a state of suspended animation
(Padilla and Roth, 2001
), and
the Arctic charr can survive several hours of anoxia, with severe metabolic
depression and a significantly reduced cardiac activity
(Pelster, 1999
). Anoxia
certainly is an extreme situation for a vertebrate embryo, but mild or chronic
hypoxia has also been shown to provoke significant physiological adaptations,
ranging from metabolic effects to ventilatory and circulatory adjustments (for
a review, see Pelster, 1997
),
and in zebrafish larvae a stimulation of cardiac activity in response to
hypoxia was observed as early as 3 d.p.f. at 28°C
(Jacob et al., 2002
). Chronic
hypoxia during embryonic and larval development may also significantly modify
differentiation and growth. In larval amphibians, for example, aquatic hypoxia
stimulates growth of respiratory surfaces and enhances the transition from
gill to lung respiration (Guimond and
Hutchison, 1976
; Burggren and
Mwalukoma, 1983
; Burggren and
Just, 1992
), but often hypoxia results in retardation of
development (Pelster, 1997
).
In our experiments the first signs of retarded development were observed at 15
d.p.f., and therefore at this age the experiment was terminated in order to
prevent a situation where physiological effects induced by hypoxia are mixed
up with effects induced by developmental retardation.
Hypoxia and red cell concentration
In mammals hypoxia usually causes an increase in the oxygen transporting
capacity of the blood, i.e. an increase in the number of circulating
erythrocytes and in hemoglobin concentration. Similar observations have been
reported for embryos of the turtle Pseudemys nelsoni
(Kam, 1993;
Jacob et al., 2002
), whereas
in chicken embryos during early development no stimulation of red cell
production was observed (Baumann and Meuer,
1992
). The results of our study clearly show that hypoxia
increases red blood cell concentration in zebrafish larvae during the second
week after fertilization, but no effect was observed until 7 d.p.f. Up to 7
d.p.f., the concentration of red cells remained fairly constant in normoxic as
well as in hypoxic animals. Weinstein et al.
(1996
) showed that the first
presumptive proerythroblast-like cells can be detected in zebrafish by the end
of the first day (20 h post-fertilization), and this primitive cohort of cells
originates in the intermediate cell mass. By transfusing fluorescently
labelled blood cells into 1.5-day-old host embryos, recording the fraction of
these labelled cells and comparing the histology of the red cells, these
authors concluded that this first cohort of primitive red cells provides the
embryo with all, or nearly all, its red blood cells for at least 4 days. A
population of new, larger and more adult-appearing erythrocytes became
predominant by 10 d.p.f.. The constant concentration of red cells observed
until 7 d.p.f. in our experiments is in line with these results, but the
severe decrease in red cell count at 12 d.p.f. was unexpected. A possible
explanation would be that the growing volume of the vascular bed is
accompanied by an increase in plasma volume, but not by an equivalent
production of erythrocytes. Accordingly, hematocrit and red cell concentration
would reduce. An increased cell volume of the newly produced erythrocytes at
constant hematocrit would also cause a decrease in red cell concentration, but
this latter explanation does not appear very likely.
In hypoxic animals this decrease in the red cell concentration was significantly less than in normoxic animals. If the decrease in red cell concentration observed between 7 d.p.f. and 12 d.p.f. is caused by an expansion of plasma volume, in hypoxic animals the increase in plasma volume would be smaller than in normoxic animals. Information about the total blood volume, however, is necessary to test this idea.
Our method provides a two-dimensional projection of the complete cast of the vascular system. A comparison of projection areas revealed no significant differences between the experimental and the control groups. Assuming a circular cross-section and similar size of vessels, this result means that there was no significant difference in the total volume of the vascular bed in the two groups. However, the acquisition of images for the visualization of the blood redistribution as compared to the acquisition of images to create a complete vascular cast required a compromise between acquisition speed and spatial resolution. Although the resolution of our images was sufficient to show changes in blood distribution, small changes in the diameter of blood vessels, and therefore changes in total blood volume, may not be detectable. For the exact determination of the total blood volume, further studies using a more refined method with higher spatial resolution are needed.
