Fluorescein Dextran Research Articles

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an approximately 40 by 10 microm surface patch was torn off, entry of fluorescein stachyose (FS; 1, 000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (approximately 5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was approximately 3 mM Ca2+ (SW is approximately 10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+ -free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

A hypothalamic projection to the turtle red nucleus: an anterograde and retrograde tracing study.

It is well known that the reptilian red nucleus lacks a descending motor cortical input to the red nucleus, but has a well-developed cerebellar input. The present study was undertaken to determine whether there is a descending rubral input that originates from the hypothalamus. Using an in vitro preparation from the turtle, injections of neurobiotin into the red nucleus resulted in retrograde labeling of neurons in the suprapeduncular nucleus of the hypothalamus. Injections of either neurobiotin or fluorescein dextran into the suprapeduncular nucleus resulted in anterograde labeling of axons and terminal boutons in the red nucleus. The majority of these terminations appeared to lie in the medial part of the red nucleus. These data have implications for the potential control of the somatic motor system of reptiles by limbic system inputs.

Interactions of ultrapure bovine hemoglobin with renal epithelial cells in vivo and in vitro.

Using an ultrapurified hemoglobin (Hb) solution, we investigated the physiological effects and cellular processing of Hb in rat kidneys and in cultured opossum kidney (OK) cells. Rats infused with < 5.0 g/kg Hb showed no change in baseline serum creatinine (SCr) values (0.58 +/- 0.05 mg/dl) over 48 h, whereas transient acute renal failure followed infusion of 7.5 g/kg Hb (SCr 3.4 +/- 1.02 mg/dl, P = 0.02). Histology of Hb-infused kidneys demonstrated tubular epithelial cell injury. Renal injury was not caused by volume or oncotic load, cardiovascular effect, or ATP depletion. After Hb infusion, heme oxygenase, the rate-limiting enzyme in Hb catabolism, was induced in an organ-specific fashion. Inhibiting heme oxygenase activity with cimetidine did not alter Hb renal injury. Using OK cells, we determined that renal epithelia process Hb by fluid-phase endocytosis. Proton permeability of fluorescein Hb endosomes was unaltered compared with fluorescein dextran controls, demonstrating that Hb does not alter endosomal membrane integrity. These data suggest that Hb renal injury in rats occurs following large doses of ultrapure Hb, does not alter early steps in Hb endosomal processing by renal epithelia, and involves a mechanism that is not heme oxygenase dependent.

A tract-tracing study of the central projections of the mesencephalic nucleus of the trigeminus in the guppy (Lebistes reticulatus, Teleostei), with some observations on the descending trigeminal tract

We studied the central projections of the mesencephalic nucleus of the trigeminal nerve (MesV) in the guppy (Lebistes reticulatus), after application of horseradish peroxidase or fluorescein dextran amine into the eye orbit. A small number (1 to 13) of large mesencephalic trigeminal neurons were solid labeled in the ipsilateral rostral mesencephalon. At the level of the trigeminal nerve entrance, the united process of each mesencephalic trigeminal cell bifurcates, giving rise to a peripheral branch that exits in the trigeminal nerve and a descending branch that runs caudally in a medial bundle separated from the descending trigeminal tract. This bundle passes close to the visceromotor nuclei of the medulla oblongata. Descending processes give rise to short collaterals to the descending nucleus of the trigeminus and the ventrolateral reticular area. Most MesV descending fibres terminate in this ventrolateral field at the transition of the medulla to the spinal cord, but one or two fibres could be followed to the C6 level, where they give rise to collaterals to the dorsal funicular nucleus. No collaterals directed to the trigeminal motor nucleus, the cerebellum, or the mesencephalic tegmentum were observed. These projections were also compared with those of the descending trigeminal tract.

Direct measurement of coupling between dendritic spines and shafts.

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

The Orientation of First Cleavage in the Sea Urchin Embryo,Lytechinus variegatus,Does Not Specify the Axes of Bilateral Symmetry

One blastomere of the two-cell stage sea urchin embryo (Lytechinus variegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientation of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two methods were used to mark the blastomere: In the first, the lipophilic carbocyanine dye DiIC16was microinjected directly into the blastomere after first cleavage was completed. In the second, caged (nonfluorescent) fluorescein–dextran was microinjected into the single-celled zygote and uncaged (made fluorescent) in one of the blastomeres at the two-cell stage using two-photon excitation microscopy (TPEM). This is the first use of TPEM for embryonic lineage tracing. In both methods the dye proved to be nontoxic and fluorescence was confined to lineally related cells. The results from both methods were similar and showed that the first cleavage furrow was variable in its orientation. Results were similar using animals obtained from different geographic locations. These results differ from those of McCain and McClay (Development,1994, 120, 395–404), who reported that the median orientation was invariant in this species. The differences between the two studies are discussed. We conclude that first cleavage does not specify nor is it predictive of the bilateral axes in this species. The technique of TPEM is proffered as a powerful new tool that will enable the marking and tracing of embryonic cell lineages with less injury and more precision than current methods.

