Physiological benefits and latent effects of an algal-salamander symbiosis.

Embryos of the salamander Ambystoma maculatum (Shaw) and the uni-cellular green alga Oophila amblystomatis (Lambert ex Wille) have evolved a resource exchange mutualism. Whereas some of the benefits of the symbiosis to embryos are known, the physiological limitations of the relationship to embryos and carry over or latent effects on larvae are not. To determine the impact of the relationship across life history stages, we measured the growth, survival, and metabolic rate in response to hypoxia of salamander embryos reared under 0-h light (algae absent), 14-h light (control - algae present, fluctuating light conditions) and 24-h light (algae present, chronic light conditions) and the resulting larvae two-weeks post hatch. Embryos reared under 0-h light demonstrated decreased growth and survival compared to 14- and 24-h light, with no effect on metabolic rates or the response of metabolic rates to declining oxygen partial pressure (pO2). Conversely, larvae from embryos reared under 0-h light exhibited compensatory growth during the two-week larval rearing period, with body sizes matching those from the 14-h light treatment. Larvae from embryos reared under 24-h light had lower wet body mass and LT50 values upon starvation compared to those reared under 14-h light. Coupled with the lowest metabolic rates under normoxic pO2 levels, this indicates the presence of negative latent effects. We discuss the findings in relation to the effect of the symbiotic relationship on hypoxia tolerance and larval fitness with respect to the presence of compensatory growth and negative latent effects.

Developmental and interactive effects of arsenic and chromium to developing Ambystoma maculatum embryos: Toxicity, teratogenicity, and whole-body concentrations.

Anthropogenic activity has contributed to elevated environmental concentrations of arsenic (As) and chromium (Cr). The spotted salamander, Ambystoma maculatum, may be useful for identifying developmental effects produced by exposure to these contaminants as adults breed and larvae develop in water that may contain As or Cr. Three sample sets among 700 developing larvae were exposed to a range of As, Cr, or 2.5:1 mixture of As:Cr concentrations, respectively. From these 700 larvae, samples containing approximately 24 larvae showed different patterns of whole-body As and Cr from individual and mixture exposure. Whole-body As concentrations were 20.27 and 45.4 µg/g dry weight for larvae exposed to 20 mg/L As and 25:10 mg/L As:Cr, respectively, while whole-body Cr concentrations were 24.8 and 22 µg/g dry weight for larvae exposed to 20 mg/L Cr and 25:10 As:Cr, respectively. Observed malformations included edema, tail kinking, facial deformities, and abnormal bending. Twelve-day lethal concentrations for As and Cr in Ambystoma maculatum larvae were 261.17 mg/L and 71.93 mg/L, respectively, while 12-d effective concentrations to induce malformations were 158.82 and 26.05 mg/L, giving teratogenic indices of 1.64 and 2.76 for individual metal exposure. Exposure to a mixture of As and Cr resulted in a response addition and yielded lower lethal and effective concentration values with a teratogenic index of 2.78, indicating that these contaminants are developmentally toxic at lower concentrations when exposed as a mixture. Data demonstrate that As and Cr affect development of amphibian larvae, and that Ambystoma maculatum may be a useful indicator of environmental toxicity for these metals.

Cells keep a memory of their tissue origin during axolotl limb regeneration.

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

Intracapsular algae provide fixed carbon to developing embryos of the salamander Ambystoma maculatum.

Each spring, North American spotted salamander (Ambystoma maculatum) females each lay hundreds of eggs in shallow pools of water. Eggs are surrounded by jelly layers and are deposited as large gelatinous masses. Following deposition, masses are penetrated by a mutualistic green alga, Oophila amblystomatis, which enters individual egg capsules, proliferates and aggregates near the salamander embryo, providing oxygen that enhances development. We examined the effects of population density of intracapsular O. amblystomatis on A. maculatum embryos and show that larger algal populations promote faster embryonic growth and development. Also, we show that carbon fixed by O. amblystomatis is transferred to the embryos, providing the first evidence of direct translocation of photosynthate from a symbiont to a vertebrate host.

The effects of atrazine on spotted salamander embryos and their symbiotic alga.

