Elimination of serotonergic cells induces a marked increase in generation of dopaminergic neurons from mesencephalic precursors.

Production of dopaminergic (DA) neurons from stem/precursor cells for transplantation in Parkinson's disease has become a major focus of research. However, the inductive signals mediating the production of DA neurons remain poorly understood, and the influence of other cell populations simultaneously generated within the cell aggregates has not been studied. We investigated whether DA phenotype (i.e. tyrosine hydroxylase-immunoreactive, TH-ir), serotonergic, floor plate (FP4-ir), and fibroblast growth factor 8 (FGF-8)-ir cells differentiate from proliferating cell aggregates obtained from rat mesencephalic precursors, and we also investigated the effects of serotonergic cells on differentiation of DA cells. We observed FP4-ir, FGF-8-ir, TH-ir and serotonergic cells within the aggregates. The TH-ir cells appeared within or in close proximity to a central FP4-ir core, and then concentrated peripherally forming a cap that surrounded the central FP4-ir area. The serotonergic cells and fibers formed a cap surrounding that of TH-ir neurons. Cell aggregates treated with an antibody against FGF-4 or with the serotonergic toxin 5,7-dyhydroxytryptamine or the serotonin synthesis inhibitor dl-p-chlorophenylalanine showed a marked decrease in the number of 5-HT-ir cells (10-20% of controls) and a marked increase in that of TH-ir neurons (700-900% of controls). The present results show that manipulation of other cell populations in the cell aggregates, particularly the serotonergic population, may be an effective method of increasing the production of DA neurons from stem/precursor cells.

Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody.

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole-mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior-posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm-inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.

[The notochord proteins of true sturgeons carrying the At1 epitope]. / Belki khordy osetrovykh ryb, nesushchie épitop At1.

Previously we reported the production of monoclonal antibodies against chordin, an acidic glycoprotein from true sturgeon notochord, carrying glycans terminating with 3-sulfoglucuronic acid. In addition, monoclonal antibodies At1, not reacting with chordin, were produced. Here we describe At1 epitope expression in sturgeon tissues and target proteins for At1 antibodies, and test interaction of these proteins with chordin and other molecules carrying glycan with 3-sulfoglucuronic acid. The expression of the antigens carrying At1 epitope during sturgeon development has also been studied.

New cell surface marker of the rat floor plate and notochord.

Regionally expressed cell surface molecules are thought to mediate contact-dependent interactions that regulate pattern formation and axon pathfinding in the developing vertebrate central nervous system (CNS). We recently isolated monoclonal antibody (mAb) CARO 2 through a screen for positional markers in the developing rat CNS. Between embryonic day (E)11.5 and E13, mAb CARO 2 specifically labels both the floor plate and notochord in the developing spinal cord. In contrast to the distribution of several well-characterized ventral midline markers, mAb CARO 2 labeling is restricted to the apical portion of the floor plate and the outer surface of the notochord. The anterior limit of mAb CARO 2 immunoreactivity corresponds to the midbrain/hindbrain border. Floor plate labeling persists throughout embryogenesis, whereas notochord labeling is not detectable after E13. During later stages of embryonic development (E16-E20) apically restricted floor plate labeling is present only in the rostral spinal cord. In postnatal rats, mAb CARO immunoreactivity is not present in any region of the CNS. Immunoblot analyses show that mAb CARO 2 recognizes an epitope on a 28-kD protein that is enriched in the floor plate, transiently expressed during embryogenesis, and membrane-associated. Consistent with the latter result, mAb CARO 2 labels the surfaces of floor plate cells. These findings suggest that the CARO 2 antigen is a new cell surface marker of the floor plate and notochord which may participate in neural cell patterning and/or axon guidance in the developing rat spinal cord.

XK endo B is preferentially expressed in several induced embryonic tissues during the development of Xenopus laevis.

XK endo B is a type I keratin that was originally identified by its preferential expression in the embryonic notochord of the amphibian Xenopus laevis. A peptide identical to a short region of its predicted amino acid sequence was used to generate antibodies against the XK endo B protein. This paper reports an immunocytochemical study of the spatial expression pattern of XK endo B during development. The protein was observed in the notochord and endoderm as predicted from previous RNA analysis. In addition, XK endo B was detected in the cement gland, in the pituitary, olfactory and pharyngeal pouch rudiments, and in a nonuniform distribution in the neural tube as well as the inner sensorial layer of the ectoderm. XK endo B expression is not limited to any germ layer or any particular cell type, but is nevertheless highly restricted in its distribution in the embryo. Its expression in several different embryonic tissues requiring inductive interactions for differentiation makes XK endo B a valuable tool with which to study the regulation of induced gene expression during embryogenesis.

