The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

The axolotl thymus: cell types of the microenvironment

Summary The three-dimensional structure of the axolotl (urodele amphibian) thymus was studied by combined scanning and transmission electron microscopy. The epithelial cell is the major component of the microenvironment forming the meshwork where thymocytes differentiate. Three different types of epithelial cells could be defined by their intracytoplasmic organelles and their localization in the subcapsular or deeper part of the organ. These epithelial cells participate in various types of lymphostromal interactions. Other stromal elements, such as interdigitating reticular cells, macrophages, eosinophil granulocytes and epithelial cysts were also defined. The absence of a true cortico-medullary differentiation in the axolotl thymus, the presence of different stromal elements and the physiological significance of these various microenvironments are discussed.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

The origin of the luteinizing hormone-releasing hormone (LHRH) neurons in newts (Cynops pyrrhogaster): the effect of olfactory placode ablation

Summary Neurons containing luteinizing hormone-releasing hormone (LHRH) are first detected in newt embryos (Cynops pyrrhogaster) in the olfactory epithelium and ventromedial portion of the olfactory nerve, after which they sequentially appear in the intracerebral course of the terminal nerve at prometamorphosis, and in the septo-preoptic area at postmetamorphosis. In adults, however, LHRH-immunoreactive cells are rarely seen in the nasal region, and their distribution shifts into the brain, suggesting their migration. In order to ascertain the origin and possible migration route of these neurons in newt larvae, the effect of unilateral or bilateral olfactory placodectomy on the LHRH neuronal system has been studied. Removal of the olfactory placode results in the absence of LHRH-immunoreactive cells in the nasal and brain regions of the operated side, whereas the subsequent growth and the LHRH-immunoreactive cellular distribution in the contralateral side are identical to those of normal larvae. Following bilateral placodectomy, no LHRH immunoreactivity is detected on either side of the olfactory-brain axis. These results suggest that LHRH neurons of the newt, Cynops pyrrhogaster, originate in the olfactory placode and then migrate into the brain during embryonic development.     

Fine structural and enzyme histochemical observations on the dorsal tail tubercle of Mertensiella caucasica (Urodela,Amphibia)

Summary Dorsal tubercle and skin of Mertensiella caucasica have been investigated with the electron microscope and enzyme histochemical methods. The epidermis of the tubercle consists of 8–9 cell layers, that of normal dorsal skin of 5–6. The tubercle is filled with large mucous glands which are surrounded by an almost complete layer of smooth muscle cells (myoepithelial cells). Their glandular cells undergo cyclical changes and are characterized by specific secretory granules, which differ from those of the relatively small mucous glands of the normal dorsal skin.In the connective tissue of the tubercle a relatively rich supply of nerve fibres has been found, which in part contain synaptic and dense core vesicles or accumulations of mitochondria. In the normal dorsal skin nerve fibres occur less frequently.The following enzymes have been demonstrated in the mucous glands of the tubercle: SDH, acid phosphatase, unspecific esterases, E 600 resistant esterase.The tubercle seems to stimulate the female cloaca chemically and mechanically.     

Prey processing in Leurognathus marmoratus and the evolution of form, and function in desmognathine salamanders (Plethodontidae)

Function and biological role of morphological specialization in desmognathine salamanders are analysed in the light of studies of feeding in Leurognatthus marmoratus. Nine morphological features uniquely characterize the Desmognathinae as compared to its sister group, the Plethodontinae, and other salamanders: (1) heavily ossified and strongly articulated skull and mandible; (2) flat, wedgelike head profile; (3) stalked occipital condyles; (4) modified atlas; (5) modified anterior trunk vertebrae; (6) atlanto-mandibular ligaments; (7) enlarged dorsal spinal muscles; (8) enlarged quadrato-pectoralis muscles; and (9) hind limbs relatively larger than forelimbs. Dorsoventral head mobility is increased at the atlanto-occipital joint by the stalked occipital condyles which simultaneously increase the mechanical advantage of the hypertrophied axial muscles that cross the joint. During head depression the atlanto-mandibular ligaments are placed in tension. Force generated by the quadrato-pectoralis muscles is transmitted directly to the mandible, creating a powerful bite with the jaws in full occlusion. Desmognathines use an efficient static pressure system for subduing and/or killing prey items held in the jaws, not a kinetic-inertial mechanism, as previously suggested. Leurognathus exhibits a behaviour ('head-tucking') unique to desmognathines that is consistent with the static-pressure hypothesis. Several desmognathine features (1, 2, 5, 7, 9) are not explicable as adaptations for feeding; these function as locomotory specializations for burrowing, especially for wedging under rocks within and alongside streams. Desmognathines use head-tucking during such wedging and burrowing movements, thus locomotory specializations act in concert with the feeding specializations. We suggest that origin of the atlanto-mandibular ligaments can be considered a 'key innovation' in that it allowed the secondary invasion of stream habitats by adults of ancestral desmognathines.     

Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltl

The development of the tectum mesencephali was studied in the frog Rana temporaria and the salamander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell proliferation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1) a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tectum.     

Organization of histamine-containing neurons in the brain of the crested newt,Triturus carnifex

The distribution of immunoreactivity for histamine was studied in the brain of the urodele Triturus carnifex using the indirect immunofluorescence method. Histamine-immunoreactive cell bodies were localized in the caudal hypothalamus within the dorsolateral walls of the infundibular recesses. These immunoreactive cell bodies were pear-shaped, bipolar and frequently of the cerebrospinal-fluid-contacting type. Histaminergic nerve fibers were detected in almost all parts of the brain. Dense innervation was seen in the telencephalic medial pallium and ventral striatum, the neuropil of the preoptic area, the septum, the paraventricular organ, the posterior commissure, the caudal hypothalamus, the ventral and lateral mesencephalic tegmentum. Medium density innervation was observed in the lateral mesencephalic tegmentum and optic tectum. Poor innervation was present in the telencephalic dorsal pallium and in the central gray of the medulla oblongata. Few fibers occurred in the olfactory bulbs and in the telencephalic lateral pallium. Double immunofluorescence staining, using an antibody against tyrosine hydroxylase, showed that histamine-immunostained somata and those containing tyrosine-hydroxylase-like immunoreactivity were co-distributed in the tuberal hypothalamus. No co-occurrence of histamine-like and tyrosine hydroxylase-like immunostaining was seen in the same neuron. The pattern of histamine-immunoreactive neurons in the newt was similar to that described in other vertebrates. Our observations, carried out on the apparently simplified brain of the newt confirm that the basic histaminergic system is well conserved throughout vertebrates.     

Noradrenergic and adrenergic systems in the brain of the urodele amphibian,Pleurodeles waltlii,as revealed by immunohistochemical methods

The distribution of noradrenaline and adrenaline in the brain of the urodele amphibian Pleurodeles waltlii has been studied with antibodies raised against noradrenaline and the enzymes dopamine--hydroxylase and phenylethanolamine-N-methyltransferase. Noradrenaline-containing cell bodies were found in the anterior preoptic area, the hypothalamic nucleus of the periventricular organ, the locus coeruleus and in the solitary tract/area postrema complex at the level of the obex. Noradrenergic fibers are widely distributed throughout the brain innervating particularly the ventrolateral forebrain, the medial amygdala, the lateral part of the posterior tubercle, the parabrachial region and the ventrolateral rhombencephalic tegmentum. Putative adrenergic cell bodies were found immediately rostral to the obex, ventral to the solitary tract. Whereas the cell bodies and their dendrites were Golgi-like stained, axons were more difficult to trace. Nevertheless, some weakly immunoreactive fibers could be traced to the basal forebrain. A comparison of these results with data previously obtained in anurans reveals not only several general features, but also some remarkable species differences.Abbreviations Acc Nucleus accumbens - AP area postrema - Apl amygdala, pars lateralis - Apm amygdala, pars medialis - ca commissura anterior - Cb cerebellum - cc central canal - Dp dorsal pallium - epl external plexiform layer - gl glomerular layer of the olfactory bulb - H ganglion habenulae - igl internal granular layer - Ip nucleus interpeduncularis - Lc locus coeruleus - Ll lateral line lobe - Lp lateral pallium - Ls lateral septum - ml mitral cell layer - Mp medial pallium - Ms medial septum - nPT nucleus pretectalis - NPv nucleus of the periventricular organ - nV nervus trigeminus - oc optic chiasm - Poa preoptic area - Ri nucleus reticularis inferior - SC nucleus suprachiasmaticus - sol solitary tract - Str striatum - thd thalamus dorsalis - thv thalamus ventralis - To tectum opticum - TP tuberculum posterius - V ventricle - VH ventral hypothalamic nucleus - III nucleus nervi oculomotorii - IXm nucleus motorius nervi glossopharyngei - Xm nucleus motorius nervi vagi     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.