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PubMed1985

Deficiency in the regulation of testicular galactolipid sulphotransferase in rats carrying the growth-and-reproduction-complex (grc) gene.

The regulation of the activity of testicular germ-cell galactolipid sulphotransferase was investigated in rats homozygous for the grc (growth and reproductive complex) gene. In the adult grc homozygotes, the activity was elevated relative to that in the wild-type animals, and a concomitant deficiency of a developmentally regulated sulphotransferase inhibitor was found. Spermatogenesis in the grc homozygotes is blocked at a stage that correlates temporally with the earliest detection of this inhibitor in the wild-type animal. In addition, there was a similar increase in the specific activity of the kidney galactolipid sulphotransferase in the grc homozygotes. This biochemical abnormality is the first to be associated with a genetically regulated, developmental defect linked to the major histocompatibility complex, and it is related to the pathogenesis of one of the major lesions controlled by the grc.

Animals↗
PubMed1998

A visuo-somatomotor pathway through superior parietal cortex in the macaque monkey: cortical connections of areas V6 and V6A.

This report addresses the connectivity of the cortex occupying middle to dorsal levels of the anterior bank of the parieto-occipital sulcus in the macaque monkey. We have previously referred to this territory, whose perimeter is roughly circumscribed by the distribution of interhemispheric callosal fibres, as area V6, or the 'V6 complex'. Following injections of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA-HRP) into this region, we examined the laminar organization of labelled cells and axonal terminals to attain indications of relative hierarchical status among the network of connected areas. A notable transition in the laminar patterns of the local, intrinsic connections prompted a sub-designation of the V6 complex itself into two separate areas, V6 and V6A, with area V6A lying dorsal, or dorsomedial to V6 proper. V6 receives ascending input from V2 and V3, ranks equal to V3A and V5, and provides an ascending input to V6A at the level above. V6A is not connected to area V2 and in general is less heavily linked to the earliest visual areas; in other respects, the two parts of the V6 complex share similar spheres of connectivity. These include regions of peripheral representation in prestriate areas V3, V3A and V5, parietal visual areas V5A/MST and 7a, other regions of visuo-somatosensory association cortex within the intraparietal sulcus and on the medial surface of the hemisphere, and the premotor cortex. Subcortical connections include the medial and lateral pulvinar, caudate nucleus, claustrum, middle and deep layers of the superior colliculus and pontine nuclei. From this pattern of connections, it is clear that the V6 complex is heavily engaged in sensory-motor integration. The specific somatotopic locations within sensorimotor cortex that receive this input suggest a role in controlling the trunk and limbs, and outward reaching arm movements. There is a secondary contribution to the brain's complex oculomotor circuitry. That the medial region of the cortex is devoted to tightly interconnected representations of the sensory periphery, both visual and somatotopic-which are routinely stimulated in concert-would appear to be an aspect of the global organization of the cortex which must facilitate multimodal integration.

Animals↗
PubMed1984

The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat.

Direct projections from the forebrain to the nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus in the rat medulla were mapped in detail using both retrograde axonal transport of the fluorescent tracer True Blue and anterograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). In the retrograde tracing studies, cell groups in the medial prefrontal cortex, lateral prefrontal cortex (primarily ventral and posterior agranular insular cortex), bed nucleus of the stria terminalis, central nucleus of the amygdala, paraventricular, arcuate, and posterolateral areas of the hypothalamus were shown to project to the NTS and in some cases also to the dorsal motor nucleus of the vagus. The prefrontal cortical areas projecting to the NTS apparently overlap to a large degree with those cortical areas receiving mediodorsal thalamic and dopaminergic input. The retrogradely labeled cortical cells were situated in deep layers of the rat prefrontal cortex. The anterograde tracing studies revealed a prominent topography in the mediolateral termination pattern of forebrain projections to the rostral part of the NTS and to the dorsal pons. The projections to the NTS were generally bilateral, except for projections from the central nucleus of the amygdala and bed nucleus of the stria terminalis which were predominantly ipsilateral. The prefrontal cortical projections to the NTS travel through the cerebral peduncle and pyramidal tract and terminate throughout the rostrocaudal extent of the NTS. Specifically, the prefrontal cortex innervates dorsal portions of the NTS (lateral part of the dorsal division of the medial solitary nucleus, dorsal part of the lateral solitary nucleus and the caudal midline region of the commissural nucleus), areas which receive relatively sparse subcortical projections. These dorsal portions of the NTS receive major primary afferent projections from the vagal and glossopharyngeal nerves. In contrast, the subcortical projections, which travel through the midbrain and pontine tegmentum, terminate most heavily in the ventral portions of the NTS, i.e., the area immediately dorsal and lateral to the dorsal motor nucleus of the vagus. Only the paraventricular hypothalamic nucleus has substantial terminals throughout the dorsal motor nucleus of the vagus. Hypothalamic cell groups innervate the area postrema and, along with the prefrontal cortex, innervate the zone subjacent to the area postrema.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗
PubMed2005

