Warren W. Burggren

Disruption of Hemoglobin Oxygen Transport Does Not Impact Oxygen-Dependent Physiological Processes in Developing Embryos of Zebra Fish (Danio rerio)

Embryonic hemoglobin circulated by the developing heart in the early vertebrate embryo is widely assumed (without substantiation) to perform the same vital role of O2 carriage that it does in fetuses and adults. In order to challenge this assumption, we measured highly O2-dependent physiological variables like O2 consumption, cardiac performance, and initial swim bladder filling in the presence and absence of functional hemoglobin in the embryos and early larvae of the zebra fish, Danio ( = Brachydanio) rerio. Functional ablation of hemoglobin by carbon monoxide or phenylhydrazine did not reduce whole-animal O2 consumption, which was approximately 85 to 90 mumol.g-1.h-1. Similarly, no differences in heart variables like ventricular pressure development or heart rate, which increased from 135 to 175 bpm between stages 36h and 96h (indicating developmental stages 36 and 96 hours after fertilization, respectively), were observed in these experiments. Initial opening of the swim bladder was not influenced in the presence of CO-occupied hemoglobin but was significantly impaired when the embryonic hemoglobin was chemically modified by incubation with phenylhydrazine. That aerobic processes continue without hemoglobin O2 transport indicates the adequacy in the embryo of simple O2 diffusion alone even in developmental stages with extensive convective blood circulation generated by the heart.

CUTANEOUS GAS EXCHANGE IN VERTEBRATES: DESIGN, PATTERNS, CONTROL AND IMPLICATIONS

1. The exchange of oxygen and carbon dioxide between skin and environment is commonplace in the vertebrates. In many lower vertebrates, the skin is the major or even sole avenue for respiration.

Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos

Chicken embryos were chronically exposed to hypoxia (P(O(2)) approximately 110 mmHg) during development, and assessed for detrimental metabolic and morphological effects. Eggs were incubated in one of four groups: control (i.e. 151 mmHg), or treated with continuous 110 mmHg (15% O(2)) during days 1-6 (H1-6), 6-12 (H6-12), or 12-18 (H12-18) with normoxia during the remaining incubation. Metabolism (V(O(2))), body mass, hemoglobin (Hb) and hematocrit (Hct) were measured in embryos on days 12 and 18 of incubation and in day-old hatchlings. Ability to maintain V(O(2)) was acutely measured during a step-wise decrease in P(O(2)) from normoxia to hypoxia (55 mmHg). On day 12, V(O(2)) of H1-6 eggs were significantly lower than in the control and H6-12 eggs. P(crit) in H6-12 eggs was lower than in control and H1-6 eggs. Body mass of H1-6 and H6-12 embryos on day 12 was significantly lower than in control embryos, while in H6-12 embryos, Hct and Hb were higher. On day 18, H6-12 embryos had significantly lower V(O(2)) than control eggs. Body mass of H6-12 and H12-18 embryos was significantly lower than control embryos. Hct and Hb did not differ between treatments. In hatchlings, mass, Hb and Hct had returned to values statistically identical to controls. However, H6-12 embryos had significantly lower V(O(2)). Long-term hypoxia altered V(O(2)) when hypoxic incubation occurred during the middle third of incubation, but not during earlier or later incubation. Thus, chronic hypoxic exposure during critical periods in development altered the developmental physiological trajectories and modified the phenotypes of the developing embryos.

Hypoxic incubation creates differential morphological effects during specific developmental critical windows in the embryo of the chicken (Gallus gallus)

Hypoxia inhibits vertebrate development, but the magnitude and timing of organ-specific effects are poorly understood. Chick embryos were exposed continuously to hypoxia (15% O2) throughout Days 1-6, 6-12, 12-18 or Days 1-18 of development, followed by morphometric measurements of major organ systems. Early hypoxic exposure reduced eye mass and beak length when measured in middle development. Liver, brain, heart, kidneys, stomach, intestines and skeletal long bones were not affected by hypoxia at any developmental stage. The chorioallantoic membrane (CAM) mass was unchanged by hypoxic exposure in early or mid-development, but CAM mass on Day 18 increased strikingly (40 and 60% in late and continuous populations, respectively) in response to hypoxic exposure. The increase in CAM mass presumably enhances oxygen delivery, thus minimizing the detrimental effects of hypoxia on development and growth. Hypoxic exposure at key critical windows in development thus results in differential effects on organ development, some of which can subsequently be repaired through additional incubation (yolk mass, eye mass, beak length).

