ABSTRACT
Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines.
ABSTRACT
Survival of the immobile embryo in response to rising temperature is important to determine a species' vulnerability to climate change. However, the collective effects of 2 key thermal characteristics associated with climate change (i.e., rising average temperature and acute heat events) on embryonic survival remain largely unexplored. We used empirical measurements and niche modeling to investigate how chronic and acute heat stress independently and collectively influence the embryonic survival of lizards across latitudes. We collected and bred lizards from 5 latitudes and incubated their eggs across a range of temperatures to quantify population-specific responses to chronic and acute heat stress. Using an embryonic development model parameterized with measured embryonic heat tolerances, we further identified a collective impact of embryonic chronic and acute heat tolerances on embryonic survival. We also incorporated embryonic chronic and acute heat tolerance in hybrid species distribution models to determine species' range shifts under climate change. Embryos' tolerance of chronic heat (T-chronic) remained consistent across latitudes, whereas their tolerance of acute heat (T-acute) was higher at high latitudes than at low latitudes. Tolerance of acute heat exerted a more pronounced influence than tolerance of chronic heat. In species distribution models, climate change led to the most significant habitat loss for each population and species in its low-latitude distribution. Consequently, habitat for populations across all latitudes will shift toward high latitudes. Our study also highlights the importance of considering embryonic survival under chronic and acute heat stresses to predict species' vulnerability to climate change.
Efectos colectivos del aumento de las temperaturas promedio y los eventos de calor en embriones ovíparos Resumen La supervivencia de los embriones inmóviles en respuesta al incremento de temperatura es importante para determinar la vulnerabilidad de las especies al cambio climático. Sin embargo, los efectos colectivos de dos características térmicas claves asociadas con el cambio climático (i. e., aumento de temperatura promedio y eventos de calor agudo) sobre la supervivencia embrionaria permanecen en gran parte inexplorados. Utilizamos mediciones empíricas y modelos de nicho para investigar cómo el estrés térmico crónico y agudo influye de forma independiente y colectiva en la supervivencia embrionaria de los lagartos en todas las latitudes. Recolectamos y criamos lagartos de cinco latitudes e incubamos sus huevos en un rango de temperaturas para cuantificar las respuestas específicas de la población al estrés por calor crónico y agudo. Posteriormente, mediante un modelo de desarrollo embrionario parametrizado con mediciones de tolerancia embrionaria al calor, identificamos un impacto colectivo de las tolerancias embrionarias al calor agudo y crónico en la supervivencia embrionaria. También incorporamos la tolerancia embrionaria crónica y aguda al calor en modelos de distribución de especies híbridas para determinar los cambios de distribución de las especies bajo el cambio climático. La tolerancia embrionaria al calor crónico (Tcrónico) permaneció constante, mientras que la tolerancia al calor agudo (Tagudo) fue mayor en latitudes altas que en latitudes bajas. La tolerancia al calor agudo ejerció una influencia más pronunciada que la tolerancia al calor crónico. En los modelos de distribución de especies, el cambio climático provocó la pérdida de hábitat más significativa para cada población y especie en su distribución de latitudes bajas. En consecuencia, el hábitat para poblaciones en todas las latitudes se desplazará a latitudes altas. Nuestro estudio también resalta la importancia de considerar la supervivencia embrionaria bajo estrés térmico crónico y agudo para predecir la vulnerabilidad de las especies al cambio climático.
Subject(s)
Climate Change , Embryo, Nonmammalian , Hot Temperature , Lizards , Animals , Lizards/physiology , Lizards/embryology , Embryo, Nonmammalian/physiology , Oviparity , Female , Models, Biological , Embryonic Development , ThermotoleranceABSTRACT
In this article, I explore Seth Farber's critique in The Spiritual Gift of Madness that the leaders of the Mad Pride movement are failing to realize his vision of the mad as spiritual vanguard of sociopolitical transformation. First, I show how, contra Farber's polemic, several postmodern theorists are well suited for this leadership (especially the Argentinian post-Marxist philosopher Ernesto Laclau). Second, I reinterpret the first book by the Icarus Project, Navigating the Space between Brilliance and Madness, by reimagining its central metaphor of Icarus in the context of late capitalism as a prison world. Finally, I conclude with four strategies derived therefrom for higher functioning mad leaders to transform our penitentiary world.