Another explanation for this observation would be that hypoxia stimulates
erythropoiesis in zebrafish larvae, as it does in fetal mammals
(Richardson and Bocking, 1998)
or in embryos of the turtle Pseudemys nelsoni
(Kam, 1993
;
Jacob et al., 2002
). Recent
molecular studies revealed that hematopoiesis in zebrafish larvae is
stimulated by VEGF, and VEGF is also known to drive angiogenesis
(Liang et al., 2001
). The
differential regulation of these processes appears to include a regulatory
loop by which VEGF controls survival of hematopoietic stem cells in mice
(Gerber et al., 2002
).
Erythropoietin (EPO), also well known to be involved in hypoxia-induced
erythropoiesis, has been shown to mediate hypoxia-induced VEGF expression in
rats (Liu et al., 1995
).
Blood distribution
A redistribution of blood in response to different metabolic demands is a
well-established physiological adaptation in many species. This is the first
study to visualize a `source and sink' pattern of blood distribution in
zebrafish larvae in response to oxygen deprivation. It is obvious that under
hypoxic conditions blood is driven from the gut (source) to the muscles in the
tail (sink), but until 7 d.p.f. no changes in blood distribution were
observed. In a previous study we observed a hypoxic stimulation of cardiac
activity as early as 3 d.p.f., which demonstrates that at about 1 day after
hatching oxygen receptors are present that respond to hypoxia, and this
information can be translated into a signal generating an increase in heart
rate and in cardiac output (Jacob et al.,
2002). Thus, the lack of oxygen can be identified by the larvae,
but until 7 d.p.f. this information is not used to induce a change in blood
distribution and in erythrocyte production. At 12 d.p.f., however, hypoxia
induces a significant increase in the perfusion of the muscle tissue, and the
so-called red-layer of muscle has been implicated in the uptake of oxygen in
early larvae and at about the time of hatching
(El-Fiky and Wieser, 1988
).
Possibly blood is redistributed towards the muscle tissue in order to enhance
oxygen uptake through the body surface under hypoxic conditions. Under
normoxic conditions, the oxygen requirements of zebrafish larvae apparently
are met by bulk diffusion until 12-14 d.p.f.
(Jacob et al., 2002
), and this
is the time when the gills with secondary lamellae are developed
(Rombough, 2002
). Also at this
time the site of oxygen uptake is shifted towards the gills, and convective
oxygen transport becomes a necessity for transporting oxygen from the gills to
the tissues. This implies that blood flow is not necessary to sustain aerobic
metabolism in normoxic larvae until about 12 d.p.f., but under hypoxic
conditions zebrafish larvae are obviously able to stimulate cardiac activity
and to redirect blood flow in order to increase perfusion of the muscle
tissue. Following the hypothesis that the red layer of muscle may be
implicated in the uptake of oxygen in early larvae
(El-Fiky and Wieser, 1988
),
this would suggest that oxygen uptake through the red layer may be enhanced,
which, given the small cross-sectional area of the larvae, is essentially
cutaneous respiration in the area of the red muscle tissue. To test this idea,
we are currently developing a method to visualize changes in hemoglobin
oxygenation in vivo by recording changes in the absorption spectrum
of the hemoglobin.
On the other hand, the redirection of blood may not be related to cutaneous
respiration, but simply reflect a maturation of control systems (e.g. hypoxic
vasodilation and hypoxic vasoconstriction). The presence of
-adrenergically controlled precapillary sphincters in the
intersegmental muscle tissue of zebrafish larvae at 8 d.p.f. has already been
shown (Schwerte and Pelster,
2000
). Furthermore, a general vasodilation in that tissue may be
caused by nitric oxide, as demonstrated by Fritsche et al.
(2000
) to already occur by 5
d.p.f. Thus, hormonal control mechanisms contributing to a redistribution of
blood flow are certainly established at this time of development.
