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M S Cooper

Publications and source records attributed to M S Cooper.

At least 19 recordsLinked to original sources
PubMed2004

11beta-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11beta-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11beta-HSD1 activity and osteoblast differentiation. 11beta-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11beta-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11beta-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11beta-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11beta-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis.

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.

Amino Acid Sequence↗
PubMed2001

Expression of 11 beta-hydroxysteroid dehydrogenase in rat osteoblastic cells: pre-receptor regulation of glucocorticoid responses in bone.

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) acts as a pre-receptor signaling mechanism for corticosteroids by regulating the access of active glucocorticoids to both glucocorticoid (GR) and mineralocorticoid receptors (MR). To examine the relationship between endogenous glucocorticoid metabolism and osteoblast function, we have characterized the expression of 11 beta-HSD isozymes in rat osteosarcoma cells. Analysis of mRNA from ROS 25/1, UMR 106 and ROS 17/2.8 cells revealed transcripts for both 11 beta-HSD type 1 (11 beta-HSD1) and type 2 (11 beta-HSD2) in all three cell lines. However, enzyme activity studies showed only high affinity dehydrogenase activity (inactivation of corticosterone (B) to 11-dehydrocorticosterone (A)), characteristic of 11 beta-HSD2; conversion of B to A was higher in ROS 25/1> UMR 106 cells>ROS 17/2.8. Although all three cell lines had similar numbers of GR (50,000/cell), glucocorticoid modulation of alkaline phosphatase activity and cell proliferation was only detectable in ROS 17/2.8 cells. Further studies showed that 11 beta-HSD2 activity in each of the cells was potently stimulated by both A and B, but not by synthetic dexamethasone. This effect was blocked by the 11 beta-HSD inhibitor, 18 beta-glycyrrhetinic acid (but not by GR or MR antagonists) suggesting direct, allosteric regulation of 11 beta-HSD2 activity. These data indicate that in osteosarcoma cells 11 beta-HSD2 plays a key role in controlling GR-mediated responses; cells with relatively high levels of 11 beta-HSD2 activity were insensitive to glucocorticoids, whilst cells with low levels showed functional responses to both dexamethasone and B. In addition to the established effects of 11 beta-HSD2 in protecting MR in the kidney and colon, our data suggest that 11 beta-HSD2 in bone represents an important pre-receptor mechanism in determining ligand availability to GR.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed2001

Morphogenetic domains in the yolk syncytial layer of axiating zebrafish embryos.

The yolk syncytial layer (YSL) of the teleostean yolk cell is known to play important roles in the induction of cellular mesendoderm, as well as the patterning of dorsal tissues. To determine how this extraembryonic endodermal compartment is subdivided and morphologically transformed during early development, we have examined collective movements of vitally stained YSL nuclei in axiating zebrafish embryos by using four-dimensional confocal microscopy. During blastulation, gastrulation, and early segmentation, zebrafish YSL nuclei display several highly patterned movements, which are organized into spatially distinct morphogenetic domains along the anterior-posterior and dorsal-ventral axes. During the late blastula period, with the onset of epiboly, nuclei throughout the YSL initiate longitudinal movements that are directed along the animal-vegetal axis. As epiboly progresses, nuclei progressively recede from the advancing margin of the epibolic YSL. However, a small group of nuclei is retained at the YSL margin to form a constricting blastoporal ring. During mid-gastrulation, YSL nuclei undergo convergent-extension behavior toward the dorsal midline, with a subset of nuclei forming an axial domain that underlies the notochord. These highly patterned movements of YSL nuclei share remarkable similarities to the morphogenetic movements of deep cells in the overlying zebrafish blastoderm. The macroscopic shape changes of the zebrafish yolk cell, as well as the morphogenetic movements of its YSL nuclei, are homologous to several morphogenetic behaviors that are regionally expressed within the vegetal endodermal cell mass of gastrulating Xenopus embryos. In contrast to the cellular endoderm of Xenopus, the dynamics of zebrafish YSL show that a syncytial endodermal germ layer can express a temporal sequence of morphogenetic domains without undergoing progressive steps of cell fate restriction.