Subject(s)
Anger , Leadership , EmotionsABSTRACT
Vertebrate embryos require access to water; however, many species nest in terrestrial habitats that vary considerably in moisture content. Oviparous, non-avian reptiles have served as models to understand how environmental factors, like moisture availability, influence development because eggs are often exposed to prevailing environments in the absence of parental care. Though much research demonstrates the importance of water absorption by eggs, many ecological factors that influence moisture availability in natural nests have received little attention. For example, the type of substrate in which nests are constructed is understudied. We experimentally incubated eggs of the brown anole lizard (Anolis sagrei) in 2 naturally occurring nest substrates that were treated with varying amounts of water to determine how natural substrates influence development at different moisture concentrations. One substrate consisted of sand and crushed seashells and the other was mostly organic material (i.e. decayed plant material). Both are common nesting substrates at our field site. When controlling for water uptake by eggs, we found that egg survival and hatchling phenotypes were similar between substrates; however, embryos developed more quickly in the sand/shell substrate than the organic substrate, indicating substrate-specific effects on embryo physiology. These results demonstrate that different natural substrates can result in similar developmental outcomes if the water available to eggs is comparable; however, some aspects of development, like developmental rate, are affected by the type of substrate, independent of water availability. Further study is required to determine how natural substrates influence embryo physiology independent of water content.
Subject(s)
Lizards , Sand , Animals , Ecosystem , Lizards/physiology , Nesting Behavior/physiology , Phenotype , Water/physiologyABSTRACT
Post-Traumatic Stress Disorder (PTSD) is not solely a psychiatric disorder; it also includes significant medical morbidity. Although there is evidence of increased risk of metabolic syndrome (MetS) in PTSD, the interpretation of previous studies is confounded by inclusion of people on antipsychotic medications, which independently cause increased MetS. In this study we investigated whether Veterans with PTSD not treated with antipsychotic medications (n=115) demonstrate increased MetS compared to an age-comparable group of people from the U.S. National Health and Nutrition Examination Survey (NHNES; n=1005). Using standardized criteria (abnormal values in 3 out of the 5 domains of obesity, hypertension, high density lipoprotein, triglyceride and fasting glucose concentrations) we compared the prevalence of MetS across groups. Relative to the NHNES group, a significantly higher proportion of the Veteran PTSD group met criteria for MetS (26.9% vs. 41.7%) with a higher proportion of abnormal values in four out of five MetS domains (excepting glucose). Our results suggest that the elevation of MetS associated with PTSD cannot be fully explained by iatrogenic effects of antipsychotic medication. We suggest that extra attention be devoted to the clinical management of metabolic risk factors for morbidity in patients with PTSD.
ABSTRACT
Aspects of global change create stressful thermal environments that threaten biodiversity. Oviparous, non-avian reptiles have received considerable attention because eggs are left to develop under prevailing conditions, leaving developing embryos vulnerable to increases in temperature. Though many studies assess embryo responses to long-term (i.e., chronic), constant incubation temperatures, few assess responses to acute exposures which are more relevant for many species. We subjected brown anole (Anolis sagrei) eggs to heat shocks, thermal ramps, and extreme diurnal fluctuations to determine the lethal temperature of embryos, measure the thermal sensitivity of embryo heart rate and metabolism, and quantify the effects of sublethal but stressful temperatures on development and hatchling phenotypes and survival. Most embryos died at heat shocks of 45°C or 46°C, which is ~12°C warmer than the highest constant temperatures suitable for successful development. Heart rate and O2 consumption increased with temperature; however, as embryos approached the lethal temperature, heart rate and CO2 production continued rising while O2 consumption plateaued. These data indicate a mismatch between oxygen supply and demand at high temperatures. Exposure to extreme, diurnal fluctuations depressed embryo developmental rates and heart rates, and resulted in hatchlings with smaller body size, reduced growth rates, and lower survival in the laboratory. Thus, even brief exposure to extreme temperatures can have important effects on embryo development, and our study highlights the role of both immediate and cumulative effects of high temperatures on egg survival. Such effects must be considered to predict how populations will respond to global change.
