Embryonic Alligators Research Articles

Temperature effects on blood gases in embryonic American alligators (Alligator mississippiensis)

Numerous studies report on the influence of temperature on blood gases in ectothermic vertebrates, but there is merely a cursory understanding of these effects in developing animals. Animals that develop in eggs are at the mercy of environmental temperature and are expected to lack the capacity to regulate gas exchange and may regulate blood gases by means of altered conductance for gas exchange. We, therefore, devised a series of studies to characterize the developmental changes in blood gases when embryonic alligators were exposed to 25, 30 and 35 °C. To determine how blood parameters were impacted by changes in embryonic temperature, blood was sampled from the chorioallantoic membrane artery. The blood in the chorioallantoic membrane artery is a mixture of oxygen-poor and oxygen-rich blood, which based on the embryonic vascular anatomy may reflect blood that perfuses the chemoreceptors of the developing animal. Our findings indicate that following a 48 h exposure to 25 °C or 35 °C, there was a positive relationship between CAM artery blood PO2, PCO2 and glucose. However, blood pH suggests embryonic alligators lack an acute regulatory mechanism for adjusting blood pH.

Short communication: Characterizing arterial and venous blood gases over the gas exchange surface, the chorioallantoic membrane, of embryonic American alligators (Alligator mississippiensis) at two points of development

Assessments of arterial and venous blood gases are required to understand the function of respiratory organs in animals at different stages of development. We measured blood gases in the arteries entering and veins leaving the chorioallantoic membrane (CAM) in embryonic alligators (Alligator mississippiensis). The CAM accounts for virtually all gas exchange in these animals, and we hypothesized that the CAM vasculature would be larger in eggs incubated in hypoxia (10% O2 for 50% or 70% of incubation), which would be reflected in a lower partial pressure of CO2 (PCO2). Contrary to this hypothesis, our measurements revealed no effects of hypoxic incubation on PCO2, and seemingly no increase in vascularization of the CAM in response to incubation in 10% O2. PCO2 was lower on the venous side, but only significantly different from arterial blood at 70% of incubation. The calculated blood flow to the CAM increased with development and was lower in both groups of alligators that had been incubated in hypoxia. Future studies should include measurements of blood parameters taken from embryos held in conditions that mirror incubation O2 levels, in combination with direct measurements of CAM artery blood flow.

Short communication: Baroreflex function in embryonic emus (Dromiceius novaehollandiae)

The baroreflex involves cardiovascular homeostatic mechanisms that buffer the system against acute deviations in arterial blood pressure. It is comprised of the cardiac limb which involves adjustments in heart rate and the peripheral limb which involves adjustments in vascular resistance. This negative feedback loop mechanism has been investigated in numerous species of adult vertebrates, however our understanding of the maturation and functional importance of the reflex in developing animals remains poorly understood. In egglaying species, our knowledge of this mechanism is limited to the domestic chicken embryo and the embryonic alligator. While each of these species possess a cardiac baroreflex prior to hatching, they differ in the timing when it becomes functional, with the embryonic chicken possessing the reflex at 90% of incubation, while the alligator possesses the reflex at 70% of incubation. In an effort to determine if bird species might share similar patterns of active baroreflex function, we studied embryonic emus (Dromiceius novaehollandiae). However, we hypothesized that emus would possess a pattern of baroreflex function similar to that of the American alligator given the emu embryo possesses functional vagal tone at 70% of incubation, possibly indicating a more mature collection of cardiovascular control mechanism than those found in embryonic chickens. Our findings illustrate that emu embryos possess a hypotensive baroreflex at 90% of incubation. Therefore, our data fail to support our original hypothesis. While only two species of birds have been studied in this context, it could indicate that baroreflex function is not essential for cardiovascular homeostasis in birds for the majority of in ovo development.