Animals↗
PubMed2001

Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by cytokines.

Patients with glucocorticoid excess develop central obesity, yet in simple obesity, circulating glucocorticoid levels are normal. We have suggested that the increased activity and expression of the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) generating active cortisol from cortisone within adipose tissue may be crucial in the pathogenesis of obesity. In this study primary cultures of human hepatocytes and adipose stromal cells (ASC) were used as in vitro models to investigate the tissue-specific regulation of 11betaHSD1 expression and activity. Treatment with tumor necrosis factor-alpha (TNFalpha) caused a dose-dependent increase in 11betaHSD1 activity in primary cultures of both sc [1743.1 +/- 1015.4% (TNFalpha, 10 ng/ml); P < 0.05 vs. control (100%)] and omental [375.8 +/- 57.0% (TNFalpha, 10 ng/ml); P < 0.01 vs. control (100%)] ASC, but had no effect on activity in human hepatocytes [90.2 +/- 2.8% (TNFalpha, 10 ng/ml); P = NS vs. control (100%)]. Insulin-like growth factor I (IGF-I) caused a dose-dependent inhibition of 11betaHSD1 activity in sc [49.7 +/- 15.0% (IGF-I, 100 ng/ml]; P < 0.05 vs. control (100%)] and omental [71.6 +/- 7.5 (IGF-I, 100 ng/ml); P < 0.01 vs. control (100%)] stromal cells, but not in human hepatocytes [101.8 +/- 15.7% (IGF-I, 100 ng/ml); P = NS vs. control (100%)]. Leptin treatment did not alter 11betaHSD1 activity in human hepatocytes, but increased activity in omental ASC [135.8 +/- 14.1% (leptin, 100 ng/ml); P = 0.08 vs. control (100%)]. Treatment with interleukin-1beta induced 11betaHSD1 activity and expression in sc and omental ASC in a time- and dose-dependent manner. 15-Deoxy-12,14-PGJ2, the putative endogenous ligand of the orphan nuclear receptor peroxisome proliferator-gamma, significantly increased 11betaHSD1 activity in omental cells [179.7 +/- 29.6% (1 microM); P < 0.05 vs. control (100%)] and sc [185.3 +/- 12.6% (1 microM); P < 0.01 vs. control (100%)] ASC, and it is possible that expression of this ligand may ensure continued cortisol generation to permit adipocyte differentiation. Protease inhibitors used in the treatment of human immunodeficiency virus infection are known to cause a lipodystrophic syndrome and central obesity, but saquinavir, indinavir, and neflinavir caused a dose-dependent inhibition of 11betaHSD1 activity in primary cultures of human omental ASC. 11betaHSD1 expression is increased in human adipose tissue by TNFalpha, interleukin-1beta, leptin, and orphan nuclear receptor peroxisome proliferator-gamma agonists, but is inhibited by IGF-I. This autocrine and/or paracrine regulation is tissue specific and explains recent clinical data and animal studies evaluating cortisol metabolism in obesity. Tissue-specific 11betaHSD1 regulation offers the potential for selective enzyme inhibition within adipose tissue as a novel therapy for visceral obesity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2001

Modulation of 11beta-hydroxysteroid dehydrogenase isozymes by proinflammatory cytokines in osteoblasts: an autocrine switch from glucocorticoid inactivation to activation.

