Stress hormones mediate developmental plasticity in vertebrates with complex life cycles

Abstract

The environment experienced by developing organisms can shape the timing and character of developmental processes, generating different phenotypes from the same genotype, each with different probabilities of survival and performance as adults. Chordates have two basic modes of development, indirect and direct. Species with indirect development, which includes most fishes and amphibians, have a complex life cycle with a free-swimming larva that is typically a growth stage, followed by a metamorphosis into the adult form. Species with direct development, which is an evolutionarily derived developmental mode, develop directly from embryo to the juvenile without an intervening larval stage. Among the best studied species with complex life cycles are the amphibians, especially the anurans (frogs and toads). Amphibian tadpoles are exposed to diverse biotic and abiotic factors in their developmental habitat. They have extensive capacity for developmental plasticity, which can lead to the expression of different, adaptive morphologies as tadpoles (polyphenism), variation in the timing of and size at metamorphosis, and carry-over effects on the phenotype of the juvenile/adult. The neuroendocrine stress axis plays a pivotal role in mediating environmental effects on amphibian development. Before initiating metamorphosis, if tadpoles are exposed to predators they upregulate production of the stress hormone corticosterone (CORT), which acts directly on the tail to cause it to grow, thereby increasing escape performance. When tadpoles reach a minimum body size to initiate metamorphosis they can vary the timing of transformation in relation to growth opportunity or mortality risk in the larval habitat. They do this by modulating the production of thyroid hormone (TH), the primary inducer of metamorphosis, and CORT, which synergizes with TH to promote tissue transformation. Hypophysiotropic neurons that release the stress neurohormone corticotropin-releasing factor (CRF) are activated in response to environmental stress (e.g., pond drying, food restriction, etc.), and CRF accelerates metamorphosis by directly inducing secretion of pituitary thyrotropin and corticotropin, thereby increasing secretion of TH and CORT. Although activation of the neuroendocrine stress axis promotes immediate survival in a deteriorating larval habitat, costs may be incurred such as reduced tadpole growth and size at metamorphosis. Small size at transformation can impair performance of the adult, reducing probability of survival in the terrestrial habitat, or fecundity. Furthermore, elevations in CORT in the tadpole caused by environmental stressors cause long term, stable changes in neuroendocrine function, behavior and physiology of the adult, which can affect fitness. Comparative studies show that the roles of stress hormones in developmental plasticity are conserved across vertebrate taxa including humans.

Keywords: Amphibian; Corticotropin-releasing factor; Developmental plasticity; Glucocorticoid; Metamorphosis; Thyroid hormone.

Fig. 1

Amphibian metamorphosis. A. Shown are the broad stages of tadpole (Xenopus laevis) metamorphosis using the terms coined by Etkin (Etkin, 1968). B. Corticotropin-releasing factor (CRF) is a thyrotropin (TSH)-releasing factor in tadpoles and other nonmammalian species. It is also a corticotropin (ACTH)-releasing factor in these species, although arginine vasotocin has a more prominent role. Neurosecretory CRF neuron cell bodies located in the anterior preoptic area (POa) respond to environmental stressors, releasing their contents into the pituitary portal system to control thyroid and interrenal (amphibian homolog of the mammalian adrenal cortex) activity. The Xenopus neuroanatomical diagrams are from Tuinhof and colleagues (Tuinhof et al., 1998). Tadpole photos by David Bay.

Fig. 2

The corticotropin-releasing factor (CRF) binding protein (CRF-BP) is expressed in tadpole tail where it modulates CRF bioavailability. Corticotropin-releasing factor is cytoprotective in the tadpole tail, helping to maintain tail viability for locomotion. At metamorphic climax the tail resorbs, and at this time the CRF-BP is upregulated under the control of thyroid hormone (TH). A. Immunoreactivity for CRF-BP can be induced preciously by treatment of premetamorphic tadpoles with TH. Nieuwkoop-Faber stage 50 tadpoles were treated with vehicle or 100 nM 3,5,3′-triiodothyronine (T₃) for 4 days by addition to their aquarium water. Shown are representative transverse cryosections (12 μm) of tadpole tail immunostained with an affinity-purified rabbit polyclonal antiserum (#3809; used at 1:30 dilution) generated against a synthetic peptide corresponding to amino acids 112–125 of Xenopus CRF-BP (FDGWIIKGEKFPSS) conjugated to keyhole limpet hemocyanin. Immune complexes were revealed using a goat anti-rabbit IgG conjugated to horse radish peroxidase using the Vectastain elite ABC kit and Vector VIP kit (both from Vector Laboratories, Inc., Burlingane, CA, USA). The right most panel shows a representative tail section from a TH-treated tadpole stained with anti-CRF-BP serum that had been preabsorbed with the synthetic peptide used as immunogen. The affinity purified antiserum was incubated with synthetic Xenopus CRF-BP peptide (100 μg/ml) in a 50 μl volume overnight at 4 °C before immunohistochemistry. All procedures involving animals were conducted in accordance with the guidelines of the University Committee on the Care and Use of Animals of the University of Michigan. B. Representative images of GFP expression in tadpole tail muscle cells (NF stage 58) in vivo after electroporation-mediated gene transfer. Muscle cells were co-electroporated with pEGFP-N3 and one of the following: TOPO-xCRF, CMV-xCRF-BP (Xenopus CRF-BP expression vector), CMV-mCRF-BP (mouse CRF-BP expression vector), or CMVneo (empty vector). Tadpoles were then reared in aquaria and GFP fluorescence imaged every two days thereafter. The graph to the right shows the quantification of the average GFP intensity in electroporated tail muscle cells over the final 4 days of the experiment. Shown are the means ± SEM (n = 6–8/treatment). * Significant difference from CMV-Neo transfected cells (Scheffe's post hoc test, P < 0.05) (reprinted from Boorse et al., 2006).