The increase in muscle tissue perfusion could also be a consequence of an
increased cardiac output, but this would not explain the decrease in gut
perfusion simultaneously recorded in our experiments. In a previous study,
Jacob et al. (2002) observed
an increase in cardiac output by 20-30% under hypoxic conditions, but in our
experiments the effect on cardiac activity was even smaller, so that a change
in cardiac activity alone cannot explain the redistribution of blood measured
in our experiments.
Another possibility is that the vascular volume in muscle tissue increased during hypoxia. As already mentioned, however, the two-dimensional projections of the complete cast of the vascular system did not reveal an increased vascular volume.
Hypoxia and tissue vascularization
Yue and Tomanek (1999)
demonstrated that coronary vessels from cultured 6-day-old quail embryo grow
faster under hypoxic conditions, while hyperoxia induced a delayed
angiogenesis. By contrast, chorioallantoic membrane capillarization of chicken
embryos has been shown to increase during hypoxia
(Dusseau and Hutchins, 1988
;
Hudlicka et al., 1992
). In
larval amphibians, aquatic hypoxia stimulates growth of respiratory surfaces
and enhances the transition from gill respiration to lung respiration
(Mwalukoma and Burggren,
1983
).
In our study, however, no significant changes in the vascular bed were
observed. There appeared to be quite a high interindividual variation in the
expression of small blood vessels like the intersegmental anastomsis or the
caudal vascular tree, and we cannot exclude the possibility that minor changes
in the expression of these vessels did occur in hypoxic animals. The overall
morphometric analysis, however, did not show any significant differences
between control and hypoxic animals. Compared with the development of
amphibians such as Xenopus or Rana, or the development of
salmonid larvae, zebrafish development is very rapid, and it may be possible
that a rearrangement of the vascular bed can only occur in later developmental
stages. On the other hand, molecular signals involved in the formation of
blood vessels have been identified in early embryonic stages. The molecular
mechanisms that lead to the extremely regular pattern in the zebrafish trunk
have been shown by Childs et al.
(2002), and VEGF up- and
down-regulation seems to be involved in this process. Whole-mount in
situ hybridization of zebrafish embryos indicated that strong expression
of VEGF had already occurred at 18 h post fertilization
(Weinstein et al., 1996
;
Liang et al., 1998
;
Tan et al., 2001
).
Physiological significance
Under normoxic conditions, oxygen supply via diffusion seems to be
sufficient to meet the metabolic demand up to 12-14 d.p.f. This changes during
hypoxia, however, because the reduced oxygen gradient cuts down diffusion of
oxygen to the tissues. The data available so far show that in this situation
convective transport can be enhanced by increasing blood flow
(Jacob et al., 2002), and the
oxygen carrying capacity of the blood can be increased. In addition, blood
flow can be redirected towards a potential site of oxygen uptake. Thus,
although under normoxic conditions convective transport is not necessary, the
larvae can use the circulatory system as a backup system to augment oxygen
distribution and oxygen supply to the tissues, which is strong evidence that
the cardiovascular system can operate as a convective transport system for
oxygen much earlier then required under normal circumstances. This situation
was called `prosynchronotropy' by Burggren and Fritsche
(1995
). A prosynchronotropic
development of convective oxygen transport appears to be very useful, because
it creates a safety belt for a situation where bulk oxygen diffusion alone
would not be sufficient to ensure oxygen supply to all tissues, and thus
widens the range of environmental conditions in which the larvae can survive.
Angiogenesis, however, seems not to play a key role in facilitating oxygen
uptake. It could be that in the small zebrafish larvae the diffusion distances
between tissue capillaries and the cells are small enough to permit an optimal
supply with oxygen, so that additional blood vessels would not yet enhance
oxygen transport to the cells.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barut, B. A. and Zon, L. I. (2000). Realizing
the potential of zebrafish as a model for human disease. Physiol.
Genomics 2,49
-51.
Baumann, R. and Meuer, H.-J. (1992). Blood
oxygen transport in early avian embryo. Physiol. Rev.