Cardiovascular responses to putative chemoreceptor stimulation of embryonic common snapping turtles (Chelydra serpentina) chronically incubated in hypoxia (10% O2)

Developmental hypoxia has been shown to result in significant changes in cardiovascular development of American alligators and common snapping turtles. These include similar effects on cardiac mass and aspects of cardiovascular function. However, given the distant phylogenetic relationship between crocodilians and chelonians, we hypothesized that snapping turtles would also exhibit differences in the effects of developmental hypoxia on cardiovascular regulation. This hypothesis was based in part on prior studies that documented differences in plasticity of vagal tone on the heart between alligators and snapping turtles incubated in hypoxic conditions. To test this hypothesis, we investigated how 10% O2 exposure over final 80% of incubation altered the heart rate and blood pressure response to two chemical manipulations of the “chemoreflex” in common snapping turtles at 70% and 90% of incubation. NaCN injections produced a dose dependent bradycardia that was mediated by cholinergic receptor stimulation. This reflex was relatively unaffected by hypoxic incubation conditions in snapping turtle embryos. Injections of the 5-HT3 agonist phenylbiguanide (PBG) caused a pronounced bradycardia that decreased in intensity at 90% of incubation in embryos from the normoxic group while the heart rate response was unchanged in the hypoxic group. This differs from the previously reported diminished heart rate response of embryonic alligators incubated in 10% O2, suggesting plasticity in this chemoreflex response differs between the species. Our data also indicate the cardiovascular response is mediated by a secondary cholinergic receptor stimulation however the inability of ganglionic blockade to inhibit the PBG response leaves the location of the receptors antagonized by PBG in question in embryonic snapping turtles. Primarily, our findings refute the hypothesis that hypoxic incubation decreases the “chemoreflex’ response of snapping turtle embryos.

The Development of Appendicular joint cartilages in Alligator mississippiensis: Evolutionary and Biomechanical Implications for Archosauria

Crocodylians and birds are the two surviving lineages within Archosauria, a clade of morphologically and ecologically diverse vertebrates that evolved highly disparate locomotor postures. The appendicular joints of crocodylians and birds differ substantially, thus complicating inferences of ancestral morphology and evolutionary transitions. Recent studies have indicated that the American alligator, an extant crocodylian, use a wide spectrum of cartilaginous and other connective tissues to construct the hip joint. However, little is known about the distribution and use of cartilage throughout other appendicular joints, as well the ontogenetic transition of the crocodilian chondro‐osseous interface during early development. In particular, the hyaline cartilage on the proximal femur of late embryonic alligators possesses a convex, cone‐like extension that inserts deep into the metaphysis. This morphology is not seen in juvenile and adults, but is superficially similar to the cartilage cone‐metaphyseal trough articulation inferred in early members of multiple archosaurian groups. The method by which alligators and other archosaurs reduced the cartilage cone during ontogeny and evolution, as well as the functional significance of the cartilage cone itself, are poorly understood.This study examined the ontogenetic transitions of proximal appendicular joints in the American alligator, spanning from in ovo stages to the first year of life. We used dissection, histology, and imaging techniques to assess the articular soft tissue anatomy at the chondro‐osseous junction of the proximal‐ and distal epiphyses of the femur and humerus. Additionally, osteological correlates for joint cartilage were identified on fully skeletonized limb bone elements and compared with bony morphology seen in fossil archosauromorphs.At the onset of hatching, the alligator did not reduce the cartilage cone via decreasing the “depth” of cartilaginous extension into the metaphysis, as inferred for early archosaurs. Instead, neonatal alligators regress the cartilage cone via multiple invasion fronts of trabecular bones, such that the subchondral growth plate of hatchlings presents a convex surface perforated by numerous cartilage canals, similar to those found in young birds. These results suggest that the ontogenetic loss of cartilage cone in the alligator is different from the evolutionary loss of cartilage cone hypothesized for early archosaurs. Instead, the role of cartilage thinning, fibrocartilage sleeve development and calcified epiphyseal cartilage may be highly associated within different sauropsid lineages in response to evolutionary changes in joint loading regimes.Support or Funding InformationThis research is supported by the Missouri State University Faculty Research Grant

Cardiovascular adjustments with egg temperature at 90% incubation in embryonic American alligators, Alligator mississippiensis.

American alligators (Alligator mississippiensis) deposit eggs in a mound nest, potentially subjecting embryos to daily variations in temperature. Whilst adult crocodilian cardiovascular responses to changes in temperature have been investigated, similar studies in alligator embryos are limited. We investigated cardiovascular function of embryonic alligators during heating and cooling as well as at different temperatures. We measured arterial blood pressure (Pm) and heart rate (fH) in response to cooling (30-26°C), heating (26-36°C), followed by a reciprocal cooling event (36-26°C) and assessed the cardiac baroreflex at 30 and 36°C. Embryonic fH increased during heating events and decreased during cooling events, while embryos were hypotensive at 26 and 36°C, although Pm did not differ between heating or cooling events. There was a clear temperature-dependent heart rate hysteresis at a given embryo's temperature, depending on whether embryos were cooling or heating. Cardiovascular regulation through the cardiac limb of the baroreflex was not affected by temperature, despite previous studies suggesting that vagal tone is present at both low and high temperatures.