What is the Purpose of the Embryonic Heart Beat? or How Facts Can Ultimately Prevail over Physiological Dogma

Embryonic physiology is often viewed as merely those processes understood for the adult but conducted on a smaller physical scale. Yet striking examples of the inaccuracy of this perspective can be identified in the embryonic cardiovascular system. For example, dogma holds that the embryonic heart begins to beat to pump blood for convective transport, just like that of the adult. This is the major assumption inherent in the hypothesis we have called “convective synchronotropy”; that is, the embryonic heart starts to beat synchronously with the need for convective blood flow. However, there is compelling evidence on many fronts that the convective flow of blood generated by the early embryonic vertebrate heart is simply not required for transport of oxygen, nutrients, metabolic wastes, or hormones, all of which can be achieved entirely by diffusion. In fact, fish, amphibian, and bird embryos lacking a functional heart (either through surgical intervention or mutation) or whose oxygen‐hemoglobin transport has been chemically eliminated nonetheless continue to function and grow in size for extended periods up to the point at which diffusion alone can no longer serve oxygen transport needs. We advocate the alternative hypothesis of “prosynchronotropy” (i.e., the heart starts to beat well before convective blood flow is needed for bulk transport). So, what is the purpose of the early embryonic heart beat? Evidence is presented herein in support of a morphogenic rationale for prosynchronotropy. Specifically, it appears that the initial rationale for the beat of the vertebrate embryonic heart may be two‐fold: to aid in subtle but significant aspects of cardiac growth, shaping, and maturation, and to facilitate cardiac maturation angiogenesis—the formation of new vessels by sprouting from vessel tips. Ultimately, the embryonic cardiovascular system provides a graphic demonstration of how adult physiological functions should not, without verification, be interpolated back to the embryo of that species.

Development of cardiovascular systems : molecules to organisms

List of contributors Foreword Constance Weinstein Introduction. Why study cardiovascular development? Warren B. Burggren and Bradley B. Keller Part I. Molecular Cellular and Integrative Mechanisms: Determining Cardiovascular Development: 1. Genetic dissection of heart development Jau-Nian Chen and Mark C. Fishman 2. Cardiac membrane characteristics Lynn Mahony 3. Development of the myocardial contractile system Anne M. Murphy 4. Vasculogenesis and angiogenesis of the developing heart Robert J. Tomanek and Anna Ratajska 5. Extracellular matrix maturation and morphogenesis Wayne Carver, Louis Terracio and Thomas K. Borg 6. Endothelial maturation Jackson Wong 7. Embryonic cardiovascular function, coupling and maturation: a species view Bradley B. Keller 8. Hormonal systems regulating the cardiovascular system Makoto Nakazawa and Fusae Kajio Part II. Species Diversity in Cardiovascular Development: 9. Evolution of cardiovascular systems: insights into ontogeny Anthony P. Farrell 10. Morphogenesis of vertebrate hearts Jose M. Icardo 11. Invertebrate cardiovascular development Brian R. McMahon, George B. Bourne and Ka Hou Chu 12. Piscine cardiovascular development Peter J. Rombough 13. Amphibian cardiovascular development Warren W. Rurggren and Regina Fritsche 14. Reptilian cardiovascular development Stephen J. Warburton 15. Avian cardiovascular development Hiroshi Tazawa and Ping-Chun Lucy Hou 16. Mammalian cardiovascular development Kent L. Thornburg, George D. Giraud, Mark D. Reller and Mark J. Morton Part III. Environment and Disease in Cardiovascular Development: 17. Oxygen, temperature and pH influences on the development of non-mammalian embryos and larvae Bernd Pelster 18. Modelling gas exchange in embryos, larvae and fetuses Alan W. Pinder 19. Principles of abnormal cardiac development Adriana C. Gittenberger-de Groot and Robert E. Poelmann 20. In utero and postnatal interventions in cardiovascular malformations V. Mohan Reddy and Frank L. Hanley 21. Insights into the future care of congenital cardiovascular malformations Edward B. Clark Epilogue. Future directions in developmental cardiovascular sciences Bradley B. Keller and Warren W. Burggren References Systematic index Subject index.