Fig. 3

Tadpoles accelerate metamorphosis when their pond dries. A. Graphic showing the acceleration of metamorphosis in response to pond drying in amphibian species that breed in arid environments, such as the Western spadefoot toad, Spea hammondii. Tadpoles that accelerate development in response to pond drying metamorphose earlier and at a smaller body size. This leads to tradeoffs between survival in the larval habitat and reduced post-metamorphic performance owing to smaller body size (graphic by Roberto Osti reprinted with permission; the graphic is based on original artwork by Leif Saul). B. Acceleration of metamorphosis of S. hammondii tadpoles caused by water volume reduction in the laboratory. The water level was maintained at either a constant high level (10 L) or decreasing (from 10 to 0.5 L; see bottom left panel). The top two panels show the frequency distributions of the two treatments for the numbers of animals metamorphosing by time since hatching. The bottom right panel shows the mean age at metamorphosis for the two treatments (mean ± SEM; n = 4 tanks/treatment; the asterisk indicates significant difference at p < 0.001; Student's unpaired t-test). C. The developmental response of S. hammondii tadpoles to a decreasing water level varied continuously in relation to the rate of change in the water level. Prometamorphic tadpoles (10 animals/tank) were exposed to different volume reduction regimes as shown in the bottom left panel (regimes 1–5). The bar graphs show mean age, body mass (BM) and snout-vent length (SVL) at metamorphosis (n = 3 tanks/treatment); error bars represent SEM (reprinted from Denver et al., 1998).

Fig. 4

Corticotropin releasing factor peptides accelerate tadpole metamorphosis. A. Acceleration of tadpole metamorphosis by the CRF peptide sauvagine (SV). Premetamorphic Spea hammondii tadpoles were injected intraperitoneally with vehicle or SV every other day for 10 days. Shown are changes in Gosner (Gosner, 1960) developmental stage (top), body weight (BW) and hind limb length (HLL). Each point is the mean + SEM (n = 10–12 animals/time point), and asterisks indicate statistically significant differences between vehicle and SV (p < 0.05). B. Injection of synthetic Xenopus CRF caused a rapid (by 6 h), dose-dependent increase in whole-body thyroxine, 3,5,3′-triiodothyronine and corticosterone content in S. hammondii tadpoles. Asterisks indicate the minimum effective dose; this dose and all doses higher were significantly different from the zero dose (p < 0.05; Student's unpaired t-test). C. Antagonism of endogenous CRF by injection of the CRF receptor antagonist a-helCRF_(9–41) (top; open symbols = a-helCRF_(9–41), closed symbols = vehicle) or rabbit antiserum to frog CRF (bottom; open symbols = anti-CRF, closed symbols = normal serum) blocked the developmental response of S. hammondii tadpoles to experimental pond drying. Dashed lines and circles indicate a decreasing water level, while solid lines and triangles indicate a constant high water level. Each point is the mean + SEM (n = 8 animals/treatment and time point). Metamorphic climax (Gosner stage 42) is indicated by the horizontal dotted line in the upper part of the graph (reprinted from Denver, 1997b).

Fig. 5

Corticosterone synergizes with thyroid hormone to accelerate tadpole metamorphosis. Tail explants from premetamorphic Xenopus laevis tadpoles were cultured for one week in the presence of 3,5,3′-triiodothyronine (T₃) or corticosterone (CORT) or both as indicated. A. Changes in the percent initial tail area over the 7 day culture period are shown. Each point is the mean ± SEM (n = 7/treatment; the statistical analysis was done on the actual tail areas; one-way ANOVA). B. Changes in the final dry weight of tadpole tail explants. The bars represent the mean ± SEM (n = 7/treatment; means with the same letter are not significantly different; Fisher's LSD post hoc test, p < 0.05). Representative images of tails from each treatment group at the 7 day time point are shown below the graphs (reprinted from Bonett et al., 2010).