Metabolic responses to chronic hypoxic incubation in embryonic American alligators (Alligator mississippiensis)

Chronic hypoxic incubation is a common tool used to study developmental changes in reduced O2 conditions, and it has been useful for identifying phenotypically plastic periods during ontogeny in laboratory settings. Reptilian embryos can be subjected to natural hypoxia due to nesting strategy, and recent studies have been important in establishing the phenotypic responses of several species to low developmental oxygen. In particular, the cardiovascular responses of American alligators (Alligator mississippiensis) to low developmental oxygen have been detailed, including a substantial cardiac enlargement that may support a higher mass specific metabolic rate. However, embryo mass-specific metabolic demands of hypoxic incubated alligator embryos have not been measured. In this study, alligator eggs were incubated in 10% O2 (H) or 21% O2 (N) environments for the entire course of embryonic development. Acute metabolic measures in 21% and 10% O2 were taken for both H and N groups. We hypothesized that acute 10% O2 exposure has no impact on metabolic rate of embryonic alligators, and that metabolic rate is unaffected by chronic hypoxic incubation when studied in embryos measured at 21% O2. Our findings suggest phenotypic changes resulting from hypoxic incubation early in incubation, in particular relative cardiac enlargement, enable embryonic alligators to sustain metabolic rate during acute hypoxic exposure.

Blood flow distribution following adrenergic blockade and nitric oxide production inhibition in embryonic American alligators, Alligator mississippiensis (879.4)

Assessment of cardiovascular function in developing egg‐laying amniotes has been primarily limited to measurements made via catheters in the chorioallantoic membrane (CAM) and potentially invasive femoral and major outflow tract flow probes. Here we assess the capacity of embryonic alligators, Alligator mississippiensis, to regulate blood flow distribution using a minimally invasive microsphere distribution technique at 70 and 90% of development, in eggs incubated in normoxia or 10% hypoxia. Measurements were made in normoxia, acute 10% hypoxia, and acute 10% hypoxia following nitric oxide inhibition or alpha‐receptor blockade. We hypothesized that the embryonic response is concomitant with a redistribution of blood flow to the brain, heart, and CAM vasculature. Embryonic alligators respond to hypoxia with an initial bradycardia followed by a hypertensive tachycardia, however chronic hypoxic incubation blunts this response. Our data suggest that cardiac output is reduced during acute hypoxia in normoxic‐incubated embryos while it is maintained the hypoxic group. In addition, tonic nitric oxide dilation is present at 70% of incubation however inhibition of this tone does not modify total cardiac output at 90% of incubation. Our data suggest that hypoxic development may modify the patterns of blood flow redistribution in embryonic alligators and the mechanisms used to achieve this.Grant Funding Source: NSF CAREER IBN IOS‐0845741 to DAC

Chronic hypoxic incubation blunts thermally dependent cholinergic tone on the cardiovascular system in embryonic American alligator (Alligator mississippiensis)

Environmental conditions play a major role in shaping reptilian embryonic development, but studies addressing the impact of interactions between chronic and acute environmental stressors on embryonic systems are lacking. In the present study, we investigated thermal dependence of cholinergic and adrenergic cardiovascular tone in embryonic American alligators (Alligator mississippiensis) and assessed possible phenotypic plasticity in a chronic hypoxic incubation treatment. We compared changes in heart rate (f H) and mean arterial blood pressure (P M) for chronically hypoxic and normoxic-incubated embryos after cholinergic and adrenergic blockade following three different acute temperature treatments: (1) 30°C (control incubation temperature), (2) acute, progressive decrease 30-24°C then held at 24°C, and (3) acute, progressive increase 30-36°C then held at 36°C. f H progressively fell in response to decreasing temperature and rose in response to increasing temperature. P M did not significantly change with decreasing temperature, but was lowered significantly with increasing acute temperature in the normoxic group at 90% of development only. Propranolol administration (β adrenergic antagonist) produced a significant f H decrease at 24, 30, and 36°C that was similar at all temperatures for all groups. For normoxic-incubated embryos at 90% of development, atropine administration (cholinergic antagonist) significantly increased f H in both 24 and 36°C treatments, but not in the 30°C control treatment. This atropine response at 24 and 36°C demonstrated acute thermally dependent cholinergic tone on f H late in development for normoxic-incubated, but not chronically hypoxic-incubated embryos. Collectively, data indicated that cardiovascular control mechanisms in embryonic alligators may be activated by thermal extremes, and the maturation of control mechanisms was delayed by chronic hypoxia.