Tissue damage by proinflammatory cytokines is attenuated at both systemic and cellular levels by counter anti-inflammatory factors such as corticosteroids. Target cell responses to corticosteroids are dependent on several factors including prereceptor regulation via local steroidogenic enzymes. In particular, two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), by interconverting hormonally active cortisol (F) to inactive cortisone (E), regulate the peripheral action of corticosteroids 11beta-HSD1 by converting E to F and 11beta-HSD2 by inactivating F to E. In different in vitro and in vivo systems both 11beta-HSD isozymes have been shown to be expressed in osteoblasts (OBs). Using the MG-63 human osteosarcoma cell-line and primary cultures of human OBs, we have studied the regulation of osteoblastic 11beta-HSD isozyme expression and activity by cytokines and hormones with established roles in bone physiology. In MG-63 cells, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) potently inhibited 11beta-HSD2 activity (cortisol-cortisone conversion) and messenger RNA (mRNA) levels in a dose-dependent manner while stimulating reciprocal expression of 11beta-HSD1 mRNA and activity (cortisone-cortisol conversion). A similar rise in 11beta-HSD1 reductase activity also was observed in primary cultures of OBs treated with 10 ng/ml TNF-alpha. Pretreatment of MG-63 cells with 0.1 ng/ml IL-1beta resulted in increased cellular sensitivity to physiological glucocorticoids as shown by induction of serum and glucocorticoid-inducible kinase (SGK; relative increase with 50 nM F but no IL-1beta pretreatment 1.12 +/- 0.34; with pretreatment 2.63 +/- 0.50; p < 0.01). These results highlight a novel mechanism within bone cells whereby inflammatory cytokines cause an autocrine switch in intracellular corticosteroid metabolism by disabling glucocorticoid inactivation (11beta-HSD2) while inducing glucocorticoid activation (11beta-HSD1). Therefore, it can be postulated that some of the effects of proinflammatory cytokines within bone (e.g., periarticular erosions in inflammatory arthritis) are mediated by this mechanism.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Immunocytochemical localization of pedal peptide in the central nervous system of the gastropod mollusc Tritonia diomedea.

Tritonia pedal ganglion peptides (TPeps) are a trio of pentadecapeptides isolated from the brain of the nudibranch Tritonia diomedea. TPeps have been shown both to increase the beating rate of ciliated cells of Tritonia and to accelerate heart contractions in the mollusc Clione limacina. Here we examine the immunocytochemical distribution of TPeps in the Tritonia central nervous system. We found the brain and buccal ganglia to be rich sources of TPep immunoreactivity. Specific cells in both structures, some of them previously identified, were immunoreactive. Moreover, immunoreactive fibers were seen connecting ganglia and exiting almost all the major nerves. In the brain, we found that the paired, ciliated statocysts apparently receive TPep innervation. In addition, we observed unstained cell bodies in each buccal ganglion with extensive TPep immunoreactive projections surrounding their somata and primary neurites. Similar projections were not observed in the brain. We also compared the TPep immunoreactivity with that of SCP(b) in the buccal ganglia. We observed many neurons and processes that were immunoreactive to both peptides. One neuron that contains both TPep- and SCP(b)-like peptides (B12) has an identified role in the Tritonia feeding network. Together, these findings suggest that TPeps may play an active role in the central nervous system of Tritonia as neurotransmitters modulating orientation, swimming, and feeding.

Animals↗
PubMed2000

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.

Animals↗
PubMed2000

Computer-assisted visualizations of neural networks: expanding the field of view using seamless confocal montaging.

Microscopic analysis of anatomic relationships within the neural networks of adult and developing tissues often requires sampling large spatial regions of neuronal architecture. To accomplish this, there are two common imaging approaches: (1) image the entire area at once with low spatial resolution; or (2) image small sections at higher magnification/resolution and then join the sections back together by mosaic reconstruction (photomontaging). Low magnification imaging is relatively rapid to perform, resulting in a visualization that encompasses a large field of view with an extended depth of field. However, for fluorescence microscopy, low magnification visualizations are often plagued by poor spatial resolution. High magnification imaging possesses superior spatial resolution, but it produces an image with limited depth of field. When creating a larger field of view, the final image is also fragmented at the boundaries where multiple images are stitched together. Using confocal microscopy as well as features of common image processing programs, we outline a new method to transform individual, spatially contiguous z-series into a montage with a seamless field of view and an extended depth of field. In addition, we show that the manual alignment of images our method requires does not introduce significant errors into the final image. We illustrate our method for visualizing neural networks using tissues from the adult gastropod mollusc, Tritonia diomedea, and the developing zebrafish, Danio rerio.

Animals↗
PubMed2000

Expression and functional consequences of 11beta-hydroxysteroid dehydrogenase activity in human bone.

Glucocorticoids have an essential role in skeletal development and function but are detrimental in excess. In several tissues, glucocorticoid action is dependent upon the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes, which interconvert active cortisol (F) and inactive cortisone (E). We previously demonstrated the expression of 11beta-HSD isozymes in human osteosarcoma cell lines, osteoblast cultures, and fetal bone. We now characterize 11beta-HSD expression in adult human bone using specific antihuman 11beta-HSD antibodies, riboprobes, and enzyme activity studies. In addition, the effect of 11beta-HSD on bone metabolism in vivo was assessed using the 11beta-HSD inhibitor carbenoxolone in eight normal male volunteers. In fresh normal human bone tissue, both 11beta-dehydrogenase (cortisol-to-cortisone conversion) and reductase (cortisone-to-cortisol conversion) activities were demonstrated. There was considerable interindividual variation in the dehydrogenase, but not reductase, activity. In bone homogenates, activity was NADP-dependent with a K(m) for F of 4.8 +/- 1.2 micromol/L, suggesting the presence of 11beta-HSD1. This was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Immunohistochemistry and in situ hybridization studies demonstrated 11beta-HSD1 isozyme expression in cells of the osteoblast lineage and in osteoclasts. The 11beta-HSD2 isozyme was expressed, but only in osteoblasts and at a low level. Ingestion of 300 mg of carbenoxolone by eight normal volunteers for 7 days resulted in a significant decrease in the bone resorption markers, pyridinoline (Pyr) and deoxypyridinoline (DPyr) (change in urinary Pyr/creatinine -1.55 +/- 0.55 [mean +/- SE], for DPyr/creatinine -0. 4 +/- 0.14 nmol/mmol; p < 0.05 for both), with no overall change in the bone formation markers C- and N-terminal propeptides of type I collagen (PICP and PINP). These data suggest that local tissue metabolism of glucocorticoids is likely to be important in determining the sensitivity of both osteoblasts and osteoclasts to glucocorticoids. In particular, variation in 11beta-HSD isozyme expression and activity may explain individual variation in susceptibility to glucocorticoid-induced osteoporosis.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed2000

Toxicity of high-activity 111In-Octreotide therapy in patients with disseminated neuroendocrine tumours.

Disseminated neuroendocrine tumours are difficult to treat and are generally not responsive to radiotherapy or chemotherapy. Nuclear medicine techniques using a radiolabelled somatostatin analogue, 111In-Octreotide, have been used for the diagnosis of neuroendocrine tumours. It has been suggested that high activities of such an agent may have a therapeutic effect. The aims of this study were to assess toxicity and to determine if there had been evidence of efficacy. Eight patients with known disseminated neuroendocrine tumours were enrolled in the study; six had carcinoid tumours, one had a medullary cell carcinoma of the thyroid and one patient had a malignant gastrinoma. Between 1.3 and 4.6 GBq of 111In-Octreotide were administered to each patient for up to five administrations over 12 months. A total of 23 administrations were given. Tests of vital signs, renal, liver and endocrine function as well as haematological markers were taken before and after treatment. The treatment was well tolerated with only one patient suffering from a sensation of flushing during the infusion but no changes in vital sings. There was a transient (up to 48 h) drop in circulating lymphocytes in four patients and platelets in two patients; no supportive therapy was needed. One patient with severe renal impairment had a slight reduction in glomerular filtration rate. We conclude that high-activity 111In-Octreotide is well tolerated with low toxicity and can be considered for use in patients with disseminated neuroendocrine tumours. Further work is now being performed to assess efficacy.

Adult↗
PubMed2000

The role of 11 beta-hydroxysteroid dehydrogenase in central obesity and osteoporosis.

Both central obesity and osteoporosis are common findings in states of glucocorticoid excess. In many tissues, including adipose tissue, hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyses the inter-conversion of active glucocorticoid, cortisol (F) and inactive cortisone (E) and regulates exposure to the glucocorticoid receptor. As such, factors which regulate 11beta-HSD1 are likely to have an important role in adipose tissue and bone physiology. Using primary cultures of human adipose stromal cells we have investigated the effect of various factors present within the adipocyte microenvironment for their effects on 11beta-HSD1 expression. IGF-1 caused a dose dependant inhibition of 11beta-HSD1 activity in both subcutaneous and omental stromal cells. Additionally, TNFalpha treatment increased 11beta-HSD1 reductase activity and mRNA expression. In adult human bone, 11beta-HSD1, but not 11beta-HSD2, expression was demonstrated using enzyme activity studies, RT-PCR and immunohistochemistry. In contrast to liver and adipose tissues, where reductase activity predominates, both reductase and dehydrogenase activities of 11beta-HSD1 were evident in bone chips and primary cultures of human osteoblasts. The action of growth factors and cytokines on glucocorticoid sensitive tissues such as adipose tissue and bone may be mediated by modulation of local glucocorticoid metabolism at a pre-receptor level.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1999

Confocal microscopic analysis of morphogenetic movements.

Confocal microscopy is an excellent means of imaging cellular dynamics within living zebrafish embryos because it provides a means of optically sectioning tissues that have been labeled with specific fluorescent probe molecules. In order to study genetically encoded patterns of cell behavior that are involved in the formation of germ layers and various organ primordia, it is possible to vitally stain an entire zebrafish embryo with one or more fluorescent probe molecules and then examine morphogenetic behaviors within specific cell populations of interest using time-lapse confocal microscopy. There are two major advantages to this "bulk-labeling" approach: (1) the applied fluorescent probe (a contrast-enhancing agent) allows all of the cells within an intact zebrafish embryo to be rapidly stained; (2) the morphogenetic movements and shape changes of hundreds of cells can then be examined simultaneously in vivo using time-lapse confocal microscopy. The neutral fluorophore Bodipy 505/515 and its sphingolipid-derivative Bodipy-C5-ceramide are particularly useful, nonteratogenic vital stains for imaging cellular dynamics in living zebrafish embryos. These photostable fluorescent probes (when applied with 2% DMSO) percolate through the enveloping layer epithelium of the embryo, and localize in yolk-containing cytoplasm and interstitial space, respectively, owing to their different physiochemical characteristics. Bodipy-ceramide, for instance, remains highly localized to interstitial fluid once it accumulates within a zebrafish embryo, allowing the boundaries of deep cells to be clearly discerned throughout the entire embryo. Through the use of either of these fluorescent vital stains, it is possible to rapidly convert a developing zebrafish embryo into a strongly fluorescent specimen that is ideally suited for time-lapse confocal imaging. For zebrafish embryos whose deep cells have been intentionally "scatter-labeled" with fluorescent lineage tracers (e.g., fluorescent dextrans), sequential confocal z-series (i.e., focus-throughs) of the embryo can be rendered into uniquely informative 3D time-lapse movies using readily available image-processing programs. Similar time-lapse imaging, combined with rapidly advancing computer-assisted visualization techniques, may soon be applied to study the dynamics of GFP-fusion proteins in vivo, as well as other types of synthetic probe molecules designed to reveal the cytological processes associated with the patterning and morphological transformations of the zebrafish's embryonic tissues.

Animals↗
PubMed1998

Stability of Adenoscan (adenosine 3 mg ml-1) in plastic syringes.

Adenosine is an effective and safe agent for myocardial stressing in nuclear medicine. It is commercially available as Adenoscan solution (adenosine 3 mg ml-1) and can be conveniently administered to patients via an anaesthesia syringe pump. The solution is dispensed in plastic syringes prior to administration to the patient. The aim of this study was to assess the stability of Adenoscan in plastic syringes to determine if it is possible to pre-dispense the solution and to store dispensed syringes ready for administration to patients. A 4 week stability analysis using high-performance liquid chromatography was carried out on Adenoscan that had been pre-dispensed into polypropylene syringes and stored at 2-8 degrees C. It was found that the concentration of adenosine in the pre-dispensed syringes remained stable during the 4 week period.

Adenosine↗
PubMed1997

Spatially distinct domains of cell behavior in the zebrafish organizer region.

To determine the sequence of cell behaviors that is involved in the morphogenesis of the zebrafish organizer region, we have examined the dorsal marginal zone of vitally stained zebrafish embryos using time-lapse confocal microscopy. During the late-blastula stage, the zebrafish dorsal marginal zone segregates into several cellular domains, including a group of noninvoluting, highly endocytic marginal (NEM) cells. The NEM cell cluster, which lies in a superficial location of the dorsal marginal zone, is composed of both enveloping layer cells and one or two layers of underlying deep cells. The longitudinal position of this cellular domain accurately predicts the site of embryonic shield formation and occupies a homologous location to the organizer epithelium in Xenopus laevis. At the onset of gastrulation, deep cells underneath the superficial NEM cell domain undergo involution to form the nascent hypoblast of the embryonic shield. Deep cells within the NEM cell cluster, however, do not involute during early shield formation, but instead move in front of the blastoderm margin to form a loose mass of cells called forerunner cells. Forerunner cells coalesce into a wedge-shaped mass during late gastrulation and eventually become overlapped by the converging lateral lips of the germ ring. During early zebrafish tail elongation, most forerunner cells are incorporated into the epithelial lining of Kupffer's vesicle, a transient teleostean organ rudiment long thought to be an evolutionary vestige of the neurenteric canal. Owing to the location of NEM cells at the dorsal margin of blastula-stage embryos, as well as their early segregation from other deep cells, we hypothesized that NEM cells are specified by an early-acting dorsalizing signal. To test this possibility, we briefly treated early-blastula stage embryos with LiCl, an agent known to produce hyperdorsalized zebrafish embryos with varying degrees of expanded organizer tissue. In Li(+)-treated embryos, NEM cells appear either within expanded spatial domains or in ectopic locations, primarily within the marginal zone of the blastoderm. These results suggest that NEM cells represent a specific cell type that is specified by an early dorsal patterning pathway.

Animals↗
PubMed1996

A cluster of noninvoluting endocytic cells at the margin of the zebrafish blastoderm marks the site of embryonic shield formation.

In zebrafish embryos, the nascent embryonic shield first appears as a thickening in the germ ring of the mid-epiboly blastoderm. This site defines the dorsal side of the developing embryo. In this paper, we report that the site of embryonic axis formation is marked earlier at the late-blastula stage by the appearance of a cluster of cells with unique endocytic activities. This cluster of cells is composed of enveloping layer epithelial cells and one to two layers of underlying deep cells. Unlike other marginal blastomeres, cells in this cluster do not participate in involution as the blastoderm undergoes epiboly. These noninvoluting endocytic marginal (NEM) cells can be selectively labeled by applying membrane impermeant fluorescent probes to pre-epiboly and mid-epiboly embryos. During embryonic shield formation, deep cells in the NEM cell cluster rearrange and are displaced forward to the leading edge of the blastoderm. As deep NEM cells move into this location, they become a group of cells known as "forerunner cells." Between 60%- and 80%-epiboly, the forerunner cells coalesce into a coherent cell cluster that forms a wedge-shaped cap at the leading edge of the blastoderm. During embryonic axis formation, deep cells migrate and converge toward the embryonic midline, which is defined by the center of the forerunner cell cluster. At approximately 90% epiboly, the forerunner cell cluster becomes overlapped by the constricting germ ring. At tailbud stage, forerunner cells form the dorsal roof of Kupffer's vesicle, which is located ventral to the nascent chordoneural hinge. On the basis of previous grafting studies and known dorsal gene expression patterns, we discuss possible roles that the NEM/forerunner cell cluster may play in teleost axis formation.

Animals↗