Biogenesis of myeloid bodies in regenerating newt (Notophthalmus viridescens) retinal pigment epithelium

Summary Myeloid bodies are believed to be differentiated areas of smooth endoplasmic reticulum membranes, and they are found within the retinal pigment epithelium in a number of lower vertebrates. Previous studies demonstrated a correlation between phagocytosis of outer segment disc membranes and myeloid body numbers in the retinal pigment epithelium of the newt. To test the hypothesis that myeloid bodies are directly involved in outer segment lipid metabolism and to further characterize the origin and functional significance of these organelles, we examined the effects on myeloid bodies of eliminating the source of outer segment membrane lipids (neural retina removal) and of the subsequent return of outer segments (retinal regeneration) in the newt Notophthalmus viridescens. Light- and electron-microscopic analysis demonstrated that myeloid bodies disappeared from the pigment epithelium within six days of neural retina removal. By week 6 of regeneration, rudimentary photoreceptor outer segments were present but myeloid bodies were still absent. However, at this time, the smooth endoplasmic reticulum in some areas of the retinal pigment epithelial cells had become flattened, giving rise to small (0.5 m long), two-to-four layer-thick lamellar units, which are myeloid body precursors. Small myeloid bodies were first observed one week later at week 7 of retinal regeneration. This study revealed that newt myeloid bodies are specialized areas of smooth endoplasmic reticulum. It also showed that a contact between functional photoreceptors and the retinal pigment epithelium is essential to the presence of myeloid bodies in the epithelial cells.     

Cannibalistic phenomena in salamandra salamandra larvae in certain water bodies and under experimental conditions

Salamandra salamandra larvae originating from a single batch (layed by one female) were raised at different temperatures and on ion-rich or distilled water. Temperature, but not the presence or absence of ions affected growth. Larvae originating from one batch or from several batches born on the same day, were kept without food for long periods of several months in order to observe cannibalistic predation under such conditions. This phenomenon is known in natural populations of salamanders inhabitating either rain pools in rocks or permanent springs poor in aquatic life. It was found that cannibalism increased under experimental conditions proportionally to the increase in size between the larvae.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

Genome size variation and species diversity in salamanders

Salamanders (Urodela) have among the largest vertebrate genomes, ranging in size from 10 to 120 pg. Although changes in genome size often occur randomly and in the absence of selection pressure, nonrandom patterns of genome size variation are evident among specific vertebrate lineages. Several reports suggest a relationship between species richness and genome size, but the exact nature of that relationship remains unclear both within and across different taxonomic groups. Here, we report (a) a negative relationship between haploid genome size (C‐value) and species richness at the family taxonomic level in salamander clades; (b) a correlation of C‐value and species richness with clade crown age but not with diversification rates; (c) strong associations between C‐value and both geographic area and climatic‐niche rate. Finally, we report a relationship between C‐value diversity and species diversity at both the family‐ and genus‐level clades in urodeles.     

Sperm storage in females of the smooth newt (Triturus v. vulgaris L.): II. Ultrastructure of the spermathecae after the breeding season

Sperm storage in cloacal spermathecae was studied in females of Triturus v. vulgaris from southern England killed at the end of the breeding season in June. This species mates and oviposits eggs in ponds from March to June. Included in the sample were 12 unmated females collected in terrestrial situations in March and mated in the laboratory. Some of these females oviposited viable eggs in the laboratory whereas others did not oviposit after mating. In addition, we examined five females with unknown mating histories that were collected from a breeding pond in June. We found that all of the specimens contained some stored sperm and were similar in spermathecal ultrastructure. The spermathecae exhibited characteristics of secretory epithelium at the end of a cycle, including irregular heterochromatic nuclei surrounded by scant cytoplasm, absence of organelles involved in synthetic activities, few secretory vacuoles, and wide intercellular canaliculi. Spermiophagy by the spermathecal epithelium was extensive. In contrast, spermathecae from females at the beginning of the breeding season as reported in our previous study were actively producing a PAS+ secretion and did not exhibit spermiophagy. Spermiophagy is a means of eliminating sperm prior to the next breeding season.     

Internuclear connections between the pretectum and the accessory optic system in Salamandra salamandra

Summary Application of horseradish peroxidase into the posterior thalamic and basal optic neuropils of Salamandra salamandra (L.) revealed strong reciprocal connections between the pretectum and the accessory optic system. Pretectal neurons located within the periventricular gray matter project to the basal optic neuropil distributing their terminals over the whole extent of this neuropil. A well developed nucleus of the basal optic neuropil, with its neurons within and medial to this neuropil, projects to the posterior thalamic neuropil. Its terminals appear to be located selectively within the core of the posterior thalamic neuropil which receives no ipsilateral retinal afferents.The pretectum and the accessory optic system are reciprocally connected to a ventral tegmental nucleus, which has not previously been described in urodeles. This nucleus is located immediately dorsal to the oculomotor and trochlear nuclei and extends from the oculomotor root to the middle of the trochlear nucleus.Dendrites of the nucleus of Darkschewitsch reach the posterior thalamic neuropil but mainly enter the rostral tegmental neuropil, while the dendrites of the nucleus of the medial longitudinal fasciculus ramify within the basal optic neuropil and the anterior tegmental neuropil with minor branches in the caudal posterior thalamic neuropil.     