Development of sympathetic cardiovascular control in embryonic, hatchling, and yearling female American alligator (Alligator mississippiensis)

We used arterial tyramine injections to study development of sympathetic actions on in vivo heart rate and blood pressure in embryonic, hatching and yearling female American alligators. Tyramine is a pharmacological tool for understanding comparative and developmental sympathetic regulation of cardiovascular function, and this indirect sympathomimetic agent causes endogenous neuronal catecholamine release, increasing blood pressure and heart rate. Arterial tyramine injection in hatchling and yearling alligators caused the typical vertebrate response — rise in heart rate and blood pressure. However, in embryonic alligators, tyramine caused a substantial and immediate bradycardia at both 70% and 90% of embryonic development. This embryonic bradycardia was accompanied by hypotension, followed by a sustained hypertension similar to the hatchling and juvenile responses. Pretreatment with atropine injection (cholinergic receptor blocker) eliminated the embryonic hypotensive bradycardia, and phentolamine pretreatment (α-adrenergic receptor blocker) eliminated the embryonic hypotensive and hypertensive responses but not the bradycardia. In addition, hexamethonium pretreatment (nicotinic receptor blocker) significantly blunted embryos' bradycardic tyramine response. However, pretreatment with 6-hydroxydopamine, a neurotoxin that destroys catecholaminergic terminals, did not eliminate the embryonic bradycardia. Tyramine likely stimulated a unique embryonic response — neurotransmitter release from preganglionic nerve terminals (blocked with hexamethonium) and an acetylcholine mediated bradycardia with a secondary norepinephrine-dependent sustained hypertension. In addition, tyramine appears to stimulate sympathetic nerve terminals directly, which contributed to the overall hypertension in the embryonic, hatchling and yearling animals. Data demonstrated that humoral catecholamine control of cardiovascular function was dominant over the immature parasympathetic nervous system in developing alligator embryos, and suggested that sympathetic and parasympathetic nerve terminals were present and developing in ovo but were not tonically active.

Characterizing calcium influx via voltage- and ligand-gated calcium channels in embryonic Alligator neurons in culture

Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarization-induced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist.

Effects of dehydration on cardiovascular development in the embryonic American alligator (Alligator mississipiensis)

Effects of dehydration on reptilian embryonic cardiovascular function are unknown. Here, we present the first morphological and physiological data quantifying the cumulative effects of four acute dehydration events on the embryonic American alligator, Alligator mississipiensis. We hypothesized that dehydration would alter embryonic morphology, reduce blood volume and augment the response to angiotensin II (Ang II), a key osmotic and blood volume regulatory response element in adult vertebrates. Drying events at 30%, 40%, 50%, and 60% of embryonic incubation reduced total egg water content by 14.43±0.37g, a 3.4 fold increase relative to controls. However, embyronic blood volume was greater in the dehydration group at 70% of embryonic incubation compared to controls (0.39±0.044mLg−1 and 0.22±0.03mLg−1, respectively), however, both groups were similar at 90% of incubation (0.18±0.02mLg−1 in the controls and 0.23±0.03mLg−1 in the dehydrated group). Dehydration altered the morphological phenotype and resulted in an overall reduction in embryonic mass at both incubation time points measured. Dehydration also altered the physiological phenotype, resulting in embryonic alligators that were relatively bradycardic at 90% of incubation. Arterial Ang II injections resulted in a dose dependent hypertension, which increased in intensity over the span of incubation studied. While progressive incubation altered the Ang II response, dehydration had no impact on the cardiovascular responses to the peptide. Quantification of Ang II type-1 receptor protein using western blot analysis illustrated that dehydration condition and incubation time point did not alter protein quantity. Collectively, our results show that dehydration during embryonic development of the American alligator alters embryonic morphology and baseline heart rate without altering arterial pressure and response to Ang II.

