bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of osteoblasts: In situ versus isolated specimens
Authors: Ries William L. , Gong Joseph K. , Sholley Milton M.
Published: February 2005

Osteoblast and osteoclasts are known to adhere tenaciously to bone surfaces even when the fibrous periosteal covering is removed. Brushsmear isolates and in situ, whole bone mounts of the osteogenic periosteum on the inner and outer calvarial tables of young adult rats were examined at the light microscopic level after exposing the cells by removing the fibrous periosteum. Based on staining, enzymatic activity, and overall morphology, most of the cells could be classified as osteogenic in nature. However, detailed analysis revealed that some cells exhibited certain unique morphologic appearances that suggested the existence of subpopulations or variant forms of the conventional or prototypic osteoblast. Three variant forms were the large nucleate, small nucleate, and multinucleate osteoblast‐like cells, the latter with nuclear numbers ranging from 2–10. The finding of all such forms in smears, short‐term cell cultures, and in situ specimens greatly lessened the probability that the unusual forms were artifactual. Phase contrast microscopic studies and sectioning of cellular isolates for study at both the light and electron microscopic levels provided additional support for the existance of the multinucleate osteoblast‐like cell. The small size of these osteoblast‐like subpopulations and the orientation of the cells to the commonly used plane of section of most histologic bone specimens, particularly with the multinucleate type, could explain why they had escaped earlier detection.
Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse
Authors: Seifert Mark F. , Marks Sandy C.
Published: February 2005

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse.

Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one‐quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three‐quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants.

The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are charcteristics of the osteosclerotic mutation which suggest that it is a unqiue csteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.
Endothelial mitosis during the initial stages of corpus luteum neovascularization in the cycling adult rat
Authors: Gaede S. D. , Sholley M. M. , Quattropani S. L.
Published: February 2005

The initial stages of neovascularization of the corpus luteum were studied in cycling adult rats using light‐microscopic autoradiography. The aim of this analysis was to determine whether endothelial mitosis is a factor in this vascular growth and whether there are differences in the amount of mitotic activity in various regions of the ovary. Ovaries were examined at two time intervals: 1–2 hr and 7–8 hr following ovulation. Animals received an intraperitoneal injection of tritiated‐thymidine 20 min prior to perfusion fixation of the ovaries. Autoradiographic demonstration of tritiated‐thymidine labeling in endothelial nuclei was considered an indication of DNA synthesis preceding mitosis. The percentage of labeled endothelial cells in the ovaries at both time intervals varied according to the region of tissue examined and the stage of differentiation of that region. Stromal vessels were less heavily labeled than thecal vessels. Thecal vessels surrounding growing follicles were more heavily labeled than those surrounding atretic follicles. The heaviest labeling was seen in the developing corpora lutea 7–8 hr following ovulation. Minimal labeling was evident in the corpora lutea which were formed in previous cycles. A regional difference was also detected in the ovarian mesothelium. The portion of the mesothelium overlying ovulated follicles and developing corporalutea had the greatest percentage of labeled cells. The major findings of this study were: (1) endothelial mitosis was elevated in the initial stages of luteal neovascularization; (2) the heightened endothelial labeling was confined to specific regions of the ovary; and (3) mesothelium in close proximity to the developing corpora lutea also displayed heightened DNA synthesis.
Restriction to endogenous plasma proteins by a fenestrated capillary endothelium: An ultrastructural immunocytochemical study of the choriocapillary endothelium
Authors: Pino Richard M.
Published: February 2005

The choriocapillaris is a fenestrated capillary bed in the eye that restricts the egress of exogenous tracer molecules with Einstein‐Stokes radii (ESR) ≥3.2 nm. The present study examined the permeability of its endothelium to the endogenous plasma proteins albumin (ESR, 3.5 nm) and IgG (ESR, 5.5 nm) using ultrastructural immunocytochemistry. Reaction product indicative of the localization of albumin and IgG (using Fab‐HRP conjugates) was high in the capillary lumen. In contrast, neither protein was localized extravascularly in Bruch's membrane, in endothelial vesicles, or in endothelial channels. The restriction was evident at the luminal side of the diaphragms spanning fenestrae, vesicles, and channels, and at the luminal front of cell junctions. In marked comparison, high levels of reaction product were localized in the extravascular space surrounding mucosal capillaries in the ileo‐jejunum. Observations of tissue subjected to postembedment staining using a protein A‐gold method were similar. These findings demonstrate for the first time the restriction of endogenous plasma proteins by a capillary endothelium identical in morphology to that of other vascular beds proven to be permeable.
Glycoconjugate localization with lectin and PA‐TCH‐SP cytochemistry in rat hypophysis
Authors: Nakagawa Fukuo , Schulte Bradley A. , Spicer Samuel S.
Published: February 2005

Glycoconjugates were localized by light microscopy with lectin‐peroxidase conjugates and by electron microscopy with the periodic acid‐thiocarbohydrazide‐silver proteinate (PA‐TCH‐SP) sequence in immunocyto‐chemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N‐glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose‐N‐acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose‐binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose‐binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA‐TCH‐SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone‐producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they from the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA‐TCH‐SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin‐containing granules as well as other cell types in the pars nervosa.
Effects of B‐retinyl acetate on human breast epithelium in explant culture
Authors: Strum Judy M. , Resau James H.
Published: February 2005

Explants of human breast (obtained aseptically from ten women ages 18–37) were examined and the vitamin A compound B‐retinyl acetate was tested (at 3 × 10−5 and 3 × 10−6 M) for its effects on the fine structure and growth of the epithelium. In the absence of B‐retinyl acetate, cells growing out from the explant (outgrowth cells) underwent squamous metaplasia, began to accumulate many intermediate filaments (tonofilaments), and revealed large desmosomes after 2 weeks in culture. In the presence of either concentration of B‐retinyl acetate, the epithelial cells were largely prevented from undergoing squamous metaplasia. The glandular epithelium inside the explant maintained a typical secretory appearance for 2 weeks in control cultures (without the retinoid) and then began to show increased numbers of lysosomes and a loss of secretory granules. These glandular epithelial cells did not undergo squamous metaplasia, but they contained increased numbers of intermediate filaments. In contrast, glandular epithelium cultured in either concentrations of B‐retinyl acetate appeared secretory for as long 6 weeks in culture, and intermediate filaments were not obvious. Autoradiographs demonstrated that both concentrations of B‐retinyl acetate inhibited cell division in the outgrowth epithelium and in the (internal) glandular epithelium. The ability of B‐retinyl acetate to reverse squamous metaplasia in the outgrowth cells was also tested. Out growth cells reversed to a more normal ductlike appearance after 6 weeks culture in standard medium followed by only 1 week in 20 μg/ml B‐retinyl acetate. After 7 weeks in standard medium and 1 week in 10 μg/ml B‐retinyl acetate, the cells showed a partial reversal of the squamous metaplasia.
Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy
Authors: Tung Hai‐Nan , Parr Earl L. , Parr Margaret B.
Published: February 2005

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer‐containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes.

To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.
Blastocyst implantation in the chinese hamster (Cricetulus griseus)
Authors: Blankenship Thomas N. , Given Randall L. , Parkening Terry A.
Published: February 2005

Embryonic development of the Chinese hamster (Cricetulus griseus) was studied from the onset of implantation to the formation of the parietal yolk sac placenta. Implantation began on day 6 of pregnancy, when the embryo became fixed to the uterine luminal epithelium. At this time there was no zona pellucida, and microvilli of the trophoblast and uterine epithelium were closely apposed. Stromal cells immediately adjacent to the implantation chamber began to enlarge and accumulate glycogen. By day 7 the mural trophoblast penetrated the luminal epithelium in discrete areas. The trophoblast appeared to phagocytize uterine epithelial cells, although epithelium adjoining the points of penetration was normal. In other areas nascent apical protrusions from the uterine epithelium indented the surface of the trophoblast. The epiblast had enlarged and both visceral and parietal endoderm cells were present. The well‐developed decidual cells were epithelioid and completely surrounded the implantation chamber. On day 8 the uterine epithelium had disappeared along the mural surface of the embryo. The embryonic cell mass was elongated and filled the yolk sac cavity. Reichert's membrane was well developed. The uterine epithelial basal lamina was largely disrupted, and the trophoblast was in direct contact with decidual cells. Primary and secondary giant trophoblast cells were present and in contact with extravasated maternal blood. The mural trophoblast formed channels in which blood cells were found in close proximity to Reichert's membrane. Decidual cells were in contact with capillary epithelium and in some cases formed part of the vessel wall. Structural changes occurring in the embryo and endometrium during implantation in the Chinese hamster are described for the first time in this report and are compared to those described for some other myomorph rodents.
An investigation of lymphatic vessels in the feline dental pulp
Authors: Bishop M. A. , Malhotra M.
Published: February 2005

The existence of lymphatic vessels in the dental pulp has been a matter of continuing controversy. We have now used light microscopy to examine semithin transverse sections of perfusion‐fixed incisors and canines in cats. Lymphatics were found in all the teeth studies. In most teeth they were present in the coronal, middlel, and apical regions of the pulp; but in a few they were lacking coronally and in the middle. Within individual teeth, lymphatics were found in the subodontoblastic zone or more centrally in the pulp; but none were found in the odontoblast layer or in the pulp horns.

Vessels located by light microscopy were subsequently examined by transmission electron microscopy. Their ultrastructural features were typical of lymphatics and included irregular, attenuated endothelium with adjacent cells joined in different ways. Occasional gaps connected the extracellular spaces with their lumens, and abluminal endothelial projections appeared to form open and bulbs. There was very little basement membrane, but anchoring filaments were found near the abluminal surface of the endothelium and near collagen fibrils.

The total cross‐sectional area of lymphatic vessels was measured in semithin sections and, with pulp area, increased from the coronal region to the middle. However, both areas decreased from the middle to the apical region suggesting either that lymph flows faster as it reaches the formens of the apical delta or that some vessels leave the tooth through lateral root canals.

Using the methods of light and transmission electron microscopy, therefore, we have shown that pulp lymphatic vessels exist. Questions remain, however, about their distribution within teeth, variations between teeth, and routes of exit from teeth.
Reappraisal of histogenesis in the bursal lymphoid follicle of the chicken
Authors: Lupetti Mario , Dolfi Amelio , Giannessi Francesco , Bianchi Francesco , Michelucci Sergio
Published: February 2005

The development of the bursal follicle and the appearance of the follicle‐associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stadied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of folicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and anumber of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that hav closed under the mesenchymal nodule become stratified and form the REp cells; they become star‐shaped because the mdedullarylymphoid cells grow between them. Finally, the cortex in formed, possibly as a result of the migration of medullary cells before they peripheralize. it is concluded that FAE cells are not specialized It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.
The role of the mesenchyme in mouse neural fold elevation. II. Patterns of hyaluronate synthesis and distribution in embryos developing in vitro
Authors: Morris‐Wiman Joyce , Brinkley Linda L.
Published: February 2005

Hyaluronate (HA) distribution patterns were examined in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Using standard image‐processing techniques, the digitized images of Alcian blue‐stained or 3H‐glucosamine‐labeled sections digested with an enzyme specific for HA, were subtracted from adjacent, undigested sections. The resultant difference picture images (DPI) accurately depicted the distribution of stained or labeled HA within the cranial mesenchyme. 3H‐glucosamine‐labeled HA was distributed uniformly throughout the cranial mesenchyme as 12, 18, and 24 hr of culture. By contrast, the mesenchyme was uniformly stained with Alcian blue at 12 hr, but stain intensity decreased in the central regions of the mesenchyme at 18 and 24 hr.

HA distribution patterns were also examined in the cranial mesenchyme of embryos cultured in the presence of diazo‐oxo‐norleucine (DON), a glutamine analogue that inhibits glycosaminoglycan and glycoprotein synthesis. In DON‐treated mesenchyme, Alcian blue staining of HA was decreased from that in controls at 12, 18, and 24 hr. However, incorporation of 3H‐glucosamine into HA was increased. The distribution of labeled HA within treated mesenchyme as 12, 18, and 24 hr resembled that in controls at 12 hr.

These results indicate that the distribution of HA within the cranial mesenchyme of normal mouse embryos during neural fold elevation and convergence is not determined solely by regional differences in HA synthesis. We propose that HA distribution patterns result from the expansion of the HA‐rich extracellular matrix of the central mesenchyme regions. This expansion may play a major role in fold elevation. These results also suggest that DON treatment reversibly inhibits HA synthesis, since treated mesenchymal cells retain the capability of synthesizing HA when provided with a glucosamine substrate. Patterns of 3H‐glucosamine incorporation by DON‐treated mesenchyme are similar to those observed in control mesenchyme prior to mesenchymal expansion at 12 hr.
Laminin expression in the mouse lung increases with development and stimulates spontaneous organotypic rearrangement of mixed lung cells
Authors: Schuger Lucia , Varani James , Killen Paul D. , Skubitz Amy P. N. , Gilbride Kevin
Published: February 2005

The recent establishment of a role for laminin in mouse lung organogenesis (Schuger et al., 1990a,b, 1991) prompted us to study its expression in the developing lung. Laminin A and B chains were detected in the murine lung from the first hours of development onward. In situ hybridization of mRNA as well as SDS‐PAGE studies of lung cells in monoculture indicated that both epithelium and mesenchyme produce complete laminin molecules. Quantitative analysis of the in situ hybridization studies showed a gradual increase in laminin expression during development which was further supported by immunohistochemistry and ELISA. The overall pattern of expression suggested that the effects of laminin in morphogenesis were not restricted to a particular stage of development. Furthermore, the increase in expression during late development supported a role for the molecule in the fetal lung, which was not previously established. We next determined whether the increase in laminin production modulated the behavior of fetal lung cells as compared with their embryonic counterparts. We previously showed that organotypic pattern formation does not occur in cultures of mixed embryonic lung cells unless exogenous laminin is added (Schuger et al., 1990b). Organotypic pattern formation is the result of cell sorting into epithelial and mesenchymal compartments and further rearrangement in a pattern resembling the tissue of origin. In the present study, we demonstrated that organotypic pattern formation occurs spontaneously in cultures of mixed fetal lung cells, which express high laminin levels. Pattern formation was abolished by antibodies to laminin. These studies suggest a correlation between laminin expression and the ability of lung cells in culture to reproduce normal tissue patterns. We conclude that laminin is critical for epithelial‐mesenchymal recognition and further morphogenic interaction during both the embryonic and fetal stages of lung development. © 1992 Wiley‐Liss, Inc.
Noncoordinate developmental regulation of N‐cadherin, N‐CAM, integrin, and fibronectin mRNA levels during myoblast terminal differentiation
Authors: MacCalman Colin D. , Bardeesy Nabeel , Holland Paul C. , Blaschuk Orest W.
Published: February 2005

N‐cadherin, N‐CAM, fibronectin, and β1‐integrins have been implicated in the control of myoblast fusion to form multinucleate myotubes, a critical step in the terminal differentiation of skeletal muscle. We have analyzed the temporal pattern of expression of mRNA transcripts encoding these adhesion molecules during the terminal differentiation of C2 mouse myoblasts. The accumulation of mRNA transcripts encoding N‐cadherin, N‐CAM, fibronectin, β5‐integrin, and β1‐integrin subunits was developmentally, but not coordinately, regulated. N‐cadherin and integrin subunit expression was maximal in profusion myoblasts and declined thereafter. In contrast, N‐CAM mRNA levels were low in prefusion myoblasts, and increased coincident with the onset of terminal differentiation. Fibronectin mRNA levels were also low in myoblasts, and they did not increase until after cell fusion had occurred. The results indicate that despite their lack of coordinate regulation maximal levels of mRNA transcripts encoding adhesion molecules are present at a stage which corresponds to the peak of the active phase of myoblast fusion. © 1992 Wiley‐Liss, Inc.
Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication‐defective retrovirus. III: Polyclonal origin of adjacent ventricular myocytes
Authors: Mikawa Takashi , Cohen‐Gould Leona , Fischman Donald A.
Published: February 2005

Replication‐incompetent variants of the avian spleen necrosis virus (SNV) encoding cytoplasmic or nuclear‐directed β‐galactosidase (β‐gal) have been used to trace the clonal growth of myocytes during left ventricular free‐wall formation. Tubular‐stage hearts were infected with a mixed suspension of both retroviruses and, after hatching, the progeny of marked cells in the ventricular wall were examined by X‐gal histochemistry. When a small number of virions was introduced individual blue patches contained myocytes with only one label type (cytoplasmic or nuclear). These results confirmed our previous conclusion that each cluster or patch represents a single clone (Mikawa et al., 1992, Dev. Dynamics, 193:11–23). Each of these clones formed a cone‐shaped patch which often extended through the entire thickness of the ventricular myocardium, but typically each patch was heterogeneous, containing a mixture of labeled and unlabeled cells. We then asked whether the two populations of myocytes in each patch were clonally related or generated from more than one progenitor. When hearts were infected with high titer viral suspensions many patches were observed in which cytoplasmic‐tagged myocytes were intermingled with nuclear‐tagged myocytes. Thus, the cone‐shaped myocyte patches in the ventricular wall are polyclones derived from separate progenitors in the precardiac mesoderm. This finding led us to examine the separation of clonally related ventricular myocytes in the developing hearts. Embryos were infected with retroviral suspensions at varying stages of development and the resulting colonies examined after hatching. Infections on embryonic days one or two (E1, E2) gave rise to transmural colonies, whereas infection at stages later than E3 resulted in smaller clusters which were not transmural, i.e., the patches filled only a layer of each cone. This result is most simply explained by myocyte migration until E2 but with progressively restricted myocyte movements after E3. © 1992 Wiley‐Liss, Inc.
Differential expression of the two δ‐crystallin genes in lens and non‐lens tissues: Shift favoring δ2 expression from embryonic to adult chickens
Authors: Li Xuan , Zelenka Peggy S. , Piatigorsky Joram
Published: February 2005

Chicken argininosuccinate lyase (ASL)/δ‐crystallin, a lens enzyme‐crystallin, is encoded in two linked genes (δ1 and δ2); only the δ2 polypeptide contains ASL activity. Here we have quantified δ1‐ and δ2‐crystallin mRNA in the lens, cornea, neural retina, heart, and brain at different stages of embryonic development and in 1‐wk‐old and 1‐yr‐old chickens by the polymerase chain reaction using internal δ1 and δ2 RNA standards. The δ1/δ2 mRNA ratio differed for every tissue and was regulated during development. In the embryo there was more δ1 than δ2 mRNA in the lens (50–100 times), cornea (3–4 times), and neural retina (2–20 times), about equal amounts of δ1 and δ2 mRNA in the heart, and more δ2 mRNA in the brain (15 times). δ1‐Crystallin mRNA differentially decreased in every tissue after hatching; by contrast, the δ2 mRNA remained about the same except for the lens, where it decreased 50‐fold between 1 wk and 1 yr after hatching. In the 1‐yr‐old chicken, the δ2/δ1 mRNA ratios were 7 in the lens, 175 in the cornea, 22 in the neural retina, 107 in the heart, and 136 in the brain, indicating that δ2‐crystallin is strongly favored in all adult tissues of the chicken. The excess of δ1 to δ2 mRNA in the embryonic lens, cornea, and neural retina is intriguing, and suggests some connection with developing transparent eye tissues. Finally, we raise the possibility that expression of both δ‐crystallin genes may create tetrameric ASL isoenzymes (perhaps with different specific activities). The unexpected predominance of δ2 mRNA in the 1‐yr‐old lens suggests that both the enzymatic and refractive functions of ASL/δ‐crystallin are operative and spatially separated, with the enzymatic role present in the cortical fibers and the refractive role in the center of the lens. © 1993 wiley‐Liss, Inc.
Crooked Neck Dwarf (cn) mutant chicken skeletal muscle cells in low density primary cultures fail to express normal α ryanodine receptor and exhibit a partial mutant phenotype
Authors: Airey Judith A. , Deerinck Thomas J. , Ellisman Mark H. , Houenou Lucien J. , Ivanenko Anna , Kenyon James L. , McKemy David D. , Sutko John L.
Published: February 2005

The Crooked Neck Dwarf (cn) mutation in chickens causes marked changes in intact embryonic skeletal muscle. We have investigated whether the cn/cn phenotype develops in vitro, and if cultured muscle cells are suitable for studies of this mutation. The properties of cn/cn muscle cells maintained in low density primary cultures (6.25 × 103 cells/cm2) are described in this report. In normal muscle cells, the α ryanodine receptor (RyR) isoform appears prior to, and at greater levels than, the βRyR, and is detected in mononucleated myocytes. The βRyR isoform appears within 24 hr after the initiation of myotube formation, which is earlier than anticipated from studies with intact embryonic muscle. Normal αRyR protein is not detected in cultured cn/cn muscle cells, whereas the βRyR, the α1‐subunit of the dihydropyridine receptor, the sarcoplasmic reticulum Ca2+‐ATPase, and calsequestrin are expressed at comparable levels in normal and mutant muscle cells. Calcium transients elicited by electrical stimulation, acetylcholine, and caffeine are similar in normal and cn/cn cultured myotubes and are blocked by ryanodine in both cell types. In addition, comparable L‐ and T‐type calcium currents are observed in normal and mutant muscle cells, suggesting that both the α1‐subunit of the dihydropyridine receptor and the βRyR in mutant muscle cells are functional. Normal and cn/cn muscle cells proliferate and form myotubes in a similar manner. These latter events do not appear to depend on sarcoplasmic reticulum calcium release, as they also occur in normal muscle cells in which calcium release is prevented by chronic treatment with 100 μM ryanodine. Both cn/cn and ryanodine‐treated normal muscle cells exhibit morphological changes similar to those observed in intact cn/cn skeletal muscle. Thus, the mutant phenotype observed in ovo is partially expressed under low density culture conditions, and neither βRyR protein nor its function appear to be capable of preventing the associated changes. © 1993 Wiley‐Liss, Inc.
Test of a model for the effects of retinoic acid on urodele limb regeneration
Authors: Ludolph David C. , Cameron Jo Ann , Stocum David L.
Published: February 2005

Previous studies have shown that in axolotls (Ambystoma mexicanum), retinoic acid (RA) treatment evokes pattern completion in limb regenrates derived from anterior and dorsal half zeugopodia (lower arms and legs), but causes regenerative failure in posterior and ventral half zeugopodia. Pattern completion in anterior and dorsal half limbs may be explained by postulating that intercalary regeneration occurs in the anteroposterior (AP) and dorsoventral (DV) axes between blastema cells that are posteriorized (anterior half limb) or ventralized (dorsal half limb) by RA, and circumferential anterodorsal cells that remain unaffected by RA and thus maintain their original positional indentities. The contrasting regenerative failure of RA‐treated posterior and ventral half zeugopodia may likewise be explained by postulating that all the blastema cells in the posterior half are posteriorized, and all the cells in the ventral half are ventralized by RA, thus eliminating differentials in transverse positional identity essential for blastema formation and outgrowth. To test these postulates we grafted blastemas derived from limbs halved in the AP and DV axes of control and RA‐treated animals to untreated whole limb stumps and analyzed the patterns of supernumerary (SN) regeneration. The site or location of SN formation will demonstrate (1) whether RA has posteriorized and ventralized the positional identity of the blastema cells and (2) if blastema cells in the periphery of the anterodorsal quadrant of the limb are resistant to these RA‐induced changes in positional identity. Our results are consistent with a model in which RA posteriorizes positional identity in both halves of the anteroposterior axis and ventralizes it in both halves of the dorsoventral axis, while circumferential anterodorsal cells maintain their original positional identities. In addition, this model can explain why systemically administered RA in urodeles does not induce transverse axial duplications in zeugopodial regenerates of whole limbs, like it does in regenerating anuran and developing chick limb buds. © 1993 Wiley‐Liss, Inc.
Cellular morphology and extracellular space at rhombomere boundaries in the chick embryo hindbrain
Authors: Heyman Isobel , Kent Andrew , Lumsden Andrew
Published: February 2005

The chick embryo hindbrain is a segmented region of the CNS characterised by repeated blocks of neuroepithelial cells, known as rhombomeres. Individual rhombomeres are polyclonal compartments, defined both by cell lineage restriction and by the restricted expression of development control genes, that later acquire specific patterns of neuronal differentiation and axon outgrowth. The interfaces between adjacent rhombomeres are defined by boundaries across which cells do not move; the boundaries contain specialised cells and are preferentially colonised at early stages of development by extending axons. In this study, routine electron microscopy and high‐pressure cryopreservation, a technique that avoids artifacts of chemical fixation, have been used to examine the morphology of rhombomere boundaries through a staged series of chick embryos. We find that the boudary regions contain enlarged extracellular spaces and that these form conduits for axons subsequently extending in the circumferential plane of the hindbrain. Labeling the ventricular surface of the neuroepithelium with DiI crystals in aqueous suspension revealed the morphology of individual cells in the intact neural tube, and demonstrated unusual fan‐shaped arrays of cells at the boundaries. These findings contribute further to the evidence that cells at rhombomere boundaries differ from those in rhombomere centres, and leads to hypotheses about both the mechanism of development of the boundaries, and the role they may play in hindbrain patterning. © 1993 Wiley‐Liss, Inc.
Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum
Authors: Reginato Anthony M. , Tuan Rocky S. , Ono Tamao , Jimenez Sergio A. , Jacenko Olena
Published: February 2005

Maintenance of chick embryos in long‐term culture without their calcareous eggshell is a useful method for studying the relationship between calcium homeostasis and cell differentiation during skeletogenesis. Previously, we have shown that in shell‐less (SL) embryos, calcium deficiency induces a cartilage‐like phenotype in osteogenic tissues, such as calvaria (Jacenko and Tuan [1986] Dev. Biol. 115:215). In this investigation, we have studied the relationship between cartilage calcification and hypertrophy, and the expression of type X collagen, a specific product of hypertrophic chondrocytes. For this study, the cephalic (calcifying) and caudal (permanently cartilaginous) regions of sterna from day 18 and day 20 normal (NL) and SL embryos were metabolically labeled with [14C]‐proline. Analysis of the biosynthetic products revealed significant differences in type X collagen expression in the cephalic region of sternal cartilage. In NL tissues, type X collagen production increased from 13.1% of total collagen at day 18 to 43.7% at day 20. In contrast, in SL embryos, type X collagen was not detectable until day 20, when it represented only 1% of total collagen. Comparison of the NL and SL embryos with respect to their serum calcium level and sternal calcium content and histology revealed a direct relationship between low systemic calcium and limited cartilage hypertrophy, undermineralization, and decreased type X collagen production in the sternal cephalic cartilage. Supplementation of CaCO3 to SL embryos increased their serum and sternal calcium, and restored cartilage hypertrophy, mineralization, and type X collagen synthesis in the cephalic portion of the sterna. These findings confirm that a critical relationship exists between calcium homeostasis, chondrocyte hypertrophy, mineralization, and type X collagen synthesis in the cephalic region of sternal cartilage. These results further demonstrate the importance of calcium in the morphogenetic events of endochondral ossification, in particular the transition from hyaline cartilage to hypertrophic cartilage, and eventually to bone. © 1993 Wiley‐Liss, Inc.
Role of satellite cells in altering myosin expression during avian skeletal muscle hypertrophy
Authors: McCormick K. M. , Schultz E.
Published: February 2005

This study examined whether satellite cells express an embryonic isoform of myosin upon fusion with hypertrophying muscle fibers. Anterior latissimus dorsi (ALD) muscle hypertrophy was induced in adult chickens by weighting one wing. One and 7 days of wing‐weighting produced significant increases in ALD muscle wet weight and in the number of mature fibers expressing ventricular‐like embryonic (V‐EMB) myosin. V‐EMB myosin expression could be an event during regeneration of fibers injured by overload or part of the hypertrophy process itself. Although there was an increase in both the number of damaged fibers and the number of mature fibers expressing embryonic myosin after wing‐weighting, results from this study suggest that these two events were not necessarily related. The apparent health of fibers expressing V‐EMB myosin and the lack of correlation between the numbers of damaged and V‐EMB myosin positive fibers (r = 0.20) suggest that embryonic myosin expression in mature fibers was likely a feature of the hypertrophy process itself. The appearance of V‐EMB myosin in mature fibers 1 day after wing‐weighting suggests that the change in myosin expression did not involve satellite cells since 24 hr is too short a time to permit more than limited satellite cell fusion. The relationship between satellite cells and embryonic myosin expression was examined more closely by labeling dividing satellite cells and their progeny with 5‐bromo‐2‐deoxyuridine, and then colocalizing labeled myofiber nuclei and embryonic myosin in consecutive transverse sections of hypertrophied ALD muscle. One week of wing‐weighting resulted in marked increases in myofiber nuclear labeling index and myofiber nuclear density compared to contralateral control. V‐EMB myosin was not expressed uniformly throughout individual fibers, but rather in discrete regions of varying length. Many V‐EMB myosin positive regions had a higher labeled nuclear density than V‐EMB myosin negative regions indicating that V‐EMB myosin expression was associated with an accumulation of satellite cell progeny in a restricted area. However, it was also clear that satellite cell progeny were not the sole source of V‐EMB myosin since labeled nuclei were completely absent from 41% of the V‐EMB positive regions. Futhermore, the presence of new nuclei did not result in obligatory expression of embryonic myosin because many V‐EMB negative regions had a high labeled nuclear density. Thus, recently incorporated nuclei arising by satellite cell division are implicated as one, but not the sole source of embryonic myosin in hypertrophying muscle. These observations suggest that the non‐uniform pattern of embryonic myosin expression in hypertrophying muscle resulted from differences in nuclear activity, not necessarily the addition of new nuclei. © 1994 Wiley‐Liss, Inc.
Forward spreading in the establishment of a vertebrate Hox expression boundary: The expression domain separates into anterior and posterior zones, and the spread occurs across implanted glass barriers
Authors: Gaunt Stephen J. , Strachan Lorna
Published: February 2005

By use of wholemount in situ hybridization, we show how expression of the chicken homeobox gene Hoxd‐4 commences in the posterior part of the primitive streak and then spreads forward, covering most of the primitive streak by the 2 somite stage, covering the entire primitive streak by the 5 somite stage, reaching the somite 1/somite 2 level of the neural tube by the 9 somite stage, and reaching the rhombomere 6/rhombomere 7 junction of the hindbrain by the 15 somite stage. Forward spreading does not depend upon cell migration, as was evidenced by vital dye (DiI) cell marking experiments. Furthermore, forward spreading does not apparently require tissue continuity since it could not be blocked by impermeable (glass) barriers surgically implanted to divide embryonic tissues. As forward spreading of chick Hoxd‐4 proceeds, the domain of expression separates, at late primitive streak stages, into “anterior” and “posterior zones,” with an intervening “intermediate zone” of weak or non‐expression. Clear anterior and posterior zones were also found for Hoxa‐3 and a‐4 expression in late primitive steak stage mouse embryos. We present evidence that the anterior zone corresponds with the “definitive” domain of Hox gene expression, as has earlier been extensively characterized in midgestation embryos. The posterior zone is transitory, probably persisting only for the duration of the primitive streak, and it is a region of intense Hox expression in primitive streak tissue, Hensen's node, and adjacent regions of neurectoderm and mesoderm. We suggest that the posterior zone marks the source of a morphogen which is the primary activator of Hox gene expression, and we discuss possible models for the mechanism of forward spreading in expression. © 1994 Wiley‐Liss, Inc.
The effect of D‐L ethionine on the pancreas of normal and pregnant hamsters
Authors: House E. L. , Jacobs M. S. , Pansky B.
Published: February 2005

A series of 49 normal male and female adult hamsters, nearly equally divided as to sex and 37 pregnant animals received ethionine subcutaneously or intraperitoneally. Normal animals were given 1.0, 0.7 or 0.4 mg per gram of weight per day over a period of 4 to 14 days. Pregnant animals received either 1.0 or 0.7 mg, beginning at different intervals during gestation: from the third to sixth; on the eighth, of from the tenth to twelfth days, the course ranging from two and one‐half to 15 days. Normal females were more sensitive than males. Among the latter, the 1.0 mg dose produced degeneration of exocrine tissue in ten days with no deaths, while in normal females, 0.7 mg gave similar results with little mortality or liver pathology. In pregnant females, the drug had adverse effects, vaginal bleeding and high mortality resulting when administered on the eighth day or later. From 11 survivors, only two litters were obtained, no damage being found in either maternal or neonatal pancreases. Pregnant animals started on the third to sixth day with the 0.7 mg dose tolerated it well. However, all but one of 17 expected litters died long before birth. The pancreases from the new‐born were normal with only slight damage found in the maternal pancreas. The route of administration was of no consequence.
Dynamic condition of proteins in the visual cells of rats and mice as shown by radioautography with labeled amino acids
Authors: Droz B.
Published: February 2005

The behavior of proteins in retinal rods was examined by injecting various labeled amino acids (methionine‐S35 and ‐H3, tyrosine‐, leucine‐, and arginine‐H3) into rats and mice, which were sacrificed at different time intervals between 10 minutes and 30 days. The retina of these animals was then radioautographed.

Within minutes after injection of any one of the listed amino acids, a radioautographic reaction appears over the inner segment of the rods. This region of the cell is therefore a site of protein synthesis. Since this synthesis may be seen at any time of the day, it must be a continuous phenomenon.

At 24 hours after injection, the radioautographic reaction appears over the junction of the inner and outer segments, and at 1.5–4 days, over the outer segment. Hence, the recently synthesized protein progressively migrates from the inner, toward and into the outer segment.

Analysis of the decay of the specific activity in the inner segment reveals that two categories of proteins are synthesized there. One is a slowly turning over protein, presumably catabolized in situ and referred to as “sedentary.” The other is a fast turning over protein, which migrates into the outer segment and is referred to as “exportable”. The exportable protein may be opsin.
Chemocytological studies on a sudanophile substance in parathyroid cells of the toad (Bufo vulgaris japonicus)
Authors: Hara Jun , Yamada Kazuyori
Published: February 2005

A sudanophile lipid was demonstrated in the parenchymal cells of the toad (Bufo vulgaris japonicus) parathyroid gland in specimens taken at the end of hibernation. A series of histochemical staining methods and extraction techniques for lipids were applied to the parathyroid tissues in an attempt to characterize chemocytologically the sudanophile substance. From its chemocytological properties such as sudanophilia and nile blue sulfate and acid hematein stainability, the substance was concluded to consist primarily of glycerides and phosphatides. The cytophysiological significance of these lipid moieties in the parathyroid cells was discussed in relation to the hibernation of the amphibian species.
Effects of radiocobalt irradiation of pregnant rabbits on the development of fetuses
Authors: Chang M. C. , Hunt Dorothy M. , Harvey Elmer B.
Published: February 2005

Female rabbits were irradiated from a radiocobalt source on various days of pregnancy and examined three to five days before delivery. Following irradiation at 400 r, on days two, four or six before implantation, 24% of the embryos implanted and 19.9% developed into apparently normal fetuses; on day seven or eight at the time of implantation or soon after, all embryos died although 61% implanted; on days 9, 10 or 11, 16.5% of the embryos developed into living fetuses with high incidences of short tail and cleft palate and with low incidences of abnormal forelimbs, fused digits of forelimbs and exencephalia; on days 12, 13 or 14, 35% of the embryos developed into fetuses with a high incidence of fused digits in the fore‐ or hind‐limbs; on days 15 to 22, 30% of the embryos developed into living fetuses without gross abnormalities.

Irradiation at 250 r on day eight did not cause death of all embryos as it did at 400 r. With irradiation at 250 r on days 8 to 14, 51 to 60% of the embryos developed into living fetuses with low incidences of abnormalities.

The size of fetuses was reduced very little following irradiation on days two to six or on days 18 to 22. But the size is distinctly reduced following the irradiation on days 11 to 16.

Necrosis of tissue, pycnosis of nuclei, and vacuolation of cells in the embryos were observed six hours after the irradiation of mothers at 400 r on days seven and eight.
Electron microscopy of the acinar cell of the that preputial gland
Authors: Beaver David L.
Published: February 2005

The rat preputial gland is a morphologically complex structure active in both lipid and protein secretion. By electron microscopy, its acinar cells contain an extensively developed endoplasmic reticulum and numerous Golgi vacuoles, where the protein secretory granules apparently arise. As the granules mature, they assume a crystalloid configuration and, after losing their surrounding membranes when the cell degenerates, are deposited in an excretory duct along with lipid and cellular debris. The granules are apparently unrelated either to mitochondria or to tonofiliments, but seem to represent a true protein secretion of the acinar cell itself.