Distribution of GABA-immunoreactive neurons in the brain of adult and developing salamanders (Pleurodeles waltli,Triturus alpestris)

The distribution of GABAergic neurons in brains of the family Salamandridae (Pleurodeles waltli, Triturus alpestris) has been investigated immunohistochemically with an antibody against gamma-aminobutyric acid (GABA). In adult animals, immunoreactive neurons, fibers, and terminals are abundantly labeled. In the telencephalon, pallial areas contain fewer GABAergic neurons and fibers than basal forebrain areas. The amygdalar complex and the habenulae have a complex pattern of GABA-immunoreactivity that is especially pronounced within the neuropil. The pretectal and basal optic systems are provided with GABAergic neurons, corroborating electrophysiological results. The dorsal thalamus and parts of the torus semicircularis are almost completely devoid of GABA-immunoreactive neurons. In the torus, magnocellular neurons known to project to the contralateral counterpart are distinctly GABA-immunoreactive. During ontogeny, GABAergic neurons arise early when the first reflexive movements occur after mechanical stimulation. At stage 28, cells are labeled initially near the nucleus of the medial longitudinal fasciculus, which is the first supraspinal tract to appear in ontogeny. At stage 30 (still before hatching), GABAergic neurons are found in the pretectum, immunoreactive neurons arising in the dorsal tegmentum slightly later. Both systems are known to mediate basic reflexes in gaze stabilization. The commissura posterior is GABAergic at early stages suggesting an important functional role in homonymous inhibition between both sides. Thus in salamanders, the neurotransmitter GABA displays a complex distribution, similar to that in other vertrebrates. This pattern emerges early in ontogeny.     

Multitubular bodies in intestinal cells of Amphiuma means/tridactylum (Urodela): ultrastructural characterization

Summary The jejunal absorptive cells of the salamander Amphiuma, when examined using transmission electron microscopy, were found to possess a unique type of intracellular vacuole containing membranous tubules. These vanoles, tentatively named multitubular bodies, were located in the cytoplasm between the nucleus and the brush-border membrane, and were seen with greatest frequency in the summer and fall. The vacuoles containing multitubular bodies had an average diameter of 0.6 m, and the membranous tubules within had an average diameter of 30 nm. The tubules differed morphologically from the vesicles in the multivesicular bodies, and from the primary lysosomes in the polylysosomal vacuoles. The tubules did not exhibit acid phosphatase activity, and were of similar diameter and membrane thickness as the Golgi saccules. In contrast to the multivesicular bodies, the multitubular bodies did not take up exogenous horseradish peroxidase. Early forms of autophagosomes resembling these vacuoles were often seen in the para-Golgi region of the cell. The multitubular bodies may represent a distinct type of autophagosome. Although the exact origin of the tubules as well as their role in cellular activity is unclear, their seasonal appearance within the multitubular bodies of the absorptive cells suggests a unique means of selective down-regulation of Golgi-like organelles.     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

The distribution of GABA-like-immunoreactive neurons in the brain of the newt,Triturus cristatus carnifex,and the green frog,Rana esculenta

Summary The distribution of -aminobutyric acid (GABA) immunoreactivity was studied in the brain of two amphibian species (Triturus cristatus carnifex, Urodela; Rana esculenta, Anura) by employing a specific GABA antiserum. A noteworthy immunoreactive neuronal system was found in the telencephalic dorsal and medial pallium (primordium pallii dorsalis and primordium hippocampi) and in the olfactory bulbs. In the diencephalic habenular nuclei there was a rich GABAergic innervation, and immunoreactive neurons were observed in the dorsal thalamus. In the hypothalamus the GABA immunoreactivity was found in the preoptic area, the paraventricular organ and in the hypothalamo-hypophysial complex. In the preoptic area of the frog some GABA-immunoreactive CSF-contacting cells were shown. In the optic tectum immunolabeled neurons were present in all the cellular layers. A rich GABAergic innervation characterized both the fibrous layers of the tectum and the neuropil of the tegmentum and interpeduncular nucleus. In the cerebellum, in addition to the Purkinje cells showing a variable immunopositivity, some immunoreactive cell bodies appeared in the central grey. Abundant immunolabeled nerve fibers in the acoustico-lateral area and some immunopositive neurons in the region of the raphe nucleus were observed. In conclusion, the GABAergic central systems, well-developed in the amphibian species studied, were generally characterized by close similarities to the pattern described in mammals.Dedicated to Professor Valdo Mazzi (Dipartimento di Biologia Animale, Università di Torino), in honor of his 70th birthday