Hematopoietic stem cells debut in embryonic lymphomyeloid tissues of elasmobranchs.

The evolutionary initiation of the appearance in lymphomyeloid tissue of the hemopoietic stem cell in the earliest (most primitive) vertebrate model, i.e. the elasmobranch (chondroichthyan) Torpedo marmorata Risso, has been studied. The three consecutive developmental stages of torpedo embryos were obtained by cesarean section from a total of six pregnant torpedoes. Lymphomyeloid tissue was identified in the Leydig organ and epigonal tissue. The sections were treated with monoclonal anti-CD34 and anti-CD38 antibodies to detect hematopoietic stem cells. At stage I (2-cm-long embryos with external gills) and at stage II (3-4 cm-long embryos with a discoidal shape and internal gills), some lymphoid-like cells that do not demonstrate any immunolabeling for these antibodies are present. Neither CD34+ nor CD38+ cells are identifiable in lymphomyeloid tissue of stage I and stage II embryos, while a CD34+CD38- cell was identified in the external yolk sac of stage II embryo. The stage III (10-11-cm-long embryos), the lymphomyeloid tissue contained four cell populations, respectively CD34+CD38-, CD34+CD38+, CD34-CD38+, and CD34-CD38- cells. The spleen and lymphomyeloid tissue are the principal sites for the development of hematopoietic progenitors in embryonic Torpedo marmorata Risso. The results demonstrated that the CD34 expression on hematopoietic progenitor cells and its extraembryonic origin is conserved throughout the vertebrate evolutionary scale.

Evolutionary intraembryonic origin of vertebrate hematopoietic stem cells in the elasmobranch spleen.

The electric ray (Torpedo Marmorata Risso) provides an animal model for the detection of early intraembryonic hemopoietic stem cells in sea vertebrates. The spleen of this bone-marrowless vertebrate appears to be the major site of hemopoietic stem cell differentiation during development and in adulthood. Splenic development in this species was investigated and hemopoietic stem cells were detected in this organ by immunocytochemistry utilizing CD34 and CD38 antibodies. At stage I (2-cm-long embryos with external gills), the spleen contains only mesenchymal cells. At stage II (3-4 cm-long embryos with a discoidal shape and internal gills), an initial red pulp was observed in the spleen, without immunostained cells. At stage III (10-11-cm-long embryos), the spleen contained well-developed white pulp, red pulp and ellipsoids. Image analysis at stage III showed four cell populations, i.e. CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38- cells. The present findings, obtained from an elasmobranch, indicate that the CD34 and CD38 phenotypes are conserved through vertebrate evolution.

Developmental regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor in Torpedo electrocyte.

Tyrosine phosphorylation is thought to play a critical role in the clustering of acetylcholine receptors (AChR) at the developing neuromuscular junction. Yet, in vitro approaches have led to conflicting conclusions regarding the function of tyrosine phosphorylation of AChR beta subunit in AChR clustering. In this work, we followed in situ the time course of tyrosine phosphorylation of AChR in developing Torpedo electrocyte. We observed that tyrosine phosphorylation of the AChR beta and delta subunits occurs at a late stage of embryonic development after the accumulation of AChRs and rapsyn in the membrane and the onset of innervation. Interestingly, in the mature postsynaptic membrane, we observed two populations of AChR differing both in their phosphotyrosine content and distribution. Our data are consistent with the notion that tyrosine phosphorylation of the AChR is related to downstream events in the pathway regulating AChR accumulation rather than to initial clustering events.

Acetylcholine receptor and myogenic factor gene expression in Torpedo embryonic development.

The mRNA levels of acetylcholine receptor (AChR) and myogenic factors were followed during embryonic development of Torpedo skeletal muscle and its homologue, the electric organ. A different developmental pattern of AChR gene expression was found in these two tissues: a slight decrease in the muscle, and a marked increase, concomitant with synapse formation, in the electric organ. However, the developmental pattern of MyoD and MRF4 mRNA levels was similar in both tissues, with no significant changes during development. This is in contrast with the sharp increase in the expression of AChR in the electric organ and may suggest that the burst in the expression of AChR during the differentiation of myotubes into electrocytes is not regulated by changes in the myogenic factor mRNA levels.

Differences between embryonic and adult Torpedo acetylcholine receptor gamma subunit.