[Expression of embryonal and differentiational proteins in chordomas and in the notochord].

The expression of the embryonal and of the differentiational proteins in 12 cases of chordoma and in the notochord of a 4-month-old and a 5-month-old human embryo have been examined immunohistologically by the ABC method using polyclonal antibodies to CEA, AFP, or S-100, and the monoclonal antibody to cytokeratin. It was found that S-100 was expressed in all cases of chordoma and in the notochords examined. CEA and cytokeratin also were found expressed in some cases of chordoma but not in the notochords. These proteins were found expressed more strongly in chordomas without a metastasis than in those with a metastasis. In the metastatic lesions, these proteins were expressed more strongly than in the primary lesions. The antibody to AFP reacted with neither the chordomas nor the notochords tested. These results suggest a possible link between the gene-expression of the tumor cells and the microenvironment in which they are harbored.

P-epitope is characteristic for the neural tissue of vertebrates.

In a search for antigens immunologically related to chordin, a notochord-specific glycoprotein of sturgeneous fishes, extracts from 55 samples of human and rabbit tissues were tested for inhibition of [125I]chordin binding to rabbit polyclonal antibodies. The strongest inhibition was observed with brain extracts of both species. Human, chicken, rabbit, and newt brain extracts also inhibited chordin binding in liquid phase to monoclonal antibodies (MAbs) against the P-epitope, the most immunogenic epitope of this glycoprotein. Immunohistochemical studies done on human and chicken embryos, newt, sterlet, and sturgeon embryos, larvae, and juveniles revealed a strong immunoreactivity of the brain, spinal cord, and tissue of the peripheral nervous system with an anti-P MAb. Other tissues, with several exceptions, showed a negative reaction in immunohistochemical experiments. The authors found that the P-epitope is ontogenetically expressed in the neural tissue of chicken, newt, and sterlet at the period of cytodifferentiation. Gel chromatography of human, chicken, and newt brain extracts showed that in each case the P-epitope was associated with a polydisperse macromolecular material of similar size. These antigens were designated as neurochordins. Prolonged pronase digestion of human and chicken brain extracts resulted in fragments with M about 3 kDa (presumably glycopeptides), which reacted with anti-P MAbs. These fragments were of the same size as corresponding glycopeptides of the pronase digest of chordin. Thus, in the present study, the P-epitope has been shown to be characteristic for the neural tissue of several vertebrate species; in the brain, it has been found in association with neurochordins, macromolecular antigens that are presumably protein conjugates with carbohydrates.

Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidian Halocynthia roretzi.

To obtain specific immunological probes for studying molecular mechanisms involved in the early embryonic development of ascidians, we have produced monoclonal antibodies directed against a homogenate of larvae of the ascidian Halocynthia roretzi. Among these, we have screened monoclonal antibodies that specifically recognize cells and/or tissues of the embryo. Characterization of six epidermis-specific monoclonal antibodies (including larval tunic-specific and larval fin-specific), three muscle-specific antibodies, two endoderm-specific antibodies, one notochord-specific antibody and two monoclonal antibodies that specifically recognize trunk-lateral cells suggests that these monoclonal antibodies may be useful as markers for analysing molecular mechanisms involved in specification of these cells. Seven monoclonal antibodies characteristically stain intercellular materials of the developing embryo and may therefore be valid for studying cellular construction of the embryo. Furthermore, monoclonal antibodies that recognize components of follicle cells, perivitelline space and sperm have also been established.

The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis.

Antibodies against various intermediate filament proteins have been used to follow cell differentiation in the early Xenopus embryo. Three new monoclonal antibodies against Xenopus cytokeratins raised against Triton-insoluble material from tadpoles (RD35/2a, RD35/3a and D3/3a), two antibodies against mammalian cytokeratins (LE65 and LP3K), monoclonal anti-(rat 200 K neurofilament protein), rabbit anti-(rat glial filament acidic protein), and rabbit antibodies to hamster and calf vimentin were used. We show that cytokeratins are present in the early central nervous system (CNS) and persist in the ependymal cells of the adult CNS. We also show that the notochord contains cytokeratin. The ontogeny of intermediate filament protein appearance in the CNS, skin and notochord between neural fold stage and swimming tadpole stage are described. These results are discussed in particular with regard to the use of the antibodies as differentiation markers.