Subject(s)
Embryo, Nonmammalian/physiology , Heat-Shock Response , Lizards/embryology , Ovum/physiology , Oxygen/administration & dosage , Thermotolerance , Animals , Female , Male , SurvivalABSTRACT
Aspects of global change result in warming temperatures that threaten biodiversity across the planet. Eggs of non-avian, oviparous reptiles (henceforth "reptiles") are particularly vulnerable to warming due to a lack of parental care during incubation and limited ability to behaviorally thermoregulate. Because warming temperatures will cause increases in both mean and variance of nest temperatures, it is crucial to consider embryo responses to both chronic and acute heat stress. Although many studies have considered embryo survival across constant incubation temperatures (i.e., chronic stress) and in response to brief exposure to extreme temperatures (i.e., acute stress), there are no standard metrics or terminology for determining heat stress of embryos. This impedes comparisons across studies and species and hinders our ability to predict how species will respond to global change. In this review, we compare various methods that have been used to assess embryonic heat tolerance in reptiles and provide new terminology and metrics for quantifying embryo responses to both chronic and acute heat stress. We apply these recommendations to data from the literature to assess chronic heat tolerance in 16 squamates, 16 turtles, five crocodilians, and the tuatara and acute heat tolerance for nine squamates and one turtle. Our results indicate that there is relatively large variation in chronic and acute heat tolerance across species, and we outline directions for future research, calling for more studies that assess embryo responses to acute thermal stress, integrate embryo responses to chronic and acute temperatures in predictive models, and identify mechanisms that determine heat tolerance.
Subject(s)
Embryo, Nonmammalian/physiology , Hot Temperature , Reptiles/embryology , Thermotolerance , Animals , Climate ChangeABSTRACT
Research on the thermal ecology and physiology of free-living organisms is accelerating as scientists and managers recognize the urgency of the global biodiversity crisis brought on by climate change. As ectotherms, temperature fundamentally affects most aspects of the lives of amphibians and reptiles, making them excellent models for studying how animals are impacted by changing temperatures. As research on this group of organisms accelerates, it is essential to maintain consistent and optimal methodology so that results can be compared across groups and over time. This review addresses the utility of reptiles and amphibians as model organisms for thermal studies by reviewing the best practices for research on their thermal ecology and physiology, and by highlighting key studies that have advanced the field with new and improved methods. We end by presenting several areas where reptiles and amphibians show great promise for further advancing our understanding of how temperature relations between organisms and their environments are impacted by global climate change.
Subject(s)
Amphibians/physiology , Body Temperature/physiology , Ecosystem , Reptiles/physiology , Amphibians/embryology , Amphibians/growth & development , Animals , Embryo, Nonmammalian/physiology , Monitoring, Physiologic , Reptiles/embryology , Reptiles/growth & developmentABSTRACT
Natural thermal environments are notably complex and challenging to mimic in controlled studies. Consequently, our understanding of the ecological relevance and underlying mechanisms of organismal responses to thermal environments is often limited. For example, studies of thermal developmental plasticity have provided key insights into the ecological consequences of temperature variation, but most laboratory studies use treatments that do not reflect natural thermal regimes. While controlling other important factors, we compared the effects of naturally fluctuating temperatures with those of commonly used laboratory regimes on development of lizard embryos and offspring phenotypes and survival. We incubated eggs in four treatments: three that followed procedures commonly used in the literature, and one that precisely mimicked naturally fluctuating nest temperatures. To explore context-dependent effects, we replicated these treatments across two seasonal regimes: relatively cool temperatures from nests constructed early in the season and warm temperatures from late-season nests. We show that natural thermal fluctuations have a relatively small effect on developmental variables but enhance hatchling performance and survival at cooler temperatures. Thus, natural thermal fluctuations are important for successful development and simpler approximations (e.g. repeated sine waves, constant temperatures) may poorly reflect natural systems under some conditions. Thus, the benefits of precisely replicating real-world temperatures in controlled studies may outweigh logistical costs. Although patterns might vary according to study system and research goals, our methodological approach demonstrates the importance of incorporating natural variation into controlled studies and provides biologists interested in thermal ecology with a framework for validating the effectiveness of commonly used methods.
