Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tigrina

 Freshwater planarians (Platyhelminthes, Turbellaria) show a great degree of morphological plasticity, making them a useful model for studying cell differentiation and pattern restoration processes during regeneration. Using confocal microscopy and a monoclonal antibody specific for muscle cells (TMUS-13), we have monitored the restoration of the body wall musculature during head regeneration in whole-mount organisms. Our results show that until the 4th day of regeneration the blastema is occupied by very disorganized muscle fibers, that from this moment become progressively organized restoring the original muscle pattern. In addition to recognizing mature muscle cells, TMUS-13 also recognizes differentiating myocytes, allowing us to trace the origin of newly formed muscle cells. We report that myocytes are detected in the postblastema region as early as day 1 of regeneration. This is the first demonstration that, in addition to serving as a proliferative zone as previously described, overt differentiation begins in the postblastema, at least for muscle cells. We also show that the TMUS13 antigen is the myosin heavy-chain gene from planarians. Received: 15 April 1997 / Accepted: 7 July 1997     

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

 To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians. Accepted: 16 September 1996     

Characterization of mouse macrophage differentiation antigens by monoclonal antibodies

Monoclonal rat antibodies to mouse macrophage antigens were prepared. For immunization phagocytic cells in the spleens of mice recovering from sublethal irradiation were used. Specificities of the monoclonal antibodies obtained were determined on cells of normal mouse cell populations as well as on cells of a panel of mouse cell lines. In an attempt to monitor expression of differentiation-related antigens two models of in vitro-induced macrophage differentiation were used: differentiation of cells of the myeloblast line Ml; CSF-1-induced differentiation of bone marrow cells. The results obtained clearly show that during maturation from undifferentiated to highly differentiated cells of the macrophage lineage expression of antigens recognized by the MIV 38, MIV 55, MV 87, and MV 114 monoclonal antibodies is enhanced. At the same time, expression of antigens recognized by the MIV 52, MIV 113, and MIV 116 monoclonal antibodies diminishes at a similar rate. The suitability of these monoclonal antibodies for the characterization of differentiation states of mouse macrophages is discussed.     

Regeneration and post-metamorphic development of the central nervous system in the protochordate Ciona intestinalis: a study with monoclonal antibodies

In this study, we use three monoclonal antibodies that recognise antigens present in the central nervous system of the ascidian Ciona intestinalis to study regeneration and post-metamorphic development of the neural ganglion. We have also used bromodeoxyuridine labelling to study generation of the neuronal precursor cells. The first antibody, CiN 1, recognises all neurones in the ganglion, whereas the second, CiN 2, recognises only a subpopulation of the large cortical neurones. Western blotting studies show that CiN 2 recognises two membrane-bound glycoproteins of apparent Mr 129 and 100 kDa. CiN 1 is not reactive on Western blots. Immunocytochemical studies with these antibodies show that CiN 1-immunoreactive neurone-like cells are present at the site of regeneration as early as 5–7 days post-ablation, a sub-population of CiN 2-immunoreactive cells being detected by 9–12 days post-ablation. The third antibody, ECM 1, stains extracellular matrix components and recognises two diffuse bands on Western blots of whole-body and ganglion homogenates. The temporal and spatial pattern of appearance of CiN 1 and CiN 2 immunoreactivity both during post-metamorphic development and in regeneration occurs in the same sequence in both processes. Studies with bromodeoxyuridine show labelled nuclei in some neurones in the regenerating ganglion. Plausibly these originate from the dorsal strand, an epithelial tube that reforms by cell proliferation during the initial phases of regeneration. A second population of cells, the large cortical neurones, do not incorporate bromodeoxyuridine and thus must have been born prior to the onset of regeneration. This latter finding indicates a mechanism involving trans-differentiation of other cell types or differentiation of long-lived totipotent stem cells.     

TCAV-1, a monoclonal antibody specific to epithelial pharyngeal cells in the planarian Dugesia (Girardia) tigrina. Application to pattern formation of the pharynx during regeneration

During regeneration in planarians, anterior (head and prepharyngeal) and posterior (postpharyngeal and tail) fragments rebuild one of the most peculiar structures of planarians: the pharynx and the pharynx cavity. Previous studies (see Brønsted, 1969, for a general review, and Asai, 1990, 1991, for anterior regeneration) have shown that within postpharyngeal pieces both structures appear in the old stump from clusters of undifferentiated cells. However, the lineage and differentiation of their elements (inner and outer epithelial cells, muscle layers, gland cells, nerve rings) and the overall pattern of growth and differentiation is not clear.     

Unilateral T cell maturation arrest in the thymus of CBA/H mice as a long-term effect after neutron irradiation

Thymuses of CBA/H mice were investigated up to 570 days after whole-body irradiation with 2.5 Gy fast fission neutrons or 6.0 Gy X rays. A number of these thymuses, observed 220-270 days after neutron irradiation, have two equal sized lobes, one of which has an abnormal T cell distribution. The present paper reports on the distribution of lymphoid and stromal cell types in these thymuses. For this purpose, we employed immunohistology using the indirect immunoperoxidase method. We incubated frozen sections of these aberrant thymuses with monoclonal antibodies directed to cell surface differentiation antigens on lymphoid cells, such as Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14; monoclonal antibodies directed to major histocompatibility complex (MHC) antigens, such as I-A and H-2K; and monoclonal antibodies directed to determinants in various thymic stromal cell types. The results of this study show a T cell differentiation arrest in only one of the two thymic lobes. T cells in the aberrant lobe express Thy-1, T-200, and MEL-14 antigens but are MT-4- and Lyt-1-. In some lobes, a weak Lyt-2 expression was observed. The observed T cell maturation arrest is mainly restricted to the cortex since in the medulla, in addition to cells with an aberrant cortical phenotype, normal T cell phenotypes are observed. This indicates that cortex and medulla have independent generation kinetics in T cell maturation. The stromal cell composition in these abnormal lobes is not different from that in the normal lobe, but the size of the medulla tends to be smaller. Furthermore, the I-A expression on the cortical epithelial cells does not reveal the characteristic reticular staining pattern that is observed in the normal lobe, since the I-A determinants are not strictly confined to the epithelial cells. In addition, cortical lymphoid and stromal cells in these lobes are slightly H-2K+. These alterations in MHC expression in the cortex are discussed in relation to the observed T cell maturation arrest.     

Production and characterization of monoclonal antibodies recognizing head activator in precursor form and immunocytochemical localization of head activator precursor and head activator peptide in the neural cell line NH15-CA2 and in hydra

A synthetic gene for the hydra neuropeptide head activator (HA) was used to produce large amounts of an HA bacterial fusion protein. From this protein an HA-containing fragment was cleaved out, attached in high copy number to carrier proteins, and used as an immunogen to produce monoclonal antibodies able to recognize head activator in precursor form. Using such antibodies and others with different specificities for HA epitopes in combination with different fixation procedures, we detected HA immunoreactivity in three locations in the HA-rich neural cell line NH15-CA2. A precursor-like HA immunoreactivity was present in the cytoplasm of cells and detected, independent of fixation procedure, by monoclonal antibodies characterized as HA-precursor-specific. With antibodies specific for the HA peptide, two immunoreactivities could be distinguished, one within cells and one at the outer cell membrane. HA was detected within differentiated cells with long processes when crosslinkers such as carbodiimide or glutaraldehyde were applied together with agents like methanol. HA peptide bound to target cells was restricted to small round cells with an undifferentiated morphology, especially to those in the process of cell division. In hydra HA precursor immunoreactivity was localized in interstitial cells and in developing nerve cells. HA peptide immunoreactivity was present in nerve cells, but was more concentrated on and in target cells such as interstitial cells and epithelial cells. In tissue sections immunoreactive cells were especially abundant in regions of high HA content such as hypostome, subhypostomal region, and the future head region of developing buds.     

The human thymic microenvironment: Thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical identification of proliferating cell types in mouse mammary gland

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.     

Planarian pharynx regeneration revealed by the expression of myosin heavy chain-A

The pharynx is a distinctive organ in the center of the body of planarians. Although the process of pharynx regeneration has been studied previously, the details and mechanism of the process remain controversial. We examined the process of regeneration of the pharynx in the planarian Dugesia japonica in detail by in situ hybridization and immunohistochemistry for myosin heavy chain-A (DjMHC-A), which is mainly expressed in the pharynx muscles and pharynx-anchoring muscles. We also monitored the behavior of the neoblasts in this process. In the regenerating posterior body fragment, the pharyngeal rudiment was formed by accumulation of cells that were probably undifferentiated cells derived from the neoblasts. The pharynx muscles appeared to differentiate in the rudiment in a manner that was coordinated with the differentiation of the pharynx-anchoring muscles in the region surrounding the rudiment. During this process, all cells containing mRNA for DjMHC-A also contained the DjMHC-A protein. These results argue against a previously proposed hypothesis that in the mesenchyme, 'pharynx-forming cells', which are committed to differentiate into the pharyngeal cells but have not yet differentiated, gather in the rudiment to form the pharynx (Agata and Watanabe, 1999). Rather, the present observations suggest that regeneration of the planarian pharynx proceeds by accumulation of cells that are probably undifferentiated cells derived from neoblasts in the rudiment, followed by their differentiation into the pharyngeal cells there.     

MUC1 (EMA) expressing plasma cells in bone marrow infiltrated by plasma cell myeloma

MUC1 (also called: epithelial membrane antigen, EMA) represents a mucin molecule strongly expressed in various epithelia and epithelial neoplasms. Its expression correlates with clinical and pathological factors as well as prognosis in some tumor types. Additionally, MUC1 was detected in normal haematopoietic cell lines and neoplasms, especially subgroups of human lymphomas including plasma cell myeloma. Therefore, the expression of MUC1 in trephine biopsies exhibiting infiltrates of plasma cell myeloma were investigated immunohistochemically. An immunoreactivity of two monoclonal antibodies (EMA and HMFG-2) was observed in about 50% of the cases. In cases exhibiting a so-called packed marrow, EMA immunoreactivity was reduced. However, MUC1 positivity did not correlate with the cytologic grade of differentiation, the fibre content of the marrow, or survival probability of the patients. However, its strong expression in a certain percentage of cases of plasma cell myeloma may be of therapeutic impact, since new therapeutic strategies include the enrichment of MUC1-specific T cells or MUC1 vaccination.     

Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.     

Intercalary muscle cell renewal in planarian pharynx

 Planarian cell renewal is achieved as a result of proliferation and differentiation of totipotent undifferentiated cells called neoblasts. The absence of mitosis within the planarian pharynx raises the question as to how cell renewal and growth occur within this organ. Two explanations have been advanced: one proposes that new cells remain close to the base of the pharynx, which then grows by distal displacement of older cells, and the other suggests that the new cells are intercalated between older cells throughout the pharynx. The second alternative, however, does not explain how new cells enter the pharynx or how they reach their final destination. In this study of myosin heavy-chain gene expression within planarian pharynx, a row of differentiating myocytes was detected all along the pharynx parenchyma. According to the hybridization pattern, all these myocytes appeared to be at early stages of differentiation. These data favour an intercalary model for muscle cell renewal within the pharynx. According to this model, neoblasts at the base of the pharynx would enter the pharynx, where they would start differentiation to myocytes, move to the subepithelial musculature and intercalate between the old muscle cells. The possible application of this intercalary model to other pharynx cell types is also discussed. Received: 30 July 1998 / Accepted: 20 November 1998     

Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.     

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.     

Influence of Enamel Matrix Derivative on Cells at Different Maturation Stages of Differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2–5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.     

Structure,histochemistry, ontogenetic development,and regeneration of the ocellus of Cladonema radiatum dujardin (cnidaria,hydrozoa, anthomedusae)

The ectodermal eyes, 45–55 μm in diameter, of the cnidarian hydrozoan Cladonema radiatum Dujardin possess a lens approximately 15 μm in diameter enveloped by an eyecup (retina). An overlying layer of intensely vacuolated distal process of the adjoining epithelial cells forms a transparent cornea. The eyecup is composed of three cell types: basal cells, melanin-containing pigment cells, and photoreceptor cells. The last two cell types occur in the ratio of approximately 2:1. Histogenesis of the eye both during ontogeny and regeneration is described from light and electron microscopic investigations. During ontogeny the cell types forming the retina are derived from a compact group of morphologically undifferentiated cells, but during regeneration a primordium is formed by regeneration cells. In both cases the lens is built from distal nonnucleated cytoplasmic portions pinched off from the pigment cells. The cornea is formed by distal lamellar processes of the ocellus adjoining the epithelial cells. Through EM-histochemical methods (silver impregnation and DOPA-oxidase reaction) the pigment of the chromatophores of the retina was identified as melanin.     

Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas

During pancreatic development, endocrine and exocrine cell types arise from common precursors in foregut endoderm. However, little information is available regarding regulation of pancreatic epithelial differentiation in specific precursor populations. We show that undifferentiated epithelial precursors in E10.5 mouse pancreas express nestin, an intermediate filament also expressed in neural stem cells. Within developing pancreatic epithelium, nestin is co-expressed with pdx1 and p48, but not ngn3. Epithelial nestin expression is extinguished upon differentiation of endocrine and exocrine cell types, and no nestin-positive epithelial cells are observed by E15.5. In E10.5 dorsal bud explants, activation of EGF signaling results in maintenance of undifferentiated nestin-positive precursors at the expense of differentiated acinar cells, suggesting a precursor/progeny relationship between these cell types. This relationship was confirmed by rigorous lineage tracing studies using nestin regulatory elements to drive Cre-mediated labeling of nestin-positive precursor cells and their progeny. These experiments demonstrate that a nestin promoter/enhancer element containing the second intron of the mouse nestin locus is active in undifferentiated E10.5 pancreatic epithelial cells, and that these nestin-positive precursors contribute to the generation of differentiated acinar cells. As in neural tissue, nestin-positive cells act as epithelial progenitors during pancreatic development, and may be regulated by EGF receptor activity.