[The antigen of the differentiated basal disc cells of Hydra].

A new antigenic marker of the differentiated basal disk cells of Hydra was characterized. An antigen named 3G11 was revealed by monoclonal antibody in granules of the basal disk gland cells of the ectoderm. The antigen appearance during budding, regeneration and ectopic foot formation evidences for the differentiation of the body column epithelial cells into basal disk gland cells. Antigen 3G11 is species-specific: among six hydra species investigated, the antigen was observed exclusively in polyps of vulgaris group which is a special taxon of the filum Hydra. Cell and tissue localization of the antigen 3G11 was similar to that of the well-established biochemical hydra marker, foot specific peroxidase, reported formerly. However, ELISA data suggest that the molecule bearing antigen 3G11 does not possess any peroxidase activity. Thus the new hydra antigenic marker 3G11 extends the number of previously used markers of differentiation and allows to improve the technique of the basal disk differentiated tissue identification.

FGF19-FGFR4 signaling elaborates lens induction with the FGF8-L-Maf cascade in the chick embryo.

The fibroblast growth factor (FGF) family is known to be involved in vertebrate eye development. However, distinct roles of individual FGF members during eye development remain largely elusive. Here, we show a detailed expression pattern of Fgf19 in chick lens development. Fgf19 expression initiated in the forebrain, and then became restricted to the distal portion of the optic vesicle abutting the future lens placode, where FGF receptor 4 (Fgfr4), a receptor for FGF19, was expressed. Fgf8, a positive regulator for L-Maf, was expressed in a portion of the optic vesicle. To examine the role of FGF19 signaling during early eye development, Fgf19 was misexpressed near the presumptive lens ectoderm; however, no alteration in the expression of lens marker genes was observed. Conversely, a secreted form of FGFR4 was misexpressed to inhibit an FGF19 signal, resulting in the induction of L-Maf expression. To further define the relationship between L-Maf and Fgf19, L-Maf misexpression was performed, resulting in ectopic induction of Fgf19 expression by Hamburger and Hamilton's stage 12/13. Furthermore, misexpression of Fgf8 induced Fgf19 expression in addition to L-Maf. These results suggest that FGF19-FGFR4 signaling plays a role in early lens development in collaboration with FGF8 signaling and L-Maf transcriptional system.

Ectodermal Wnt function as a neural crest inducer.

Neural crest cells, which generate peripheral nervous system and facial skeleton, arise at the neural plate/ectodermal border via an inductive interaction between these tissues. Wnts and bone morphogenetic proteins (BMPs) play roles in neural crest induction in amphibians and zebrafish. Here, we show that, in avians, Wnt6 is localized in ectoderm and in vivo inhibition of Wnt signaling perturbs neural crest formation. Furthermore, Wnts induce neural crest from naive neural plates in vitro in a defined medium without added factors, whereas BMPs require additives. Our data suggest that Wnt molecules are necessary and sufficient to induce neural crest cells in avian embryos.

Mesoglia & microglia--a historical review of the concept of mononuclear phagocytes within the central nervous system.

More than a century and a half has elapsed since the first accounts of mesodermal phagocytic elements were proposed within the central nervous system. Over the intervening decades, body and substance were added to this concept through the advancement of histological techniques at the disposal of the researcher and the acute and keen-minded skills of the pathologist. Notable among these pioneering efforts were the contributions of W. Ford Robertson, Santiago Ramon y Cajal, Pio del Rio-Hortega and Wilder Penfield amongst an entire cavalcade of other noteworthy figures. The term 'mesoglia' and 'third element of the nervous system' was bestowed upon these cells towards the beginning of the twentieth century to account for their separate origins from neurons and macroglia. It was later amended by del Rio-Hortega in 1919, to 'microglia' in order to further discriminate between true mesodermal elements and oligodendrocytes, previously regarded as a component of 'mesoglia'. This particular contention sparked much controversy among del Rio-Hortega's peers and resulted in an escalation of fruitful research throughout Europe that eventually declined up to the outbreak of the Second World War. The post-war years were a period of the 'dark ages' that cast doubt on the very existence and nature of microglia, until the 'renaissance' of research was once again rejuvenated in the 1960s, by a new cohort of intrigued minds: Cammermeyer, Blinzinger, Kreutzberg and others who saw in the 'third element' the potential that is now commonly ascribed to microglia: the intrinsic immune effector cells of the CNS. It is now universally accepted that microglia are involved as the first line of rapid defence in any pathology of the nervous system, and as such, present a diagnostic tool for the neuropathologist. Although our knowledge of microglia stems from an extensive body of work conducted over the last two decades, much of the earlier work (pre-1960s) has remained somewhat obscure. This is partly accountable due to the limited availability of translated works, and additionally to the lack of a compendium of these articles. This paper will present a comprehensive overview of the pioneering research on mononuclear phagocytes within the central nervous system, which has direct bearing on our present-day understanding of the concept of microglia.

Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere.

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16-cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCl-treated whole embryos developed into exo-gastrulae but Hpoe restriction in ectoderm occurred in these exo-gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.

Immunohistochemical characterization of monoclonal antibodies (PDs) as markers of the periderm in the developing chicken embryo.

The outermost surface cell layer of the developing embryo, the periderm, arises from the initial single layer of ectoderm and is eventually exfoliated from the stratified epidermis, which has the same ectodermal origin. In this study, monoclonal antibodies against chicken limb bud ectoderm were generated and screened for those which stained the periderm. Four separate antibodies termed PD2, 3, 7 and 9 were obtained from 180 mixed hybridomas. These PD antibodies stained the periderm selectively at all stages examined (stage 20-42). By correlating the results of immunohistochemistry with observations made by transmission electron microscopy, it was revealed that PD antibodies stained both the squamous periderm at an early stage and rounded bulging peridermal cells just before exfoliation. Therefore we feel that PD antibodies may be useful in further systematic investigations of the development and function of the chicken embryonic periderm.

The mouse chimera during intrauterine stages: immunohistochemical analysis with the C3H strain-specific antibody.

Our objective was to establish an immunohistological method for analysis of chimerism in mouse chimeras at embryonic stages with an anti-C3H strain-specific antigen (CSA) antibody. We developed an effective new method to retain CSA antigenicity with good morphology of embryonic tissues by using microwave irradiation (MWI) for pre-fixation, 95% ethanol/1% acetic acid as post-fixative solution, and polyester wax as embedding material. We used a biotinylated mouse monoclonal anti-CSA antibody, peroxidase-avidin, and silver amplification. These procedures were successful in demonstrating the chimerisms in various tissues of C3H<-->Balb/c chimeras at different embryonic stages and postnatal days. In chimeras at Days 7 and 7.5 post coitum (p.c.), both genotypes were clearly identified and well intermingling in every embryonic tissue (embryonic ectoderm, mesoderm, extra-embryonic ectoderm, ectoplacental cone, amnion, and chorion). Chimerisms at Day 14.5 p.c. were also clearly observed in mesencephalon, neural retina, spinal cord, lung, kidney, and liver. We concluded that the present immunohistological procedures for analysis of chimerism during embryonic periods will give us insightful information about dynamic histological changes such as cell proliferation, migration, selection, and death during organogenesis.

Immunoelectron microscopic analysis of a novel carbohydrate differentiation antigen (CDA-3C2) in the developing rat olfactory and otic systems.

A carbohydrate differentiation antigen (CDA-3C2) exhibits a highly specific and restricted pattern of expression during rat embryogenesis. In the periphery of the embryo, this antigen is associated transiently with the lateral ectoderm but is retained only in the olfactory and otic epithelium throughout morphogenesis. At the light microscopic level, CDA-3C2 immunoreactivity appears mostly along cell periphery and in the extracellular matrix. The aim of the present study was to determine the specific cellular and subcellular distribution of CDA-3C2 in vivo in order to identify potential sites of cellular and tissue function of the antigen during embryogenesis. There was a strikingly similar subcellular distribution of CDA-3C2 in the developing otic and olfactory systems, found mostly along cell membranes, microvillar projections and acellular secretions of the epithelium. Mature sensory components of the epithelia were not immunoreactive, whereas supportive cells and their secreted structures were densely stained. The highly coincident nature of CDA-3C2 in both sensory epithelia suggests that this carbohydrate epitope, and possibly its carrier macromolecule, participate in a morphogenetic function common to these two sensory epithelia.

Dermal cylindroma. Expression of intermediate filaments, epithelial and neuroectodermal antigens.