Premeiotic origin of teratomas: is meiosis required for differentiation into mature tissues?

By virtue of meiotic cell division, primordial germ cells with heterozygous alleles develop into postmeiotic germ cells with homozygous alleles. Female and male germ cells may develop tumors--so-called teratomas--with a unique coexistence of a variety of histological elements from all three embryonic germ layers. In particular, mature teratomas consist exclusively of developmentally mature tissues whereas immature teratomas contain variable amounts of mature and immature tissues. In this study, we report genetic analysis of individual tissue components from mature and immature teratomas. The majority of mature teratomas showed consistent and concordant homozygous alleles in all selectively procured tissue components. In a small subset of mature teratomas, we observed discordant homozygous alleles. In contrast, immature teratomatous tissue revealed a heterozygous genotype. Remarkably, mature tissue components within immature teratoma revealed homozygosity. The findings suggest that immature teratomas and at least a subset of mature teratomas may originate from premeiotic cells, and implicate that meiosis may be required for differentiation into mature tissues.

Alleles↗
PubMed1985

Clonal analysis of early mammalian development.

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.

Amnion↗
PubMed1999

Ontogeny and regional variability of keratin 2e (K2e) in developing human fetal skin: a unique spatial and temporal pattern of keratin expression in development.

Keratin 2e (K2e) is expressed in the upper spinous and granular cells of adult epidermis. A highly specific polyclonal antibody was made against a C-terminal peptide of K2e and used to observe K2e expression at different developmental stages. At 12.5 weeks estimated gestational age (EGA) K2e was detected in trunk skin in scattered cells in the intermediate layer. At 13.5 weeks EGA, greater numbers of intermediate cells were stained with variable intensity, and staining in this pattern increased with age. Epidermal sheets from 14 weeks EGA showed that K2e + cells were excluded from developing hair follicles. At 135 days EGA, the following regional patterns were observed: in cheek, trunk, dorsal and ventral knee, elbow and dorsal hand there was moderate to intense staining of upper intermediate keratinocytes excluding cells of the hair canals and sweat ducts. The periumbilical region distinctly lacked K2e staining, while more distal areas showed increasing numbers of K2e + cells. The earliest expression of K2e was at 10 weeks EGA in the presumptive nail bed of developing digits. By 13.5 weeks EGA this pattern had shifted to the proximal nail fold, and K2e was absent in the nail bed. K2e was excluded from developing sweat glands and ducts and from developing hair follicles at the hair germ and early peg stages. By 15 weeks EGA in the fetal hair follicle small groups of cells were K2e + and by 19 weeks K2e + cells were seen at the level of the matrix. Some overlap in staining was detected for K2e with K10, and in palmar skin with K9; however, mostly the filamentous staining patterns for these keratins were distinctive. This study shows that the complex patterns of temporal and regional expression of K2e differ from known patterns for other epidermal keratins and suggest different regulation and function for this epidermal keratin.

Embryonic and Fetal Development↗
PubMed2004

Integrin-dependent functions of the angiogenic inducer NOV (CCN3): implication in wound healing.