The pulmonary circulation of the chelonian reptile: Morphology, haemodynamics and pharmacology

Summary1.Blood pressures measured in lightly anaesthetized turtles (Pseudemys scripta) and tortoises (Testudo graeca) indicate that pressures throughout the ventricle are superimposable, but vascular impedances to blood flow in the pulmonary outflow tract and especially in the large extrinsic pulmonary arteries result in slightly lower pulmonary than systemic arterial systolic pressures in both species.2.Pulmonary outflow tract impedance is increased by vagal stimulation and acetylcholine and decreased by adrenaline. However, the pulmonary outflow tract apparently contributes little to the overall pulmonary impedance changes which occur during intermittent breathing.3.An analysis of pulmonary arterial impedance suggests that a large central arterial reservoir actively fills during systole and passively empties through a functionally single peripheral resistance during diastole. Morphological examination as well as in vitro compliance measurements and perfusion with drugs of the extrinsic pulmonary arteries corroborate these data by revealing a highly distensible central arterial reservoir nearly devoid of smooth muscle and vasomotor responses. The more distal pulmonary arteries are much less compliant, contain much smooth muscle, and show marked vasoconstriction in response to acetylcholine and vagal stimulation.4.Data on pulmonary impedance, morphology and pharmacology are incorporated into a classic ‘Windkessel’ haemodynamic model of the chelonian pulmonary circulation.

Egg yolk environment differentially influences physiological and morphological development of broiler and layer chicken embryos

SUMMARY Maternal effects are important in epigenetic determination of offspring phenotypes during all life stages. In the chicken (Gallus gallus domesticus), transgenerational transfer of egg yolk factors may set the stage for morphological and physiological phenotypic differences observed among breeds. To investigate the effect of breed-specific yolk composition on embryonic broiler and layer chicken phenotypes, we employed an ex ovo, xenobiotic technique that allowed the transfer of broiler and layer chicken embryos from their natural yolks to novel yolk environments. Embryonic day two broiler embryos developing on broiler yolk culture medium (YCM) had significantly higher heart rates than layer embryos developing on layer YCM (176±7 beats min–1 and 147±7 beats min–1, respectively). Broiler embryos developing on layer YCM exhibited heart rates typical of layer embryos developing normally on layer YCM. However, layer embryo heart rates were not affected by development on broiler YCM. Unlike O2 consumption, development rate and body mass of embryos were significantly affected by exposure to different yolk types, with both broiler and layer embryos displaying traits that reflected yolk source rather than embryo genotype. Analysis of hormone concentrations of broiler and layer egg yolks revealed that testosterone concentrations were higher in broiler yolk (4.63±2.02 pg mg–1 vs 3.32±1.92 pg mg–1), whereas triiodothyronine concentrations were higher in layer yolk (1.05±0.18 pg mg–1 vs 0.46±0.22 pg mg–1). Thus, a complex synergistic effect of breed-specific genotype and yolk environment exists early in chicken development, with yolk thyroid hormone and yolk testosterone as potential mediators of the physiological and morphological effects.

Epigenetics as a source of variation in comparative animal physiology – or – Lamarck is lookin' pretty good these days

Considerable variation is inherent both within and between comparative physiological data sets. Known sources for such variation include diet, gender, time of day and season of experiment, among many other factors, but a meta-analysis of physiological studies shows that surprisingly few studies report controlling for these factors. In fact, less than 3% of comparative physiological papers mention epigenetics. However, our understanding of epigenetic influences on physiological processes is growing rapidly, and it is highly likely that epigenetic phenomena are an additional ‘hidden’ source of variation, particularly in wild-caught specimens. Recent studies have shown epigenetic inheritance of commonly studied traits such as metabolic rate (water fleas Daphnia magna; emu, Dromaius novaellandiae), hypoxic tolerance, cardiac performance (zebrafish, Danio rerio), as well as numerous morphological effects. The ecological and evolutionary significance of such epigenetic inheritance is discussed in a comparative physiological context. Finally, against this context of epigenetic inheritance of phenotype, this essay also provides a number of caveats and warnings regarding the interpretation of transgenerational phenotype modification as a true epigenetic phenomenon. Parental effects, sperm storage, multiple paternity and direct gamete exposure can all be confounding factors. Epigenetic inheritance may best be studied in animal models that can be maintained in the laboratory over multiple generations, to yield parental stock that themselves are free of epigenetic effects from the historical experiences of their parents.

A Three-Dimensional Functional Assessment of Heart and Vessel Development in the Larva of the Zebrafish (Danio Rerio)

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart’s irregular shape and its significant change in morphology during the first 6 d of development. By use of a threedimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross‐sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%–600% increase in cardiac output (from 10 to 50–60 nL min−1) occurs on days 5 and 6 postfertilization in Danio rerio. Cross‐sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between‐individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross‐sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three‐dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.