Subject(s)
Lizards , Animals , Cold Temperature , Phenotype , Seasons , TemperatureABSTRACT
Seasonal changes in reproduction have been described for many taxa. As reproductive seasons progress, females often shift from greater energetic investment in many small offspring towards investing less total energy into fewer, better provisioned (i.e. larger) offspring. The underlying causes of this pattern have not been assessed in many systems. Two primary hypotheses have been proposed to explain these patterns. The first is an adaptive hypothesis from life-history theory: early offspring have a survival advantage over those produced later. Accordingly, selection favours females that invest in offspring quantity early in the season and offspring quality later. The second hypothesis suggests these patterns are not intrinsic but result from passive responses to seasonal changes in the environment experienced by reproducing females (i.e. maternal environment). To disentangle the causes underlying this pattern, which has been reported in brown anole lizards (Anolis sagrei), we performed complementary field and laboratory studies. The laboratory study carefully controlled maternal environments and quantified reproductive patterns throughout the reproductive season for each female. The field study measured similar metrics from free ranging lizards across an entire reproductive season. In the laboratory, females increased relative effort per offspring as the reproductive season progressed; smaller eggs were laid earlier, larger eggs were laid later. Moreover, we observed significant among-individual variation in seasonal changes in reproduction, which is necessary for traits to evolve via natural selection. Because these patterns consistently emerge under controlled laboratory conditions, they likely represent an intrinsic and potentially adaptive adjustment of reproductive effort as predicted by life-history theory. The field study revealed similar trends, further suggesting that intrinsic patterns observed in the laboratory are strong enough to persist despite the environmental variability that characterizes natural habitats. The observed patterns are indicative of an adaptive seasonal shift in parental investment in response to a deteriorating offspring environment: allocating greater resources to late-produced offspring likely enhances maternal fitness.
Subject(s)
Life History Traits , Lizards , Animals , Female , Laboratories , Reproduction , SeasonsABSTRACT
Most studies of thermal tolerance use adults, but early-life stages (e.g. embryos) are often more sensitive to thermal agitation. Studies that examine effects on embryos rarely assess the potential for thermal tolerance to change with ontogeny or how effects differ among sympatric species, and often utilize unrealistic temperature treatments. We used thermal fluctuations from nests within the urban-heat island to determine how thermal tolerance of embryos changes across development and differs among two sympatric lizard species (Anolis sagrei and Anoliscristatellus). We applied fluctuations that varied in frequency and magnitude at different times during development and measured effects on embryo physiology and survival, and hatchling morphology, growth and survival. Thermal tolerance differed between the species by â¼2°C: embryos of A. sagrei, a lizard that prefers warmer, open-canopy microhabitats, were more robust to thermal stress than embryos of A. cristatellus, which prefers cooler, closed-canopy microhabitats. Moreover, thermal tolerance changed through development; however, the nature of this change differed between the species. For A. cristatellus, thermal tolerance was greatest mid-development. For A. sagrei, the relationship was not statistically clear. The greatest effects of thermal stress were on embryo and hatchling survival and embryo physiology. Hatchling morphology and growth were less affected. Inter-specific responses and the timing of stochastic thermal events with respect to development have important effects on embryo mortality. Thus, research that integrates ecologically meaningful thermal treatments, considers multiple life-history stages and examines interspecific responses will be critical to make robust predictions of the impacts of global change on wildlife.