72,941
-965.
Burggren, W. and Fritsche, R. (1995). Cardiovascular measurements in animals in the milligram range. Braz. J. Med. Biol. Res. 28,1291 -1305.[Medline]
Burggren, W. and Mwalukoma, A. (1983). Respiration during chronic hypoxia and hyperoxia in larval and adult bullfrogs (Rana catesbeiana) I. Morphological responses of lungs, skin and gills. J. Exp. Biol. 105,191 -203.[Abstract]
Burggren, W. W. and Just, J. J. (1992). Developmental changes in physiological systems. In Environmental Physiology of the Amphibians (ed. M. E. Feder and W. W. Burggren), pp. 467-530. Chicago: The University of Chicago Press.
Burggren, W. W. and Keller, B. B. (1997). Development of Cardiovascular Systems: Molecules to Organisms. Cambridge: Cambridge University Press.
Childs, S., Chen, J. N., Garrity, D. M. and Fishman, M. C. (2002). Patterning of angiogenesis in the zebrafish embryo. Development 129,973 -982.[Medline]
Dusseau, J. W. and Hutchins, P. M. (1988). Hypoxia-induced angiogenesis in chick chorioallantoic membranes: a role for adenosine. Respir. Physiol. 71, 33-44.[CrossRef][Medline]
El-Fiky, N. and Wieser, W. (1988). Life styles and patterns of development of gills and muscles in larval cyprinids (Cyprinidae; Teleostei). J. Fish Biol. 33,135 -145.
Feder, M. E. and Booth, D. T. (1992). Hypoxic boundary layers surrounding skin-breathing aquatic amphibians: Occurrence, consequences and organismal responses. J. Exp. Biol. 166,237 -251.
Fritsche, R., Schwerte, T. and Pelster, B. (2000). Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio. Am. J. Physiol Regul. Integr. Comp. Physiol. 29,R2200 -R2207.
Gerber, H. P., Malik, A. K., Solar, G. P., Sherman, D., Liang, X. H., Meng, G., Hong, K., Marsters, J. C. and Ferrara, N. (2002). VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature 417,954 -958.[CrossRef][Medline]
Gielen, J. L. and Kranenbarg, S. (2002). Oxygen balance for small organisms: an analytical model. Bull. Math. Biol. 64,175 -207.[CrossRef][Medline]
Guimond, R. W. and Hutchison, V. H. (1976). Gas exchange of the giant salamanders of North America. In Respiration of Amphibious Vertebrates (ed. G. M. Hughes), pp.313 -338. London, New York, San Francisco: Academic Press.
Hastings, D. and Burggren, W. (1995). Developmental changes in oxygen consumption regulation in larvae of the South African clawed frog Xenopus laevis. J. Exp. Biol. 198,2465 -2475.[Medline]
Hudlicka, O., Brown, M. and Egginton, S.
(1992). Angiogenesis in skeletal and cardiac muscle.
Physiol. Rev. 72,369
-417.
Isogai, S., Horiguchi, M. and Weinstein, B. M. (2001). The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev. Biol. 230,278 -301.[CrossRef][Medline]
Jacob, E., Drexel, M., Schwerte, T. and Pelster, B.
(2002). Influence of hypoxia and of hypoxemia on the development
of cardiac activity in zebrafish larvae. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 283,R911
-R917.
Kam, Y.-C. (1993). Physiological effects of hypoxia on metabolism and growth of turtle embryos. Respir. Physiol. 92,127 -138.[CrossRef][Medline]
Liang, D., Chang, J. R., Chin, A. J., Smith, A., Kelly, C., Weinberg, E. S. and Ge, R. (2001). The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development. Mech. Dev. 108, 29-43.[CrossRef][Medline]
Liang, D., Xu, X., Chin, A. J., Balasubramaniyan, N. V., Teo, M. A., Lam, T. J., Weinberg, E. S. and Ge, R. (1998). Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio. Biochim. Biophys. Acta 1397,14 -20.[Medline]
Liu, Y. X., Cox, S. R., Morita, T. and Kourembanas, S. (1995). Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells: identification of a 5' enhancer. Circ. Res. 7,638 -643.