Worldwide amphibian declines have been a concern for biologists for the past several decades. The causes of such declines may include habitat loss, invasive species, pathogens, and man-made chemicals. Agricultural herbicides, in particular, are known to interfere with reproduction in amphibians and are likely contributing to population declines. We tested the effects of the herbicide atrazine on developing spotted salamanders (Ambystoma maculatum) and their symbiotic green alga Oophila amblystomatis. We exposed spotted salamander egg masses to atrazine at concentrations of 0 microg/L (control), 50, 100, 200, and 400 microg/L. Algae were eliminated in all atrazine treatments. Hatching success was significantly lower for atrazine-treated egg masses than for the controls, and was inversely related to atrazine concentration. The highest developmental stage reached by the embryos was significantly lower in the atrazine treatments than in the controls, and was inversely related to atrazine concentration. These results indicate that atrazine exposure affected spotted salamanders both directly by causing pathologies and mortality in embryos and indirectly by eliminating their symbiotic alga.

Role of tropomyosin in actin filament formation in embryonic salamander heart cells.

Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.

Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.

Acid precipitation and embryonic mortality of spotted salamanders, Ambystoma maculatum.

Spotted salamanders breed in temporary pools formed in early spring by melted snow and rain. Many of these pools reflect the low pH of precipitation in the northeastern United States. Egg mortality is low (less than 1 percent) in pools near neutrality, but high (greater than 60 percent) in pools more acid than pH 6. Developmental anomalies and the embryonic stage at which death occurs are the same in field situations as at corresponding pH's in laboratory experiments.

Gap junctional conductance is a simple and sensitive function of intracellular pH.

The pH of the cytoplasm (pHt) measured with pH-sensitive microelectrodes in cleavage-stage blastomeres of amphibian (Ambystoma) and teleost (Fundulus) embryos is about 7.7. In electrotonically coupled cell pairs, junctional conductance is rapidly and reversibly reduced by acidification of the cytoplasm. The relation between junctional conductance and pHi is the same for increasing and decreasing pH and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 nM (pK = 7.3) and n = 4 to 5. The closure of gap junction channels at low pHi appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow pHi changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.

Normal anterior endoderm corrects the heart defect in cardiac mutant salamanders (Ambystoma mexicanum).

Recessive mutant gene c in axolotl embryos results in an absence of heart function. Normal (+/+) anterior endoderm cultured with mutant (c/c) hearts totally corrects the defect.

Voltage dependence of junctional conductance in early amphibian embryos.

Isolated pairs of blastomeres from early amphibian embryos (Ambystoma, Rana, Xenopus) are electrontonically coupled. Junctional conductance and permeability to the dye Lucifer Yellow are markeldy and reversibly decreased by moderate transjunctional polarization in either direction. The relationship between junctional conductance and transjunctional voltage is sufficiently steep that a physiological role in regulation of intercellular communication is plausible.

Evidence for abnormal heart induction in cardiac-mutant salamanders (Ambystoma mexicanum).

Homozygosity for simple recessive gene c in axolotl embryos results in the absence of a heartbeat. Gene c alters the morphology of the mutant anterior endoderm - the primary heart inductor.

Effect of road deicing salt on the susceptibility of amphibian embryos to infection by water molds.

Some causative agents of amphibian declines act synergistically to impact individual amphibians and their populations. In particular, pathogenic water molds (aquatic oomycetes) interact with environmental stressors and increase mortality in amphibian embryos. We documented colonization of eggs of three amphibian species, the wood frog (Rana sylvatica), the green frog (Rana clamitans), and the spotted salamander (Ambystoma maculatum), by water molds in the field and examined the interactive effects of road deicing salt and water molds, two known sources of mortality for amphibian embryos, on two species, R. clamitans and A. maculatum in the laboratory. We found that exposure to water molds did not affect embryonic survivorship in either A. maculatum or R. clamitans, regardless of the concentration of road salt to which their eggs were exposed. Road salt decreased survivorship of A. maculatum, but not R. clamitans, and frequency of malformations increased significantly in both species at the highest salinity concentration. The lack of an effect of water molds on survival of embryos and no interaction between road salt and water molds indicates that observations of colonization of these eggs by water molds in the field probably represent a secondary invasion of unfertilized eggs or of embryos that had died of other causes. Given increasing salinization of freshwater habitats on several continents and the global distribution of water molds, our results suggest that some amphibian species may not be susceptible to the combined effects of these factors, permitting amphibian decline researchers to devote their attention to other potential causes.

Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas.

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.

Expression of the axolotl homologue of mouse chaperonin t-complex protein-1 during early development.