Morphology of the embryonic and hatchling american alligator ductus arteriosi and implications for embryonic cardiovascular shunting

The ductus arteriosi (DA) are embryonic blood vessels found in amniotic vertebrates that shunt blood away from the pulmonary artery and lungs and toward the aorta. Here, we examine changes in morphology of the right and left DA (LDA), and right and left aorta (LAo) from embryonic and hatchling alligators. The developing alligator has two-patent DA that join the right and LAo. Both DA exhibit a muscular phenotype composed of an internal smooth muscle layer (2-4 cells thick). At hatching, the lumen diameter of both DA decreases as the vessels begin to close within the first 12 h of posthatch life. Between day 1 and day 12 posthatching, the vessel becomes fully occluded with endothelial and smooth muscle cells filling the lumen. A number of DA from hatchlings contained blood clots along their length. The lumen of the full term alligator DA is reduced in comparison with the full term chicken DA. The developing alligator embryo has an additional right-to-left shunt pathway in the LAo arising from the right ventricle. The embryonic LAo diameter is twice the diameter of either the right DA or LDA, providing a lower resistance pathway for blood leaving the right ventricle. On the basis of these findings, we propose that the paired DA of the embryonic alligator have a reduced role in the embryonic right-to-left shunt of blood from the right ventricle when compared with the avian DA.

Hypoxic alligator embryos: Chronic hypoxia, catecholamine levels and autonomic responses of in ovo alligators

Hypoxia is a naturally occurring environmental challenge for embryonic reptiles, and this is the first study to investigate the impact of chronic hypoxia on the in ovo development of autonomic cardiovascular regulation and circulating catecholamine levels in a reptile. We measured heart rate (f H) and chorioallantoic arterial blood pressure (MAP) in normoxic (‘N21’) and hypoxic-incubated (‘H10’; 10% O 2) American alligator embryos ( Alligator mississippiensis) at 70, 80 and 90% of development. Embryonic alligator responses to adrenergic blockade with propranolol and phentolamine were very similar to previously reported responses of embryonic chicken, and demonstrated that embryonic alligator has α and β-adrenergic tone over the final third of development. However, adrenergic tone originates entirely from circulating catecholamines and is not altered by chronic hypoxic incubation, as neither cholinergic blockade with atropine nor ganglionic blockade with hexamethonium altered baseline cardiovascular variables in N21 or H10 embryos. In addition, both atropine and hexamethonium injection did not alter the generally depressive effects of acute hypoxia — bradycardia and hypotension. However, H10 embryos showed significantly higher levels of noradrenaline and adrenaline at 70% of development, as well as higher noradrenaline at 80% of development, suggesting that circulating catecholamines reach maximal levels earlier in incubation for H10 embryos, compared to N21 embryos. Chronically elevated levels of catecholamines may alter the normal balance between α and β-adrenoreceptors in H10 alligator embryos, causing chronic bradycardia and hypotension of H10 embryos measured in normoxia.

Chronic hypoxic incubation blunts a cardiovascular reflex loop in embryonic American alligator (Alligator mississippiensis)

Hypoxia is a naturally occurring environmental challenge for embryonic non-avian reptiles, and this study is the first to investigate the impact of chronic hypoxia on a possible chemoreflex loop in a developing non-avian reptile. We measured heart rate and blood pressure in normoxic and hypoxic-incubated (10% O(2)) American alligator embryos (Alligator mississippiensis) at 70 and 90/95% of development. We hypothesized that hypoxic incubation would blunt embryonic alligators' response to a reflex loop stimulated by phenylbiguanide (PBG), a 5-HT(3) receptor agonist that stimulates vagal pulmonary C-fiber afferents. PBG injection caused a hypotensive bradycardia in 70 and 95% of development embryos (paired t tests, P<0.05), a response similar to mammals breathing inspired air (all injections made through occlusive catheter in tertiary chorioallantoic membrane artery). Hypoxic incubation blunted the bradycardic response to PBG in embryos at 95% of development (two-way ANOVA, P<0.01). We also demonstrated that the vagally mediated afferent limb of this reflex can be partially or completely blocked in ovo with a 5-HT(3) receptor blockade using ondansetron hydrochloride dihydrate (OHD), with a ganglionic blockade using hexamethonium, or with a cholinergic blockade using atropine. Atropine eliminated the hypotensive and bradycardic responses to PBG, and OHD and hexamethonium significantly blunted these responses. This cardiovascular reflex mediated by the vagus was affected by hypoxic incubation, suggesting that reptilian sympathetic and parasympathetic reflex loops have the potential for developmental plasticity in response to hypoxia. We suggest that the American alligator, with an extended length of time between each developmental stage relative to avian species, may provide an excellent model to test the cardiorespiratory effects of prolonged exposure to changes in atmospheric gases. This extended period allows for lengthy studies at each stage without the transition to a new stage, and the natural occurrence of hypoxia and hypercapnia in crocodilian nests makes this stress ecologically and evolutionarily relevant.

