Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

Changes in intracellular pH following egg activation and during the early cell cycle of the amphibian Pleurodeles waltlii, coincide with changes in MPF activity.

Previous work on Xenopus laevis suggests a temporal coincidence between inactivation of the M-phase promoting factor (MPF) and intracellular pH (pHi) increase during egg activation. In addition, we recently showed that during the early cell cycle of Xenopus eggs, MPF activity cycling and pHi oscillations were temporally and functionally related. In the present work, using eggs of another amphibian, Pleurodeles waltlii, which has a natural cell cycle considerably longer than that of Xenopus laevis, we show a temporal coincidence between MPF activity and pHi changes, both at the time of egg activation and at each of the following cell cycles. Egg activation-induced pHi changes in Pleurodeles did not involve classical plasma membrane ion exchangers, and were not due to the activation of a H+ conductance. On the other hand, the pHi oscillations intervening at each cell cycle were suppressed by inhibitors of protein synthesis or phosphorylation, as were their counterparts in Xenopus eggs. We propose that physiological pHi changes in Pleurodeles and Xenopus eggs might have a metabolic origin, in direct relation with the cascade of phosphorylations-dephosphorylations of proteins implicated in the control of the cell cycle.     

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.     

Hemoglobin switching in the salamanderPleurodeles waltlii

Summary In order to study the cellular distribution of larval and adult hemoglobins during larval development ofPleurodeles waltlii a double specific immunofluorescent labelling technique was developed.Rabbit antibodies specific for larval and adult hemoglobin components were prepared and conjugated with tetramethyl-rhodamine isothiocyanate for the anti-larval antibodies and fluorescein isothiocyanate for the anti-adult hemoglobin antibodies.Both simultaneous and sequential staining with the two types of fluorescent antibodies indicated that larval and adult hemoglobins were never observed within the same erythrocyte during development. The results provide evidence that two distinct cell populations exist, one synthesizing exclusively larval hemoglobins which is progressively replaced by the other one synthesizing exclusively adult hemoglobins. It remains to be determined if these two populations arise from two distinct types of stem cells (adult and larval) or from the same stem cell type.