Subject(s)
Embryo, Nonmammalian/physiology , Hot Temperature , Islands , Lizards/physiology , Sympatry/physiology , Thermotolerance/physiology , Animals , Cities , Female , Heart Rate/physiology , Lizards/anatomy & histology , Models, Biological , Probability , Sample Size , Survival AnalysisABSTRACT
The evolution of reproductive strategies depends on local environmental conditions. When environments are seasonal, selection favors individuals that align changes in key reproductive traits with seasonal shifts in habitat quality. Offspring habitat quality can decline through the season, and increased maternal provisioning to late-produced offspring may compensate. This shift, however, may depend on environmental factors that influence reproduction and are, themselves, subject to temporal changes (e.g., food abundance). We studied the brown anole lizard (Anolis sagrei) to demonstrate how prey abundance modifies seasonal changes in key reproductive traits. We bred lizards in controlled laboratory conditions across the reproductive season and manipulated the availability of food by providing some breeding pairs high prey availability and some low. Halfway through the season, we switched half of the breeding pairs to the opposite treatment. We measured growth of male and female lizards as well as latency to oviposit, fecundity, egg size, egg content (yolk, water, shell mass), and egg quality (steroid hormones, yolk caloric content) over this period. Higher prey availability enhanced lizard growth and some key reproductive traits (egg size, fecundity) but not others (egg content and quality). Moreover, we found that seasonal patterns of reproduction were modified by prey treatment in ways that have consequences for offspring survival. Our results demonstrate that seasonal changes in reproduction are dependent on fluctuations in local environmental conditions. Moreover, researchers must account for seasonal shifts in environmental factors and reproductive traits (and their interactions) when designing experiments and drawing conclusions about how the environment influences reproduction.
Subject(s)
Food Chain , Life History Traits , Lizards/physiology , Animals , Female , Florida , Male , Random Allocation , Reproduction , SeasonsABSTRACT
Effects of global change (i.e. urbanization, climate change) on adult organisms are readily used to predict the persistence of populations. However, effects on embryo survival and patterns of development are less studied, even though embryos are particularly sensitive to abiotic conditions that are altered by global change (e.g. temperature). In reptiles, relatively warm incubation temperatures increase developmental rate and often enhance fitness-relevant phenotypes, but extremely high temperatures cause death. Due to the urban heat island effect, human-altered habitats (i.e. cities) potentially create unusually warm nest conditions that differ from adjacent natural areas in both mean and extreme temperatures. Such variation may exert selection pressures on embryos. To address this, we measured soil temperatures in places where the Puerto Rican crested anole lizard (Anolis cristatellus) nests in both city and forest habitats. We bred anoles in the laboratory and subjected their eggs to five incubation treatments that mimicked temperature regimes from the field, three of which included brief exposure to extremely high temperatures (i.e. thermal spikes) measured in the city. We monitored growth and survival of hatchlings in the laboratory for 3â months and found that warmer, city temperatures increase developmental rate, but brief, thermal spikes reduce survival. Hatchling growth and survival were unaffected by incubation treatment. The urban landscape can potentially create selection pressures that influence organisms at early (e.g. embryo) and late life stages. Thus, research aimed at quantifying the impacts of urbanization on wildlife populations must include multiple life stages to gain a comprehensive understanding of this important aspect of global change.
Subject(s)
Embryonic Development/physiology , Hot Temperature/adverse effects , Lizards/physiology , Mortality , Animals , Cities , Climate Change , Embryo, Nonmammalian/physiology , Florida , Lizards/embryologyABSTRACT
Many studies of phenotypic plasticity alter environmental conditions during embryonic development, yet only measure phenotypes at the neonatal stage (after embryonic development). However, measuring aspects of embryo physiology enhances our understanding of how environmental factors immediately affect embryos, which aids our understanding of developmental plasticity. While current research on reptile developmental plasticity has demonstrated that fluctuating incubation temperatures affect development differently than constant temperatures, most research on embryo physiology is still performed with constant temperature experiments. In this study, we noninvasively measured embryonic heart rates of the brown anole (Anolis sagrei), across ecologically relevant fluctuating temperatures. We incubated eggs under temperatures measured from potential nests in the field and examined how heart rates change through a diel cycle and throughout embryonic development. We also evaluated how experimental design (e.g., repeated vs. single measures designs, constant vs. fluctuating temperatures) and different protocols (e.g., removing eggs from incubators) might influence heart rate. We found that heart rates were correlated with daily temperature and increased through development. Our findings suggest that experimenters have reasonable flexibility in choosing an experimental design to address their questions; however, some aspects of design and protocol can potentially influence estimations of heart rates. Overall, we present the first ecologically relevant measures of anole embryonic heart rates and provide recommendations for experimental designs for future experiments.