The novel angiogenic inducer CCN3 (NOV, nephroblastoma overexpressed) is a matricellular protein of the CCN family, which also includes CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN3 is broadly expressed in derivatives of all three germ layers during mammalian development, and its deranged expression is associated with vascular injury and a broad range of tumors. We have shown that CCN3 promotes proangiogenic activities in vascular endothelial cells through integrin receptors and induces neovascularization in vivo (Lin, C. G., Leu, S. J., Chen, N., Tebeau, C. M., Lin, S. X., Yeung, C. Y., and Lau, L. F. (2003) J. Biol. Chem. 278, 24200-24208). In this study, we show that CCN3 is highly expressed in granulation tissue of cutaneous wounds 5-7 days after injury and is capable of inducing responses in primary fibroblasts consistent with wound healing. Purified CCN3 supports primary skin fibroblast adhesion through integrins alpha(5)beta(1) and alpha(6)beta(1) and induces fibroblast chemotaxis through integrin alpha(v)beta(5). We show that CCN3 is a novel ligand of alpha(v)beta(5) in a solid phase binding assay. Although not mitogenic on its own, CCN3 also enhances basic fibroblast growth factor-induced DNA synthesis. Furthermore, CCN3 up-regulates MMP-1 and PAI-1 expression but interacts with TGF-beta1 in an antagonistic or synergistic manner to regulate the expression of specific genes. These findings, together with its angiogenic activity, support a role for CCN3 in cutaneous wound healing in skin fibroblasts and establish its matricellular mode of action through integrin receptors.

Cell Adhesion↗
PubMed1992

Substance P-containing pyramidal neurons in the cat somatic sensory cortex.

Light and electron microscopic immunocytochemical methods were used to verify the possibility that neocortical pyramidal neurons in the first somatic sensory cortex of cats contain substance P. At the light microscopic level, substance P-positive neurons accounted for about 3% of all cortical neurons, and the vast majority were nonpyramidal cells. However, 10% of substance P-positive neurons had a large conical cell body, a prominent apical dendrite directed toward the pia, and basal dendrites, thus suggesting they are pyramidal neurons. These neurons were in layers III and V. At the electron microscopic level, the majority of immunoreactive axon terminals formed symmetric synapses, but some substance P-positive axon terminals made asymmetric synapses. Labelled dendritic spines were also present. Combined retrograde transport-immunocytochemical experiments were also carried out to study whether substance P-positive neurons are projection neurons. Colloidal gold-labelled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase was injected either in the first somatic sensory cortex or in the dorsal column nuclei. In the somatic sensory cortex contralateral to the injection sites, a few substance P-positive neurons in layers III and V also contained black granules, indicative of retrograde transport. This indicates that some substance P-positive neurons project to cortical and subcortical targets. We have therefore identified a subpopulation of substance P-positive neurons that have most of the features of pyramidal neurons, are the probable source of immunoreactive axon terminals forming asymmetric synapses on dendritic spines, and project to the contralateral somatic sensory cortex and dorsal column nuclei. These characteristics fulfill the criteria required for classifying a cortical neuron as pyramidal.

Amino Acid Sequence↗
PubMed1995

Mouse embryonic hematopoiesis.

The hematopoietic system of vertebrates is derived from the mesodermal germ layer in early embryogenesis. Various animal models have been used for the study of hematopoiesis, from early stages in the visceral yolk sac or its analog, to the later stages where hematopoiesis is observed in intraembryonic areas surrounding the aorta, genital ridge and pro/mesonephros. Using the mouse as a model, we describe what is known about mammalian embryonic hematopoiesis and put it in the context of hematopoietic cell formation in avian, amphibian and fish embryos. Evolutionary comparisons and recent experimental evidence show that there are two embryonic sites of developing hematopoietic activity in the mouse before fetal liver hematopoiesis and suggest that, during ontogeny, two successive waves of hematopoietic activity may contribute to the blood system of the adult.

Animals↗
PubMed1993

Characterization of Candida albicans cell wall antigens with monoclonal antibodies.