We report on immunohistochemical staining patterns in so-called apocrine tumors of skin with special emphasis on the dermal cylindroma. The results were compared with apocrine tubular adenoma, syringocystadenoma papilliferum and the normal eccrine sweat gland. A relationship of dermal cylindroma to the apocrine gland is suggested by expression of lysozyme and alpha 1-antichymotrypsin. The tumor shares keratin, epithelial membrane antigen (EMA) and EGF-receptor expression with eccrine and apocrine glands. The presence of intermingled cells with a coexpression of keratin and vimentin argues for a partial myoepithelia-like differentiation. Neuroectodermal antigens are missing. Therefore, dermal cylindroma is classified as an adnexal tumor of skin with a variable rate of cells of apocrine secretory, myoepithelial and undifferentiated phenotypes.

Embryonal germ-layer antigens: target for autoimmunity.

Histopathological analysis of some systemic autoimmune diseases and syndromes led us to the conclusion that the common feature of the organs involved might be their embryonal origin. We suggest that organs derived from the same germ layer express common germ-layer-specific antigens. Such antigens could serve as target antigens for the autoimmune response.

Immunohistochemical localization of the carbohydrate antigen 4C9 in the mouse embryo: a reliable marker of mouse primordial germ cells.

Expression of 4C9, a Lex[Gal beta 1----4(Fuc alpha 1----3)GlcNAc] antigen, during mouse embryogenesis was studied by immunohistochemical methods. Distribution of 4C9 was similar to, but not identical with that of SSEA-1 (stage-specific embryonic antigen-1). Notably, 4C9 was detected in some of the inner cell mass cells of late blastocysts, ectoderm cells migrating from the primitive streak to the mesoderm space and primordial germ cells just formed from the migrating cells. Thus, 4C9 was considered to be continuously expressed in the cell lineage starting at the totipotent 8 cell stage and leading to primordial germ cells. While 4C9 gradually decreased from the surface of primordial germ cells after they have settled in the gonad, the antigen remained in cytoplasmic granules for some period in a sex determined manner. In male gonads, cytoplasmic granules positive for 4C9 tended to be polarized to one side of cytoplasm. The 4C9 reactive material completely disappeared from male germ cells by day 16 of gestation. In female gonads, granules scattered throughout the cytoplasm and cell surface were positive for 4C9. On day 16 of gestation the cell surface antigenicity was lost, but some cytoplasmic antigenicity still remained. As above, 4C9 is a reliable marker to study the origin, migration and differentiation of primordial germ cells, and to distinguish male and female germ cells. By immunoelectron microscopy, 4C9 was detected at the plasma membrane, the Golgi apparatus, and dense-cored vesicles in primordial germ cells on 10-11 days of gestation.

[The distribution of competence for adenohypophysis development in the ectoderm of chick embryos]. / Raspredelenie kompetentsii k adenogipofizarnomu razvitiiu v éktoderme kurinykh zarodyshei.

The ability of various zones of the cephalic and trunk ectoderm to differentiate into adenohypophysis after the contact with the bottom of the prosencephalon was studied in tissue culture of chick embryos as the stage of 10-13 somites. Stomodeal presumptive lens ectoderm and lateral cephalic ectoderms were shown to be competent for development into adenohypophysis. In all cases adenohypophyseal cords were formed in the zones of ectoderm contact with the brain. The cords contained antigens A-2, A-3 specific for chicken adenohypophysis as well as ACTH and beta-lopotropin. Trunk ectoderm proved to be incapable to differentiate into adenohypophysis.

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.

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.

An axon growth associated antigen is also an early marker of neuronal determination.

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.

CA 125 in the epithelium closely related to the embryonic ectoderm: the periderm and the amnion.

The amnion is believed to be derived from either cytotrophoblastic cells or embryonic ectoderm. However, it produces and secretes CA 125, which is considered a differentiation antigen of fetal coelomic epithelium derived from the mesoderm of germ cells. To verify this, the immunohistochemical localization of CA 125 in human fetal tissues (between 7 and 23 weeks of gestation) derived from the ectoderm, endoderm, or mesoderm, and in the fetal membranes and placenta was studied. Among the mesoderm-derived tissues, only the fetal coelomic epithelium-related tissues were positive for anti-CA 125 from 15 weeks of gestation. The endoderm-derived tissues did not react with anti-CA 125. However, among the ectoderm-derived tissues, only the periderm reacted with anti-CA 125 from 7 weeks until it sloughed from the stratum intermedium by 23 weeks of gestation. Among the fetal membranes and placenta, only the amnion reacted with anti-CA 125 from 9 weeks to term. These findings indicate that the amnion and the periderm, both of which constitute the epithelia covering the amniotic cavity, in addition to the fetal coelomic epithelium-related tissues, produce CA 125.