Fig. 6

Ranid tadpoles release an alarm pheromone from their skin when attacked by a predator that causes rapid freezing behavior and suppression of the HPI axis in conspecifics. A. An alarm pheromone is produced by ranid tadpoles and is released via a stimulus-secretion coupled pathway. The graphs show the mean time spent swimming of tadpoles exposed to the different treatments. (1) Tadpoles reduced activity when exposed to predator-conditioned water (dragon fly larvae fed live tadpoles) but not by euthanized tadpoles homogenized in water. (2) Tadpoles reduced activity when exposed to water conditioned by tadpoles poked with a hypodermic needle, but not by dragon fly larvae fed dead tadpoles. (3) Tadpoles reduced activity when exposed to water conditioned by tadpoles that had been immersed in 5 mM potassium chloride (KCl), but not by tadpoles immersed in water alone or the KCl alone. (4) Tadpoles reduced activity when exposed to a homogenate made with euthanized tadpoles in 1% Triton X-100, but not tadpoles homogenized in water alone. Pred — predator; Tad — tadpole; homog — tadpole homogenate; Triton — Triton X-100. The bars show the mean ± SEM; means with the same letter within an experiment are not significantly different (p < 0.05). B. Exposure of tadpoles to alarm pheromone reduced whole-body corticosterone (CORT) content in a time and dose-dependent manner. Tadpoles were exposed to the tadpole Triton X-100 homogenate and then sacrificed at different times for analysis of whole-body CORT content (top graph). The alarm pheromone caused a dose-dependent suppression of tadpole whole-body CORT content (bottom graph). Tadpoles were exposed to the homogenate for 4 h before sacrifice and analysis of whole-body CORT content. C. Reversing the decline in endogenous CORT caused by exposure to the alarm pheromone through treatment with CORT partially blocked the anti-predator behavior. Corticosterone was added to the aquarium water to a final concentration of 50 nM. Controls received an equal volume of ethanol vehicle (final concentration 0.001%). Triton X-100 tadpole homogenate was added to the tanks in 20 μl aliquots at 15 min intervals. Shown is the mean time tadpoles spent swimming±SEM. Asterisks indicate significant differences between the CORT treated and control groups at the indicated doses (p < 0.05) (reprinted from Fraker et al., 2009).

Fig. 7

Exposure to predators induced anti-predator morphology in wood frog tadpoles. A. Gray treefrog tadpoles exposed to predators developed tail coloration that discourages predation (an aposematic response), and changes in body morphology that enhances predator avoidance (photo by Michael Benard). B. Exposure of tadpoles to the non-lethal presence of predators caused a biphasic response in whole-body CORT content. Tadpoles were exposed to caged aeshnid predators fed conspecific tadpoles in mesocosms and sampled at the indicated times for measurement of whole-body CORT content. The tadpoles initially reduced whole-body CORT content at 4 h after exposure, but then elevated CORT content by 4 days after exposure (time × treatment interaction: p < 0.001; one-way ANOVA). Each point represents the mean ± SEM (n = 10 animals/treatment and time point). C. Exposure of tadpoles to alarm pheromone or CORT generated similar anti-predator morphology, and the effect of the alarm pheromone was blocked by co-treatment with the corticosteroid synthesis inhibitor metyrapone (MTP). Tadpoles were treated with alarm pheromone (Pred), CORT (125 nM) or Pred plus MTP (110 mM). Tail height (top graph) and trunk length (bottom graph) were corrected for body weight. Both measures were significantly affected by the treatments (p < 0.05). Tadpoles treated with alarm pheromone or CORT both developed deeper tails and shorter trunks compared with controls, and were not significantly different from each other in either measure. Tadpoles treated with alarm pheromone plus MTP had shallower tails and longer trunks than tadpoles treated with alarm pheromone alone. Each point is the mean ± SEM (n = 16/treatment) (reprinted from Middlemis-Maher et al., 2013).

Fig. 8

Treatment of premetamorphic Xenopus laevis tadpoles with CORT to mimic a stress response decreased brain glucocorticoid receptor (GR), increased anxiogenic behavior and increased brain corticotropin-releasing factor (CRF) in juvenile frogs. Prometamorphic tadpoles (NF stage 56–57) were treated with vehicle (0.001% ethanol) or corticosterone (CORT; 100 nM) for 5 days by addition to their aquarium water as described (Hu et al., 2008). All analyses were done two months after metamorphosis. A. Treatment of prometamorphic tadpoles with CORT decreased GR-ir in the brains of juvenile frogs, (GR-ir analyzed on cryosections of frog brain as described by Yao et al., 2008a; n = 5/treatment). Shown are the anterior preoptic area (POa) and medial pallium (homolog of mammalian hippocampus). Similar changes in GR-ir were seen in the amygdala and bed nucleus of the stria terminals (BNST; not shown). B. Treatment of tadpoles with CORT increased anxiogenic behavior of juvenile frogs. Frogs were placed individually into tanks (n = 7/treatment), the baseline behavior was monitored for one hr (which did not differ between treatments – data not shown), then individual frogs were subjected to a single negative stimulus (tapping on the tank with a pen every two seconds for one minute with enough force to startle the frog). Frog behavior was recorded for one hr using a closed-circuit camera, and activity level was scored (time spent stationary or moving). C. Treatment of prometamorphic tadpoles with CORT caused a large increase in CRF-ir in the region of the medial amygdala (MeA) and BNST of the brains of juvenile frogs. The CRF-ir was analyzed on cryosections of frog brain as described (Yao et al., 2004; n = 5/treatment). * p < 0.05 Student's unpaired t-test.