The antigenic composition of Candida albicans is very complex. In order to study the antigenic relationship between blastoconidia and germ tubes of C. albicans, we produced several monoclonal antibodies and analyzed their reactivity against cell wall antigens either in intact cells or in cells treated with dithiothreitol. Overall, four types of reactivity were found. Monoclonal antibodies 3D9 and 15C9 stained the germ tubes only when tested by indirect immunofluorescence. However, they showed a different reactivity by immunoblotting. Monoclonal antibody 3D9 reacted with antigens with molecular masses of > 200 and 180 kDa specifically expressed in the germ tube. Monoclonal antibody 15C9 reacted with antigens of 87, 50, and 34 kDa present in the germ tube extract and with antigens of 92, 50, 34, and 32 kDa present in the blastoconidium extract. The reactivity of blastoconidia treated for different times with dithiothreitol with these monoclonal antibodies was also studied by enzyme-linked immunosorbent assay. The reactivity of monoclonal antibody 3D9 did not significantly change during the cell wall extraction. However, the reactivity of monoclonal antibody 15C9 was increased for blastoconidia extracted for 60 min and decreased markedly for blastocondia extracted for 120 min. Monoclonal antibody G3B was nonreactive by indirect immunofluoresence but reacted with antigens of 47 and 38 kDa present in the germ tube extract and with an antigen of 47 kDa present in the blastoconidium extract. Monoclonal antibody B9E stained both morphological phases by indirect immunofluorescence. By immunoblotting, it reacted with antigens of > 70 kDa present in the germ tube extract and with antigens of > 63, 56, 47, and 38 kDa present in the blastoconidium extract. Based on the results presented in this study, four types of antigens are described. Type I antigens are expressed on the outermost layers of the germ tube cell wall only. Type II antigens are expressed both on the germ tube cell wall surface and within the blastoconidium cell wall. Type III antigens are found within the cell wall of both blastoconidia and germ tubes. Type IV antigens are expressed on both the blastoconidium and germ tube surface. Two types more can be hypothesized for antigens expressed on the blastoconidium cell surface and within the germ tube cell wall (type V) and for those expressed on the blastoconidium surface only (type VI).

Animals↗
PubMedDate not supplied

In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body.

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.

Animals↗
PubMed1993

Visceral yolk sac-derived tumors.

Externalization of the visceral yolk sac, after fetectomy, induces the development of extra-embryonal fetal tumors in rodents. These tumors are either benign teratomas that appear 3 to 4 weeks after the displacement of the yolk sac or malignant tumors, i.e. yolk sac carcinomas. The latter appear 4 to 8 months after the surgery. If however, Mouse Sarcoma Virus (MSV) is injected in the placentas at the time of fetectomy (day 12 of pregnancy) the malignant tumors develop much earlier (2 to 3 months after surgery) and some display characteristics of embryonal carcinoma. Whether virus induced or not, the yolk sac carcinomas that develop from the displaced visceral yolk sac possess the same morphological and biological characteristics. They are composed of both parietal and visceral yolk sac structures and sometimes trophoblast. The tumors metastasize, grow in ascites form and kill their host. They are readily transplantable in syngeneic rats and grow in tissue culture as an epithelial-like sheet of cells. On the other hand, the benign teratomas are composed of various well differentiated adult tissues. In these tissues, derivatives of all three germ layers are observed. Numerous experiments prove that the stem cells for these various adult tissues are not germ cells. Instead the stem cells are multipotential cells that arise in the displaced yolk sac by a process of dedifferentiation. These poorly differentiated cells originate from the endoderm of the displaced visceral yolk sac. By redifferentiation they give rise to the various adult tissues characteristic for benign teratomas. The multipotential poorly differentiated cells are also likely to be the target cells for malignant transformation. Malignant transformation of these cells, whether induced by a virus or spontaneously occurring in the displaced yolk sac, leads not only to the development of yolk sac carcinomas and eventually embryonal carcinoma but also, although rarely, to choriocarcinoma. The latter tumor is transplantable in allogeneic hosts. It is hormonally active since it secretes lactogen and progesterone. The extra-embryonal fetal tumors and in particular the rat yolk sac carcinomas and choriocarcinoma proved to be a good source for the detection of oncofetal antigens. At least two different oncofetal endodermal antigens were detected with monoclonal antibodies (mab) made after immunization with yolk sac carcinoma. Another mab, made against choriocarcinoma, was found to react specifically with the cytotrophoblast both in the normal placenta and in the tumor. No other placental cells showed a positive reaction.

Animals↗
PubMed1988

Intracortical connections and their physiological correlates in the primary auditory cortex (AI) of the cat.

We studied the functional and anatomical properties of the intrinsic connections in the primary auditory cortex (AI) of the cat by using physiological mapping and retrograde tracing methods. Our results revealed that a focal microinjection of tracer labeled as many as five intracortical patches in AI. The patches contained labeled pyramidal and non-pyramidal cell types, most of which were clustered in the middle layers. A densely distributed anterograde-like reaction product was present in the superficial layers above the labeled cells. The distribution of the patches was anisotropic, with most patches occurring dorsal, ventral, and anterior to the injection site. We examined the correlation between the characteristic frequency (CF) and binaural response properties of the injected and labeled regions. We found local labeling in regions possessing CFs equivalent to or slightly greater than that of the injected area. This appears to be a specific connection since we were able to predict the general location of many of the patches on the basis of the organization of the isofrequency domains. Patches were more numerous dorsoposterior to the injection site when the isofrequency contours ran obliquely (i.e., dorsoposterior to ventroanterior) across AI. The binaural response properties of the injected and labelled regions, however, were unrelated.