Molecular chaperones assist in the folding of proteins, but their role during development is not well understood. Here we report the temporal and spatial expression pattern of the axolotl homologue of mouse chaperonin TCP-1 during normal amphibian embryogenesis and in several models of abnormal embryogenesis. A partial axolotl TCP-1 cDNA (646 bp; 519 coding bp) isolated by 3' RACE PCR shows considerable homology to mouse TCP-1. Developmental Northerns and RT-PCR analyses of whole axolot1 embryos revealed a low level of maternal TCP-1 transcripts in fertilized eggs. The maternal transcripts were down-regulated to a non-detectable level in early gastrulae. Zygotic TCP-1 transcripts first appeared during gastrulation. They were mainly expressed in mid-neurula and later stage embryos. Whole-mount in situ hybridization studies showed abundant TCP-1 transcripts in the blastopore at the mid-gastrula stage and in the brain and spinal cord beginning at the neurula stage, and in the somites (myotomes) at the tailbud stage. RT-PCR analysis of TCP-1 expression in axolotl embryos treated with either high salt (causing exogastrulation) or ultraviolet (UV) irradiation (causing ventralization) substantiated the correlation between TCP-1 expression and neural and somitic development. In high salt-induced exogastrulated embryos TCP-1 mRNA was detectable in the ectoderm part (with neural tissues) but not in its exogastrulated endoderm part. Lower levels of TCP-1 expression were detected in UV-irradiated, ventralized embryos with smaller head and reduced neural and somitic tissues. Normal levels of TCP-1 expression were detected in embryos with double axes/heads. These studies provide strong evidence that at the transcript level axolotl chaperonin TCP-1 is regulated both temporally and spatially during embryogenesis, especially in neural and somitic development.

Automated 3-D reconstruction of the surface of live early-stage amphibian embryos.

Although three-dimensional (3-D) reconstructions of the surfaces of live embyos are vital to understanding embryo development, morphogenetic tissue movements and other factors have prevented the automation of this task. Here, we report an integrated set of software algorithms that overcome these challenges, making it possible to completely automate the reconstruction of embryo surfaces and other textured surfaces from multiview images. The process involves: 1) building accurate point correspondences using a robust deformable template block matching algorithm; 2) removing outliers using fundamental matrix calculations in conjunction with a RANSAC algorithm; 3) generating 3-D point clouds using a bundle adjustment algorithm that includes camera position and distortion corrections; 4) meshing the point clouds into triangulated surfaces using a Tight Cocone algorithm that produces water tight models; 5) refining surfaces using midpoint insertion and Laplacian smoothing algorithms; and 6) repeating these steps until a measure of convergence G, the rms difference between successive reconstructions, is below a specified threshold. Reconstructions were made of 2.2-mm diameter, neurulation-stage axolotl (amphibian) embryos using 44 multiview images collected with a robotic microscope. A typical final model (sixth iteration) contained 3787 points and 7562 triangles and had an error measure of G = 5.9 microm.

Morphogenesis of the axolotl pronephric duct: a model system for the study of cell migration in vivo.

Pronephric duct (PND) morphogenesis is a critical early event in the development of the vertebrate excretory system. This structure is the exit channel for both pronephric and mesonephric filtrate, forms the ureteric bud of the metanephros and gives rise to the ductus deferens of the testis. In addition, the PND and ureteric bud epithelia induce terminal differentiation of the mesonephric and metanephric mesenchyme, respectively. Elongation of the PND in all vertebrates involves active cell migration of the primordium. In urodele embryos--unlike in some anuran, avian and mammalian embryos--elongation of the PND occurs solely by cell migration. In the axolotl embryo, the PND primordium segregates as an ovoid tissue mass from the anterodorsal flank mesoderm directly beneath somites 3-7. The primordium then extends caudally along the ventral border of the developing somites until it reaches the cloaca. The ease with which these embryos can be manipulated microsurgically makes the PND system ideal for the study of the mechanisms controlling cell migration in vivo. This review summarizes the progress that has been made in characterizing the environmental cues and the cell surface recognition systems that drive this tightly regulated migration event.

Surface contraction and expansion waves correlated with differentiation in axolotl embryos. II. In contrast to urodeles, the anuran Xenopus laevis does not show furrowing surface contraction waves.

We have observed a number of contraction waves traversing the axolotl (Ambystoma mexicanum) embryo (a urodelan amphibian) from the midblastula transition up to at least neural tube closure, and wished to learn if similar "differentiation waves" appear on the popular laboratory anuran amphibian, the South African clawed toad, Xenopus laevis. Time lapse video microscopy showed that no contraction waves are visible on the surface of Xenopus from gastrulation through neurulation. It is possible that cell intercalations in the double-layered ectoderm of the Xenopus embryo are homologous to the surface waves in the single layered ectoderm of the axolotl embryo. In any case, a simple, universal correspondence between surface waves and induction phenomena and differentiation does not exist.