Role of the left aortic arch and blood flows in embryonic American alligator (Alligator mississippiensis)

All embryonic and fetal amniotes possess a ductus(i) arteriosus(i) that allows blood to bypass the pulmonary circulation and the non-functional lungs. The central hemodynamic of embryonic reptiles are unique, given the additional systemic aorta that allows pulmonary circulatory bypass, the left aorta (LAo). The LAo exits in the right ventricle or ‘pulmonary side’ of reptilian hearts in both embryos and adults, but its functional significance in ovo is unknown. This study investigated the role of the LAo in embryonic American alligators by surgically occluding the LAo and measuring oxygen consumption and, in addition, measured hemodynamic responses to hypoxia in embryonic alligators. We measured systemic cardiac output and primary chorioallantoic membrane (CAM) artery blood flow for normoxic and hypoxic-incubated (10% O2) American alligator embryos (Alligator mississippiensis). Chronic blood flow (1–124 h) in the primary CAM artery for hypoxic-incubated embryos (92 ± 26 ml min−1 kg−1) was elevated when compared with normoxic-incubated embryos (29 ± 14 ml min−1 kg−1, N = 6; P = 0.039). For hypoxic-incubated embryos, acute LAo blood flow (49.6 ± 24.4 ml min−1 kg−1) was equivalent to the combined flow of the three systemic great vessels that arise from the left ventricle, the right aorta, common carotid and subclavian arteries (43.6 ± 21.5 ml min−1 kg−1, N = 5). Similarly, for normoxic-incubated embryos, LAo blood flow (27.3 ± 6.6 ml min−1 kg−1) did not statistically differ from the other three vessels (18.4 ± 4.9 ml min−1 kg−1, N = 5). This study contains the first direct test of LAo function and the first measurements of blood flow in an embryonic reptile. These data support the hypotheses that embryonic alligators utilize the LAo to divert a significant amount of right ventricular blood into the systemic circulation, and that CAM blood flow increases following chronic hypoxic conditions. However, surgical occlusion of the LAo did not affect egg dot{V}_{{text{O}}_{2}}, supporting the hypothesis that the LAo of reptiles is not critical to maintain in ovo oxygen consumption.Electronic supplementary materialThe online version of this article (doi:10.1007/s00360-010-0494-6) contains supplementary material, which is available to authorized users.

Cardiovascular plasticity during hypoxic incubation of alligator (Alligator mississippiensis)

The vertebrate cardiovascular (CV) system becomes operational early in development and must function and mature both anatomically and physiologically. Deviations in developmental environment create challenges that impact CV function and maturation. Reptilian embryos may be particularly impacted given features of development in egg‐laying species, such as hypoxia. Using chronic hypoxic incubation as a stressor (10% O2 beginning at 20% of development), the plasticity of the CV system was assessed in American alligator embryos. Compared to normoxic‐incubated embryos, chronic hypoxic incubation resulted in bradycardia at 90% of incubation, but differing effects on arterial pressure. Assessments of both acute hypoxic and chemically induced chemoreflex indicated maturation of this regulatory mechanism is plastic during embryonic development and blunted by chronic hypoxic incubation. In addition, roles of the sympathetic and parasympathetic nervous systems are altered by hypoxia. These data indicate that maturation of CV function can exhibit a significant degree of phenotypic plasticity in embryonic alligators. NSF Career award IBN IOS‐0845741 to DAC and NSF IOS‐0922756 to JWH.