Animals↗
PubMed1978

Canine distemper virus-associated encephalitis: modification by passive antibody administration.

Gnotobiotic puppies were intraperitoneally infected with R252-canine distemper virus (CDV), and treated with normal serum (NS) or CDV-specific antibody. Resultant central nervous system (CNS) lesions were evaluated by light and immunofluorescence microscopy. Lesions in NS-treated dogs, if present, consisted of small foci of neuronal degeneration and accompanying microgliosis, restricted to grey matter of the frontal lobe. Dogs receiving anti-CDV antibody, in contrast, had extensive lesions in the brain. Laminar cortical necrosis, gliosis and astrocytic activation of the grey matter of the frontal lobe was the most consistent lesion observed. In addition, foci of cerebellar granule cell layer degeneration and necrosis was seen along with segmental poliomalacia of the spinal cord. In several dogs, eosinophilic intracytoplasmic and intranuclear viral inclusion bodies were observe in neurons. The lesions observed in these dogs are compared to previously reported lesions of CDV infection in dogs.

Animals↗
PubMed1989

Retrovirus-induced insertional mutation in Mov13 mice affects collagen I expression in a tissue-specific manner.

In the Mov13 mouse mutant, transcription of the alpha 1 (1) collagen gene is blocked by a retroviral insert in the first intron. We now report that teeth derived from homozygous embryos produce a dentin layer containing normal amounts of collagen 1. In situ hybridization and RNAase protection experiments indicate that the mutant allele is efficiently transcribed in odontoblasts, in contrast to other cell types. Correct splicing of the primary transcript containing the viral sequence results in a functional alpha 1 (1) collagen mRNA. The absence of a mutagenic effect in odontoblasts, as opposed to fibroblasts, suggests that the retroviral insert interferes with tissue-specific transcriptional control of the alpha 1 (1) collagen gene, most likely by inactivating cell-type-specific cis-acting regulatory elements.

Animals↗
PubMedDate not supplied

Rewiring of transcortical projections to middle suprasylvian cortex following early removal of cat areas 17 and 18.

Retrograde tracers were injected into middle suprasylvian (MS) cortex of two groups of experimental adult cats that had incurred removal of visual areas 17 and 18 on either the day of birth (P1), or at 1 month of age (P28). Tracers were also injected into the same region of intact and adult ablated control cats. The locations and numbers of labeled neurons in the experimental and control groups were compared. Following lesions on P1, but at no other age, increased numbers of neurons projected to MS cortex. Virtually all of the additional neurons were located in the superficial layers of the ventral posterior suprasylvian (vPS) cortex. These results demonstrated that (1) neurons with ipsilateral transcortical axons have the potential to reconfigure their projections after early, localized cortical damage elsewhere in the cortex of the same hemisphere; (2) this reconfiguration involves expansion of specific projections and is not a generalized capacity of all cortical neurons; (3) the expansion is modality specific; and finally, (4) the ability of cortical neurons to reorganize projections is limited in time. The expanded projection from vPS to MS cortex may contribute to neuronal compensations and the sparing of visually guided behaviors previously demonstrated in cats with neonatal visual cortex damage, and is a testament to the latent capacities immature cerebral cortex neurons possess to establish new projections following restricted damage to the cerebral cortex early in life.

Animals↗
PubMed1994

Expression of transforming growth factor-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo.

Specific cDNA probes and antibodies for chicken transforming growth factor (TGF)-beta 2 and beta 3 were used to study expression of TGF-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo. Expression of the mRNAs for both TGF-beta isoforms was detected by day 1.5 of incubation (Hamburger and Hamilton stage 10) by RNA Northern blot analysis and increased with developmental age. Expression of TGF-beta 2 and beta 3 mRNAs was detected in every embryonic tissue examined, with the level of expression of both isoforms being high in heart, brain and muscle and low in kidney and liver. Coordinate unidirectional upregulation of expression of TGF-beta 2 and beta 3 mRNAs occurred in most embryonic tissues with development except the heart, where the steady-state level of expression of TGF-beta 2 mRNA decreased with age, while that of TGF-beta 3 mRNA increased. In situ hybridization analysis detected TGF-beta 2 and beta 3 mRNAs as early as the definitive primitive streak stage (stage 4). During neurulation (stage 10), TGF-beta 2 and beta 3 mRNAs were detected in cells of all three germ layers; TGF-beta 3 mRNA was detected in neurectoderm as well. Following neurulation, TGF-beta 3 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome and dermomyotome at stage 16; expression of TGF-beta 2 mRNA was not as prominent as TGF-beta 3 mRNA in these structures. By stage 29, both TGF-beta 2 and beta 3 mRNAs were localized in several tissues including heart, lung, gizzard and feathers. Immunohistochemical staining analysis detected immunoreactive TGF-beta 2 and beta 3 proteins in all three germ layers of stage 4 embryos. Staining for TGF-beta 2 and beta 3 proteins was detected in several cell types and tissues in the early developing embryo frequently in the same locations as TGF-beta 2 and beta 3 mRNAs, with staining for TGF-beta 2 being less intense than TGF-beta 3. However, in some cases, localization of TGF-beta 2 and beta 3 proteins was different from that of the mRNAs, indicating that a complex mechanism of transcription, translation and secretion exits during chicken embryogenesis. These and other results suggest that TGF-beta 2 and beta 3 may play important roles and act through both autocrine and paracrine mechanisms in the development of many tissues in the chicken.

Animals↗
PubMed1993

Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumor-derived cell line, NCCIT.

BACKGROUND: Germ cell tumors are empirically divided into seminomas and nonseminomatous germ cell tumors (NSGCT). Some authorities consider seminomas to be the precursors of NSGCT, whereas others consider them as distinct and unrelated neoplasms. Here, we report that the human NSGCT-derived stem cell line, NCCIT has hybrid features of seminoma and embryonal carcinoma, and suggest that this cell line could be useful for studying the relationship of seminoma to NSGCT. EXPERIMENTAL DESIGN: NCCIT, a developmentally pluripotent permanent cell line derived from a mediastinal NSGCT was karyotyped and characterized morphologically, immunochemically, and biochemically. The cells were grown under standard tissue culture conditions and were also exposed to retinoic acid to induce differentiation. RESULTS: The dividing NCCIT stem cell populations consist of vimentin-positive, keratin-negative cells that do not express desmoplakin or cadherin E (uvomorulin) and are not interconnected with one another. These cells have a high nucleocytoplasmic ratio and contain few cytoplasmic organelles, except for free ribosomes and a small number of mitochondria. Lacto- and globoseries oligosaccharide antigens recognized with antibodies to murine stage specific antigens 1, 3 and 4 (SSEA-1, SSEA-3 and SSEA-4), and human teratocarcinoma mucin-like antigen TRA-1-60 and TRA-1-81 are coexpressed on the cell membranes of a considerable number of stem cells. On most cells alkaline phosphatase can be detected by enzyme histochemistry. The placental isoenzyme of alkaline phosphatase was demonstrated by Western blotting in cell extracts. The liver/bone/kidney isoenzyme of alkaline phosphatase is immunochemically detected on 40% of cells. The culture supernatants also contain chorionic gonadotropin and alpha-fetoprotein, presumably derived from trophoblastic and yolk sac-like cells. The cells are hyperdiploid (chromosome range from 54 to 64) and show prominent structural chromosomal aberrations, mostly deletions and isochromosomes. Retinoic acid treatment inhibited the growth of NCCIT cells and induced stem cell differentiation into keratin, glial fibrillary acid protein, and neurofilament-positive somatic cells. The differentiation was associated with the disappearance of oligosaccharide surface antigens typical of the undifferentiated stem cells; a loss of proteins typical of undifferentiated cells and the appearance of new proteins; and the deposition of extracellular matrix. CONCLUSIONS: NCCIT is a developmentally pluripotent cell line that can differentiate into derivatives of all three embryonic germ layers (i.e., ectoderm, mesoderm, and endoderm) and extraembryonic cell lineages. We suggest that this cell line could be a malignant replica of human cleavage stage embryonic cells with features intermediate between seminoma and embryonal carcinoma.

Adult↗