Mwalukoma, A. and Burggren, W. W. (1983). Respiration during chronic hypoxia and hyperoxia in larval and adult bullfrogs (Rana catesbeiana). I. Morphological responses of lungs, skin and gills. J. Exp. Biol. 105,191 -203.[Abstract]
Padilla, P. A. and Roth, M. B. (2001). Oxygen
deprivation causes suspended animation in the zebrafish embryo.
Proc. Natl. Acad. Sci. USA
98,7331
-7335.
Pelster, B. (1997). Oxygen, temperature, and pH influences on the development of nonmammalian embryos and larvae. In Development of Cardiovascular Systems (ed. W. W. Burggren and B. B. Keller), pp. 227-239. Cambridge: Cambridge University Press.
Pelster, B. (1999). Environmental influences on the development of the cardiac system in fish and amphibians. Comp. Biochem. Physiol. A 124,407 -412.
Pelster, B. and Bemis, W. E. (1991). Ontogeny of heart function in the little skate Raja erinacea. J. Exp. Biol. 156,387 -398.
Pelster, B. and Burggren, W. W. (1996).
Disruption of hemoglobin oxygen transport does not impact oxygen-dependent
physiological processes in developing embryos of zebra fish (Danio
rerio). Circ. Res.
79,358
-362.
Pinder, A. W. and Feder, M. E. (1990). Effect of boundary layers on cutaneous gas exchange. J. Exp. Biol. 154,67 -80.
Richardson, B. S. and Bocking, A. D. (1998). Metabolic and circulatory adaptations to chronic hypoxia in the fetus. Comp. Biochem. Physiol. A 119,717 -723.
Rombough, P. (2002). Gills are needed for
ionoregulation before they are needed for O2 uptake in developing
zebrafish, Danio rerio. J. Exp. Biol.
205,1787
-1794.
Schmid-Schönbein, H. (1988). Fluid dynamics and hemorheology in vivo: The interactions of hemodynamic parameters and hemorheological `properties' in determining the flow behavior of blood in microvascular networks. In Clinical Blood Rheology, Vol. 1 (ed. G. D. O. Lowe), pp.129 -219. Boca Raton: CRC Press.
Schwerte, T. and Pelster, B. (2000). Digital
motion analysis as a tool for analysing the shape and performance of the
circulatory system in transparent animals. J. Exp.
Biol. 203,1659
-1669.
Tan, D. C., Kini, R. M., Jois, S. D., Lim, D. K., Xin, L. and Ge, R. (2001). A small peptide derived from Flt-1 (VEGFR-1) functions as an angiogenic inhibitor. FEBS Lett. 494,150 -156.[CrossRef][Medline]
Territo, P. R. and Altimiras, J. (1998). The ontogeny of cardio-respiratory function under chronically altered gas compositions in Xenopus laevis. Respir. Physiol. 111,311 -323.[CrossRef][Medline]
Territo, P. R. and Burggren, W. W. (1998).
Cardio-respiratory ontogeny during chronic carbon monoxide exposure in the
clawed frog Xenopus laevis. J. Exp. Biol.
201,1461
-1472.
Warren, K. S. and Fishman, M. C. (1998).
`Physiological genomics': mutant screens in zebrafish. Am. J.
Physiol. 275,H1
-H7.
Weinstein, B. M., Schier, A. F., Abdelilah, S., Malicki, J.,
Solnica-Krezel, L., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Driever,
W. and Fishman, M. C. (1996). Hematopoietic mutations in the
zebrafish. Development
123,303
-309.
Yue, X. and Tomanek, R. J. (1999). Stimulation of coronary vasculogenesis/angiogenesis by hypoxia in cultured embryonic hearts. Dev. Dyn. 216,28 -36.[CrossRef][Medline]
Related articles in JEB: