Robert J. Denver

ADAPTIVE PLASTICITY IN AMPHIBIAN METAMORPHOSIS: RESPONSE OF SCAPHIOPUS HAMMONDIITADPOLES TO HABITAT DESICCATION

Amphibians exhibit extreme plasticity in the timing of metamorphosis, and several species have been shown to respond to water availability, accelerating metamorphosis when their ponds dry. In this study we analyzed the plasticity of the developmental response to water volume reduction in the western spadefoot toad, Scaphiopus hammondii. Also, we attempted to identify the environmental cue(s) that may signal a desiccating larval habitat. We spawned adults in the laboratory and raised tadpoles in aquaria in a controlled environmental chamber. Water levels of aquaria were gradually reduced by removing water at the rate of 0.5–1 L/d; water in control aquaria was similarly disturbed but not removed. Tadpoles subjected to water volume reduction showed significant acceleration of metamorphosis. The developmental acceleration depended on the rate of reduction of the water level; i.e., tadpoles exhibited a continuum of response. This developmental response did not result from thermal differences between treatments...

Environmental Stress as a Developmental Cue: Corticotropin-Releasing Hormone Is a Proximate Mediator of Adaptive Phenotypic Plasticity in Amphibian Metamorphosis

Environmentally induced phenotypic plasticity allows developing organisms to respond adaptively to changes in their habitat. Desert amphibians have evolved traits which allow successful development in unpredictable environments. Tadpoles of these species can accelerate metamorphosis as their pond dries, thus escaping mortality in the larval habitat. This developmental response can be replicated in the laboratory, which allows elucidation of the underlying physiological mechanisms. Here I demonstrate a link between a classical neurohormonal stress pathway (involving corticotropin-releasing hormone, CRH) and the developmental response to habitat desiccation. Injections of CRH-like peptides accelerated metamorphosis in western spadefoot toad tadpoles. Conversely, treatment with two CRH antagonists, the CRH receptor antagonist alpha-helical CRH(9-41) and anti-CRH serum, attenuated the developmental acceleration induced by habitat desiccation. Tadpoles subjected to habitat desiccation exhibited elevated hypothalamic CRH content at the time when they responded developmentally to the declining water level. CRH injections elevated whole-body thyroxine, triiodothyronine, and corticosterone content, the primary hormonal regulators of metamorphosis. In contrast, alpha-helical CRH(9-41) reduced thyroid activity. These results support a central role for CRH as a neurohormonal transducer of environmental stimuli into the endocrine response which modulates the rate of metamorphosis. Because in mammals, increased fetal/placental CRH production may initiate parturition, and CRH has been implicated in precipitating preterm birth arising from fetal stress, this neurohormonal pathway may represent a phylogenetically ancient developmental regulatory system that allows the organism to escape an unfavorable larval/fetal habitat.

Stress hormones mediate environment-genotype interactions during amphibian development.

Environments experienced by organisms during early development shape the character and timing of developmental processes, leading to different probabilities of survival in the developmental habitat, and often profound effects on phenotypic expression later in life. Amphibian larvae have immense capacity for plasticity in behavior, morphology, growth and development rate. This creates the potential for extreme variation in the timing of, and size at metamorphosis, and subsequent phenotype in the juvenile and adult stage. Hormones of the neuroendocrine stress axis play pivotal roles in mediating environmental effects on animal development. Corticotropin-releasing factor, whose secretion by hypothalamic neurons is induced by environmental stress, influences the timing of amphibian metamorphosis by controlling the activity of the thyroid and interrenal (adrenal; corticosteroids) glands. At target tissues, corticosteroids synergize with thyroid hormone to promote metamorphosis. Thus, environmental stress acts centrally to increase the activity of the two principle endocrine axes controlling metamorphosis, and the effectors of these axes synergize at the level of target tissues to promote morphogenesis. While stress hormones can promote survival in a deteriorating larval habitat, costs may be incurred such as reduced tadpole growth and size at metamorphosis. Furthermore, exposure to elevated corticosteroids early in life can cause permanent changes in the expression of genes of the neuroendocrine stress axis, leading to altered physiology and behavior in the juvenile/adult stage. Persistent effects of stress hormone actions early in life may have important fitness consequences.

CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3-derived CLE ligand. The biochemical role of the receptor-like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2-CRN heteromultimeric and CLV1-BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane-localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3-derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand-binding receptor complexes controlling stem cell specification in Arabidopsis.

Structural and Functional Evolution of Vertebrate Neuroendocrine Stress Systems

The vertebrate hypothalamus–pituitary–adrenal (HPA; or interrenal) axis plays pivotal roles in animal development and in physiological and behavioral adaptation to environmental change. The HPA, or stress axis, is organized in a hierarchical manner, with feedback operating at several points along the axis. Recent findings suggest that the proteins, gene structures, and signaling pathways of the HPA axis were present in the earliest vertebrates and have been maintained by natural selection owing to their critical adaptive roles. In all vertebrates studied, the HPA axis is activated in response to stressors and is controlled centrally by peptides of the corticotropin‐releasing factor (CRF) family of which four paralogous members have been identified. Signaling by CRF‐like peptides is mediated by at least two distinct G protein‐coupled receptors and modulated by a secreted binding protein. These neuropeptides function as hypophysiotropins and as neurotransmitters/neuromodulators, influencing stress‐related behaviors, such as anxiety and fear. In addition to modulating HPA activity and behavioral stress responses, CRF‐like peptides are implicated in timing key life history transitions, such as metamorphosis in amphibians and birth in mammals. CRF‐like peptides and signaling components are also expressed outside of the central nervous system where they have diverse physiological functions. Glucocorticoids are the downstream effectors of the HPA axis, playing essential roles in development, energy balance and behavior, and feedback actions on the activity of the HPA axis.

Evolution of leptin structure and function.

Leptin, the protein product of the obese(ob or Lep) gene, is a hormone synthesized by adipocytes that signals available energy reserves to the brain, and thereby influences development, growth, metabolism and reproduction. In mammals, leptin functions as an adiposity signal: circulating leptin fluctuates in proportion to fat mass, and it acts on the hypothalamus to suppress food intake. Orthologs of mammalian Lep genes were recently isolated from several fish and two amphibian species, and here we report the identification of two Lep genes in a reptile, the lizard Anolis carolinensis. While vertebrate leptins show large divergence in their primary amino acid sequence, they form similar tertiary structures, and may have similar potencies when tested in vitro on heterologous leptin receptors (LepRs). Leptin binds to LepRs on the plasma membrane, activating several intracellular signaling pathways. Vertebrate LepRs signal via the Janus kinase (Jak) and signal transducer and activator of transcription (STAT) pathway. Three tyrosine residues located within the LepR cytoplasmic domain are phosphorylated by Jak2 and are required for activation of SH2-containing tyrosine phosphatase-2, STAT5 and STAT3 signaling. These tyrosines are conserved from fishes to mammals, demonstrating their critical role in signaling by the LepR. Leptin is anorexigenic in representatives of all vertebrate classes, suggesting that its role in energy balance is ancient and has been evolutionarily conserved. In addition to its integral role as a regulator of appetite and energy balance, leptin exerts pleiotropic actions in development, physiology and behavior.

Thyroid Hormone-dependent Gene Expression Program for Xenopus Neural Development

Although thyroid hormone (TH) plays a significant role in vertebrate neural development, the molecular basis of TH action on the brain is poorly understood. Using polymerase chain reaction-based subtractive hybridization we isolated 34 cDNAs for TH-regulated genes in the diencephalon of Xenopus tadpoles. Northern blots verified that the mRNAs are regulated by TH and are expressed during metamorphosis. Kinetic analyses showed that most of the genes are up-regulated by TH within 4-8 h and 13 are regulated by TH only in the brain. All cDNA fragments were sequenced and the identities of seven were determined through homology with known genes; an additional five TH-regulated genes were identified by hybridization with known cDNA clones. These include five transcription factors (including two members of the steroid receptor superfamily), a TH-converting deiodinase, two metabolic enzymes, a protein disulfide isomerase-like protein that may bind TH, a neural-specific cytoskeletal protein, and two hypophysiotropic neuropeptides. This is the first successful attempt to isolate a large number of TH-target genes in the developing vertebrate brain. The gene identities allow predictions about the gene regulatory networks underlying TH action on the brain, and the cloned cDNAs provide tools for understanding the basic molecular mechanisms underlying neural cell differentiation.

Characterization of an alarm pheromone secreted by amphibian tadpoles that induces behavioral inhibition and suppression of the neuroendocrine stress axis.

Many species assess predation risk through chemical cues, but the tissue source, chemical nature, and mechanisms of production or action of these cues are often unknown. Amphibian tadpoles show rapid and sustained behavioral inhibition when exposed to chemical cues of predation. Here we show that an alarm pheromone is produced by ranid tadpole skin cells, is released into the medium via an active secretory process upon predator attack, and signals predator presence to conspecifics. The pheromone is composed of two components with distinct biophysical properties that must be combined to elicit the behavioral response. In addition to the behavioral response, exposure to the alarm pheromone caused rapid and strong suppression of the hypothalamo-pituitary-adrenal (HPA) axis, as evidenced by a time and dose-dependent decrease in whole body corticosterone content. Reversing the decline in endogenous corticosterone caused by exposure to the alarm pheromone through addition of corticosterone to the aquarium water (50 nM) partially blocked the anti-predator behavior, suggesting that the suppression of the HPA axis promotes the expression and maintenance of a behaviorally quiescent state. To our knowledge this is the first evidence for aquatic vertebrate prey actively secreting an alarm pheromone in response to predator attack. We also provide a neuroendocrine mechanism by which the behavioral inhibition caused by exposure to the alarm pheromone is maintained until the threat subsides.

Endocrinology of complex life cycles: Amphibians

A complex life cycle, where an animal begins life as a larva then undergoes a metamorphosis to the adult form, is thought to represent the ancestral vertebrate life history mode. Most amphibian species exhibit complex life cycles, and the timing of metamorphosis is a central life history trait that is controlled by the interplay of developmental progression, body size and condition, and environmental signals. These different processes and signals are integrated by the neuroendocrine system to regulate production of hormones by the thyroid gland. Thyroid hormone (TH) is the primary morphogen controlling metamorphosis, while corticosteroids (CS) produced by the interrenal glands synergize with TH to promote metamorphic changes. The actions of TH are modulated by monodeiodinase enzymes expressed in TH target tissues. Corticosteroids act by sensitizing tissues to the actions of TH via the upregulation of TH receptors and monodeiodinases. The increase in thyroid gland activity during metamorphosis is controlled by the hypothalamus and pituitary gland. The hypothalamo–pituitary–thyroid and hypothalamo–pituitary–interrenal axes are regulated at multiple levels. Hypothalamic corticotropin-releasing hormone (CRH) functions as a common, central regulator of pituitary thyroid stimulating hormone (TSH) and adrenocorticotropic hormone (ACTH) secretion in tadpoles. CRH neurons transduce the signals of environmental change (e.g., pond drying, resource availability) on metamorphic timing through CRH actions on TSH and ACTH secretion, and subsequently the production of TH and CS. The proximate mechanisms for the evolution of body size at metamorphosis, and the timing of transformation among amphibian lineages involved the modification of neuroendocrinological pathways and hormone action.

Basic Transcription Element-binding Protein (BTEB) Is a Thyroid Hormone-regulated Gene in the Developing Central Nervous System EVIDENCE FOR A ROLE IN NEURITE OUTGROWTH

Thyroid hormone (3,5,3′-triiodothyronine; T3) is essential for normal development of the vertebrate brain, influencing diverse processes such as neuronal migration, myelin formation, axonal maturation, and dendritic outgrowth. We have identified basic transcription element-binding protein (BTEB), a small GC box-binding protein, as a T3-regulated gene in developing rat brain. BTEB mRNA levels in cerebral cortex exhibit developmental regulation and thyroid hormone dependence. T3 regulation of BTEB mRNA is neural cell-specific, being up-regulated in primary cultures of embryonic neurons (E16) and in neonatal astrocytes (P2), but not in neonatal oligodendrocytes (P2). T3 rapidly up-regulated BTEB mRNA in neuro-2a cells engineered to express thyroid hormone receptor (TR) β1 but not in cells expressing TRα1, suggesting that the regulation of this gene is specific to the TRβ1 isoform. Several lines of evidence support a transcriptional action of T3 on BTEB gene expression. Overexpression of BTEB in Neuro-2a cells dramatically increased the number and length of neurites in a dose-dependent manner suggesting a role for this transcription factor in neuronal process formation. However, other T3-dependent changes were not altered;i.e. overexpression of BTEB had no effect on the rate of cell proliferation nor on the expression of acetylcholinesterase activity.