Antibodies to a synthetic peptide corresponding to residues 346-359 of the Torpedo acetylcholine receptor (AChR) gamma subunit, were employed to compare the adult and embryonic receptor. This peptide contains a consensus phosphorylation site for cAMP-dependent protein kinase (PKA). The anti-peptide antibodies discriminated between adult and embryonic AChRs, and reacted preferentially with the adult gamma form. These observed immunological differences did not seem to stem from different phosphorylation states. Our results suggest that the embryonic Torpedo AChR may have a gamma-like subunit that differs from the known adult form of this subunit, at least in the specific region that contains the phosphorylation site for PKA.

Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes.

The expression of the postsynaptic 43-kDa and 58-kDa proteins and actin during development of the Torpedo marmorata electric organ was compared to that of nicotinic acetylcholine receptors (AChRs). Western blot analysis demonstrates that AChRs and proteins of 43 kDa (43K protein) and 58 kDa (58K protein) are all present prior to synaptogenesis. Subsequently, levels of all 3 synaptic proteins increase dramatically during differentiation and innervation of electrocytes. In contrast, actin is present in relatively high concentrations at early times and decreases thereafter. The equimolar ratio of AChRs and the 43K protein found in the adult electric organ is established early in development. Furthermore, the AChR and 43K protein share a common postsynaptic localization in electrocytes following synapse formation. Aggregates of the AChR that form at the ventral pole of the oval-shaped electrocytes prior to innervation, however, show no detectable immunofluorescence staining with anti-43K monoclonal antibodies. Therefore, in some cases, aggregation of AChRs occurs without the 43K protein.

Development of the electromotor system in Torpedo marmorata: cationic staining of the electric organ.

The electric organs of embryonic Torpedo marmorata have been reacted with three cationic stains to evaluate the appearance and distribution of anionic sites. Ruthenium red, alcian blue and lysozyme were used at different pHs and found to react in a time-related manner to anionic components within the interelectrocyte space. The basal lamina covering the ventral electrocyte surface possesses the greatest number of anionic sites whereas growth cone, presynaptic terminal and glial membranes displayed almost no staining. Since this lamina serves as the exclusive substrate for ingrowing neurites during synaptogenesis, the results are consistent with the idea that charge distribution on the membrane surface may provide a necessary cue for neurite motility, extension and eventual synaptogenesis.

Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adult Torpedo ocellata.

The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G2 form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G2 form in back muscle is sensitive to PIPLC, whereas the G4 tetramer of Torpedo brain is completely resistant.

Creatine phosphokinase: isoenzymes in Torpedo marmorata.

Creatine phosphokinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) is the major constituent of the "low-salt-soluble" proteins of the electric organ from Torpedo marmorata. The denatured subunits of the enzyme have an apparent Mr of 43 000 and isoelectric points ranging between pH 6.2 and pH 6.5. Identical properties are found for the creatine phosphokinase from Torpedo muscle tissue. Anti-(electric organ creatine phosphokinase) antibodies are specific for the muscle-type enzyme and do not cross-react with enzymes present in Torpedo brain and electric lobe tissue. Biochemical and immunochemical properties of the enzyme associated with acetylcholine-receptor-enriched membranes show that this enzyme is as the "low-salt-soluble" electric organ enzyme of the muscle-specific type. In vitro translation of electric organ poly(A)-rich mRNA in a reticulocyte lysate reveals the abundance of mRNA specific for muscle creatine phosphokinase. During embryonic development of the electrocyte a continuous increase of translatable amounts of this mRNA is observed. No brain-type polypeptides are synthesized. The subunits of the brain-specific enzyme differ in molecular mass (Mr approximately equal to 42000) and isoelectric properties (pI approximately equal to 7.0-7.2). The unexpected finding that the brain forms are more basic than the muscle-specific enzyme is supported by agarose and cellulose acetate electrophoresis and ion-exchange chromatography properties.

Torpedo electromotor system development: developmentally regulated neuronotrophic activities of electric organ tissue.

Explant cultures of electric lobe from 45-60 mm stage Torpedo embryos and both ganglionic and dissociated cell cultures prepared from 8-day chick ciliary ganglia have been used to determine whether the electric organs of Torpedo marmorata contain developmentally regulated neuronotrophic activity. Electric lobe explants were evaluated by measuring their neurone density, choline acetyltransferase (CAT0, and low salt, Triton X-100-soluble protein contents. Addition of soluble extracts prepared from the electric organs of late stage embryos (85-105 mm) to standard medium results in the maintenance of nearly theoretical neurone densities in electric lobe explants during a 7-day culture period. Soluble electric organ extracts from early embryonic stages (42-59 mm) do not increase neurone density relative to control cultures but cause an elevation in the CAT content of the explants over control values. On the basis of this analysis it is concluded (1) that late embryonic stage and adult electric organs contain neuronotrophic activity that allows electromotor neurones to survive in vitro and (2) that activity increases rapidly in the electric organs between the 59 nd 72 mm stages of development at a time when rapid increases in postsynaptic membrane markers in the electric organs occur and when peripheral synaptogenesis begins. The activity of late stage embryonic electric organs is heat stable and lost on dialysis. Using ciliary ganglion explants and evaluating both the initial fibre outgrowth and the CAT content after 4 days in vitro, trophic activity is found to be maximal at early embryonic stages (45-55 mm) and to decline thereafter. It is shown that the decline in activity is not due to an increase in toxicity. Using established dissociated ganglionic cell survival assays the specific activity of neuronotrophic factors allowing survival is constant between the 45 and 73 mm stages in the electric organs and then rapidly declines, but activity per electric organ increases rapidly between the 45 and 73 mm stages and then remains at a constant level. The use of poly-dl-ornithine substrates coated with heart-conditioned medium for the cell survival assay results in up to tenfold increase in the trophic titre of the electric organ extracts. The neuronotrophic activity supporting survival of ciliary motorneurones present in embryonic electric organs is heat labile and retained on dialysis. It is concluded that developing electric organs contain at least two neuronotrophic factors that have different properties and are differently regulated. Both factors may contribute during development to bringing naturally occurring electromotor neurone cell death to an end.

The developmental morphology of Torpedo marmorata: the electric lobes.

The development of the electric lobes of Torpedo marmorata has been investigated using light and electron microscopical techniques. The lobe Anlagen become visible in the rhombencephalon along the floor of the 4th ventricle at the 10-mm stage. Many of the neuroepithelial cells in the Anlagen differentiate, Becoming postmitotic and axonic by the 24 mm stage. Proliferative zones of neuroepithelial cells disappear from the electric lobes by the 30-mm stage. After their initial, early differentiation the electromotor neurons remain monopolar until the 40-mm stage when dendrite formation begins. The differentiation of the electromotor neuron from a mono- to an immature multi polar form occurs between the 40- and 55-mm stages and involves, in addition to dendrite formation, a change from a pear-shaped to a spherical cell body, a dramatic increase in cytoplasmic volume, a centralization of the nucleus, an enlargement of the nucleolus and its migration away from the nuclear membrane, and differentiation of the axon hillock. The electric lobes are invaded by sinusoids at the 24-mm stage but formation of the capillary network by sprouting cords of endothelial cells begins later at the 40-mm stage. Neuronal cell death (26-74-mm stages) appears to be mainly an autolytic process and the debris is removed by immature glial cells. Afferent fiber growth cones are first recognized in the lobes at the 60-mm stage but synapses are not observed until the 78-mm stage. Myelination begins in the electric lobes concomitantly with the onset of synaptogenesis. A twofold increase in dendrite length occurs over the period when synapses begin to form in the lobes but dendritic maturation is not complete until the neonatal (120-mm) stage. The results are discussed in relation to the development of the electric organs.

The developmental morphology of Torpedo marmorata: electric lobe-electromotoneuron proliferation and cell death.

Electromotoneuron proliferation and cell death have been quantitatively studied in the electric lobe of Torpedo marmorata from an embryonic body-length stage of 26-mm to adult animals. These neurons project to the electric organ and form synapses with electrocytes which possess a remarkably large postsynaptic target surface. For this reason cell death would not be predicted to occur if synaptic competition were to be hypothesized as the cause. Isolated observations at the ultrastructural level suggested, however, that cell death was indeed taking place and therefore it seemed appropriate to examine this question in detail. Our findings show first that neuron production appears to be a continuous process throughout the period studied, generating totals of over 70,000 electromotoneurons per lobe by adulthood. Second, two waves of cell death were identified, one occurring early in embryogenesis (stage 30 mm), well before the onset of synaptogenesis, and a second coincident with the onset of synaptogenesis (stages 55--74 mm). It is difficult to reconcile this latter wave with the hypothesis of synaptic competition as the postsynaptic surface at this time of development is largely devoid of synaptic contacts. We conclude that in the electromotor system of Torpedo, synaptic competition is probably not the mechanism of cell death.