Subject(s)
Adaptation, Physiological , Embryo, Nonmammalian/physiology , Heart Rate/physiology , Lizards/embryology , Temperature , Animals , Embryonic Development/physiologyABSTRACT
The halteres of flies are mechanosensory organs that provide information about body rotations during flight. We measured haltere movements in a range of fly taxa during free walking and tethered flight. We find a diversity of wing-haltere phase relationships in flight, with higher variability in more ancient families and less in more derived families. Diverse haltere movements were observed during free walking and were correlated with phylogeny. We predicted that haltere removal might decrease behavioural performance in those flies that move them during walking and provide evidence that this is the case. Our comparative approach reveals previously unknown diversity in haltere movements and opens the possibility of multiple functional roles for halteres in different fly behaviours.
Subject(s)
Diptera/physiology , Animals , Behavior, Animal , Biomechanical Phenomena , Flight, Animal/physiology , Mechanoreceptors/physiology , Movement , Phylogeny , Walking/physiology , Wings, Animal/physiologyABSTRACT
Emerging evidence supports the notion of disrupted sleep as a core component of Posttraumatic Stress Disorder (PTSD). Effective treatments for nighttime PTSD symptoms are critical because sleep disruption may be mechanistically linked to development and maintenance of PTSD and is associated with significant distress, functional impairment, and poor health. This review aimed to describe the state of science with respect to the impact of the latest behavioral and pharmacological interventions on posttraumatic nightmares and insomnia. Published studies that examined evidence for therapeutic effects upon sleep were included. Some behavioral and pharmacological interventions show promise, especially for nightmares, but there is a need for controlled trials that include valid sleep measures and are designed to identify treatment mechanisms. Our ability to treat PTSD-related sleep disturbances may be improved by moving away from considering sleep symptoms in isolation and instead conducting integrative studies that examine sequential or combined behavioral and/or pharmacological treatments targeting both the daytime and nighttime aspects of PTSD. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Subject(s)
Dreams/psychology , Sleep Initiation and Maintenance Disorders/therapy , Stress Disorders, Post-Traumatic/therapy , Antidepressive Agents/therapeutic use , Antipsychotic Agents/therapeutic use , Behavior Therapy/methods , Dreams/drug effects , Humans , Hypnotics and Sedatives/therapeutic use , Sleep Initiation and Maintenance Disorders/drug therapy , Sleep Initiation and Maintenance Disorders/psychology , Stress Disorders, Post-Traumatic/drug therapy , Stress Disorders, Post-Traumatic/psychology , Treatment OutcomeABSTRACT
Taste buds on the anterior part of the tongue develop in conjunction with epithelial-mesenchymal specializations in the form of gustatory (taste) papillae. Sonic hedgehog (Shh) and Bone Morphogenetic Protein 4 (BMP4) are expressed in developing taste papillae, but the roles of these signaling molecules in specification of taste bud progenitors and in papillary morphogenesis are unclear. We show here that BMP4 is not expressed in the early tongue, but is precisely coexpressed with Shh in papillary placodes, which serve as a signaling center for both gustatory and papillary development. To elucidate the role of Shh, we used an in vitro model of mouse fungiform papillary development to determine the effects of two functional inhibitors of Shh signaling: anti-Shh (5E1) antibody and cyclopamine. Cultured E11.5 tongue explants express Shh and BMP4(LacZ) in a pattern similar to that of intact embryos, localizing to developing papillary placodes after 2 days in culture. Tongues cultured with 5E1 antibody continue to express these genes in papillary patterns but develop more papillae that are larger and closer together than in controls. Tongues cultured with cyclopamine have a dose-dependent expansion of Shh and BMP4(LacZ) expression domains. Both antibody-treated and cyclopamine-treated tongue explants also are smaller than controls. Taken together, these results suggest that, although Shh is not involved in the initial specification of papillary placodes, Shh does play two key roles during pmcry development: (1) as a morphogen that directs cells toward a nonpapillary fate, and (2) as a mitogen, causing expansion of the interplacodal epithelium and underlying mesenchyme.