Phylogeny of the Telencephalic Subventricular Zone in Sauropsids: Evidence for the Sequential Evolution of Pallial and Subpallial Subventricular Zones

The telencephalon of birds and placental mammals harbors a proliferative subventricular zone (SVZ) in the subpallium as well as the pallium. Turtles, which are phylogenetically intermediate between bird, and mammals, exhibit at best a rudimentary SVZ. This suggests that SVZs evolved independently in mammals and birds, but it is not clear whether subpallial and pallial SVZs evolved with the origin of birds or in some earlier, non-avian sauropsid ancestor. To answer this question, we examined the brains of embryonic alligators (Ferguson stages 15–22) because crocodilians are the closest extant sister group to birds. To visualize the SVZ we labeled mitotic cells with antibodies against phosphorylated histone-3 (pH3) and proliferating cells with antibodies against proliferating cell nuclear antigen (PCNA). We found that the telencephalon of alligators contains an SVZ only in the subpallium. Because turtles, lizards and amphibians seem to lack SVZs, our finding suggests that a subpallial SVZ evolved in the last common ancestor of birds and crocodilians. Given that placental mammals and birds, but not marsupial mammals or reptiles, possess an SVZ within their pallium, we conclude that a pallial SVZ probably evolved independently in birds and placental mammals.

Effects of incubation temperature and estrogen exposure on aromatase activity in the brain and gonads of embryonic alligators.

During embryogenesis, incubation temperature and the hormonal environment influence gonadal differentiation of some reptiles, including all crocodilians. Current evidence suggests that aromatase, the enzyme that converts androgens to estrogens, has a role in sexual differentiation of species that exhibit temperature-dependent sex determination (TSD). During the temperature-sensitive period (TSP) of sex determination, we compared aromatase activity in the brain and gonads of putative male and female alligator embryos to determine if aromatase activity in the embryonic brain could provide the hormonal environment necessary for ovarian development in a TSD species. In addition, we assessed the pattern of aromatase activity in the brain and gonads of embryos treated with estradiol-17beta (E(2)) and incubated at male-producing temperatures to compare enzyme activity in E(2) sex-reversed females to control males and females. This has particular significance regarding wildlife species living in areas contaminated with suspected environmental estrogens. Gonadal aromatase activity remained low during the early stages of the TSP in both sexes and increased late in the TSP only in females. Aromatase activity in the brain increased prior to gonadal differentiation in both sexes. These results suggest that aromatase activity in the brain is not directly responsible for mediating differentiation of the gonad. E(2) exposure at male-producing temperatures resulted in sex-reversed females that had intermediate gonad function and masculinized brain activity. This study indicates the need to examine multiple end points and to determine the persistence of developmental alterations in contaminant-exposed wildlife populations.

Scale morphogenesis and ultrastructure of dermis during embryonic development in the alligator (Alligator mississippiensis, Crocodilia, Reptilia)

AbstractFormation of scales in different body regions of embryonic alligators is described using light and electron microscopy. Transformation of the skin surface to produce scales takes place between stages 19 and 23, after which the shape of scales is complete over most of the embryonic surface. Scalation is not synchronous; different regions develop scales at different rates. Initially scales are formed on the back and dorsal side of the proximal tail and appear as undulations of the epidermis which form symmetrical (bumps) or asymmetrical (serrated) scale anlagen. No dermal condensations are apparent beneath the epidermis, although in some areas of the skin (belly, limbs) mesenchymal cells are more numerous within the bumps than in other areas. At stage 21, scalation has spread to the neck and belly but is absent or poorly developed over most areas of the flank, gular, jaw, limb and head regions. Grooves form between the outer edges of adjacent scales or interbump regions. A superficial denser dermis and a reticulated deep subdermis are visible in many scales from stage 21. The dermis forms a superficial loose and a deep dense layer from stage 22. Both loose and deep dermis, and sometimes the deep reticulate subdermis, move towards the surface to form the dermal core of scales, although the mechanism of this movement is not known. Bundles of collagen fibrils, with almost no elastic fibrils, are progressively deposited, especially in the denser dermis. At stage 22, the flank, gular and proximal areas of limbs form scales, but the head, jaw, distal limbs and digits still lack scales. The digits become scaled at stage 23 when scalation is well advanced in the other regions. By stage 24 most of the body is scaled and subsequent scale modifications occur only by growth. Five main types of scales are recognized by their shape: symmetrical scutes, asymmetrical scutes, overlapping scutes, tuberculate scales, and elevated asymmetrical scutes (tail verticils). Pigmentation, mainly due to epidermal melanocytes, is visible at embryonic stage 23 and progresses through stages 24 and 25.