Phenotypic analysis of four human medulloblastoma cell lines and transplantable xenografts.

An extensive panel of monoclonal antibodies (MAb) and monospecific antisera reactive against neuroectodermal-, neuronal-, glial-, and lymphoid-associated antigens, extracellular matrix, HLA, and cell-surface receptors was used to characterize the phenotype of four continuous, karyotypically distinct medulloblastoma cell lines and transplantable xenografts. All four cell lines demonstrated significant reactivity with anti-neuroectodermal-associated MAb. No apparent pattern of reactivity with anti-lymphoid MAb was seen; notably, there was a uniform absence of detectable Thy-1. Review of the complete antibody reactivity profile revealed a dichotomy between lines TE-671 and Daoy and lines D283 Med and D341 Med, which have been previously shown to express neurofilament protein in culture and xenografts, and to exhibit neuroblastic morphological features in biopsy and xenograft tissue sections. TE-671 and Daoy reacted with the MAb directed against tenascin, epidermal growth factor (EGF) receptor, HLA-A,B epitopes, beta 2-microglobulin and 5/8 of the glioma-associated antigens, but did not react with the anti-neurofilament protein (NFP) MAb. D283 Med and D341 Med expressed NFP but did not react with MAb against tenascin, EGF receptor, HLA-A,B epitopes, beta 2-microglobulin or 6/8 and 7/8 (respectively) of the glioma-associated antigens. The observed phenotypic differences provide a conceptual framework for investigating basic differences in the biological behavior of medulloblastoma. Moreover, the subdivisions can be evaluated for prospective value in tissue diagnosis, cerebrospinal fluid cytology and antibody-mediated imaging and therapy.

Intrasplenic immunization for production of monoclonal antibodies against mouse blastocysts.

Applying the intrasplenic immunization method monoclonal antibodies were raised against trophectoderm of mouse blastocysts. Adhesive C57BL/6 blastocysts, obtained 18 h after estrogen reactivation from an experimental delay of implantation, and irradiated with 5000 rad were used as immunogen. Male DBA/2 mice were immunized by four intrasplenic depositions of about ten blastocysts each. The sensitized spleen cells were fused with mouse plasmacytoma cells on the 5th day after the last booster, followed by isolation of hybridoma clones by conventional monoclonal antibody procedures. 82 hybridoma clones were obtained of which two produced IgM antibodies recognizing trophoblast determinants. Absorbing the monoclonal antibodies with C57BL/6 splenic leukocytes followed by immunolabelling of blastocysts demonstrated that the antibodies recognized neither MHC nor TLX antigens. Pre- and peri-implantation stages were mapped by indirect immunofluorescence microscopy. Morulae were negative while blastocysts were positively labeled. Adhesive blastocysts labeled more strongly than delayed blastocysts. Cultured blastocysts showed an intense labeling of some of the trophoblast cells, while other trophoblast cells were unlabeled.

Neuroectoderm-associated antigens on Ewing's sarcoma cell lines.

The histogenesis of Ewing's sarcoma, the second most frequent primary bone tumor in humans, remains controversial. Ten Ewing cell lines were analyzed by immunological methods. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. They included ganglioside GD2, a marker of neuroectodermal tissues and tumors, and an acidic glycolipid detected by monoclonal antibody HNK-1 in the nervous system. The P61 rat monoclonal antibody that reacts with a peptide moiety of neural cell adhesion molecule (N-CAM) and a rabbit antiserum raised to purified mouse N-CAM also stained Ewing cells. Flow cytometry analysis performed using these reagents allowed the definition of four distinct Ewing phenotypes: all reagents equally stained group 1 lines; group 2 lines were strongly reactive with anti-N-CAM reagents, by contrast with a fainter staining with HNK-1 and anti-GD2 antibodies; all reagents but P61 were strongly reactive with group 3 lines; in group 4, Ewing lines were stained by P61 but only poorly by the anti-N-CAM antiserum. Several antibodies to melanoma and neuroblastoma associated antigens including two monoclonal antibodies to the nerve growth factor receptor were also found to react with Ewing cells. By contrast, all antibodies detecting antigens specifically expressed in hematopoietic cell lineages were totally unreactive. HLA class II antigens were never detected while the level of expression of class I antigens varied to a large extent. Ewing cells are characterized by a specific t(11;22)(q23-24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor. Thus, Ewing's sarcoma cells share antigenic and karyotypic features with derivatives of the neuroectoderm possibly indicating a related histogenesis.