Glycine minimizes reperfusion injury in a low-flow, reflow liver perfusion model in the rat

This study investigated the effects of glycine on reperfusion injury in a low-flow, reflow liver perfusion model. With this protocol, livers were perfused at low flow rates of approximately 1 ml.g-1. min-1 for 75 min, which caused cells in pericentral regions of the liver lobule to become anoxic because of insufficient delivery of oxygen. When normal flow rates (approximately 4 ml.g-1.min-1) were restored for 40 min, an oxygen-dependent reperfusion injury occurred. Upon reflow, lactate dehydrogenase (LDH), a cytosolic enzyme, and malondialdehyde (MDA), an end product of lipid peroxidation, were released into the effluent perfusate. LDH increased from basal levels of approximately 1-35 IU.g-1.h-1 in livers from control rats. Glycine (0.06-2.00 mM) minimized enzyme release in a dose-dependent manner (half-maximal decrease = 133 microM), with maximal values only reaching 5 IU.g-1.h-1 when glycine was increased to 2 mM. Reflow for 40 min after 75 min of low-flow hypoxia caused death in approximately 30% of previously anoxic parenchymal cells in pericentral regions; however, infusion of glycine (2 mM) decreased cell death to less than 10%. Strychnine (1 mM), which was found to mimic the cytoprotective effect of glycine in proximal renal tubules, also reduced LDH release to 11 IU.g-1.h-1 in this study. Bile was released at rates of approximately 42 microliters.g-1.h-1 in livers from control rats, but values were not altered significantly by glycine. Maximal MDA production during reperfusion decreased by 35% with 0.6 mM of glycine. Trypan blue distribution time, an indicator of hepatic microcirculation, was reduced significantly by glycine at 5 and 40 min after reflow, but changes were about twofold greater at later time points compared with earlier ones (half-maximal decrease = 225 microM). Time for oxygen to reach steady state upon reflow was reduced by glycine in a dose-dependent manner, and the rates of entry and exit of a dye confined to vascular space (fluorescein dextran) were increased two- to threefold by glycine, respectively. Taken together, these data indicate that a reperfusion injury that occurs in previously hypoxic pericentral regions of the liver upon reintroduction of oxygen is minimized by glycine, possibly by action on a glycine-sensitive anion channel to improve microcirculation during the reperfusion period.

Efferent projections from the retrochiasmatic area to the median eminence and to the pars nervosa of the hypophysis with special reference to the A15 dopaminergic cell group in the sheep.

Anterograde tracers, viz. Phaseolus vulgaris leucoagglutinin and fluorescein dextran, were used in conjunction with tyrosine hydroxylase immunohistochemistry to study the projections of the A15 dopaminergic cell group towards the median eminence and pituitary in sheep. After injection of the tracers in the retrochiasmatic area, which contains the cell group A15, fibres containing anterograde tracer were observed in the internal zone of the median eminence and in the pars nervosa of the pituitary. Numerous tyrosine hydroxylase immunoreactive fibres were present in the external zone of the median eminence and in the pars intermedia and the pars nervosa of the pituitary, with characteristic patterns of organisation in each area. Most tyrosine hydroxylase-immunoreactive fibres containing fluorescein dextran were located in the pars nervosa, whereas only a few were observed in the internal zone of the median eminence. It was concluded that at least part of the dopaminergic innervation of the pars nervosa originated from the A15 group. These results provide morphological evidence for (1) the role of dopaminergic neurons of the A15 cell group in the seasonal control of prolactin secretion via the release of dopamine in the pars nervosa, and (2) putative physiological interactions between dopamine and the secretion of neurohypophysial hormones in sheep.

The effect of the low blink rate in rabbits on topical drug penetration.

The low rate of blinking in the rabbit as compared to the human suggests that there may be a greater contact time for drugs applied topically in the animal. However, there have been no experiments performed to examine this belief directly. A small drop containing either fluorescein or FITC Dextran was applied to the eye of a rabbit without the use of tranquilizer or anesthetic. Subsequently, the change in the fluorescence of the precorneal tear film was followed with a noninvasive fluorometer. The animal did not blink for 20-30 min. after the instillation. With either fluorophore, the tear film fluorescence remained constant after the first few minutes and fell precipitously at the first blink. Comparison with the loss kinetics in the human eye, an exponential decline with an average rate constant of 0.16 min-1, indicates that the AUC will be 3 times greater in the rabbit, and the penetration of a drug can be overestimated to the same degree from the low blink rate alone. There was a rise in the corneal fluorescence, attributable to penetration of the fluorophore, after the eye was washed at the end of the experiment. This was used to estimate the epithelial permeability which averaged 1.5 x 10(-4) cm hr for fluorescein and 1.9 x 10(-5) cm hr-1 for FITC Dextran. For most drugs, however, the epithelial permeability is sufficiently high that all the material present in the tear film will partition into the cornea in a few minutes. Considerations of contact time become irrelevant under these circumstances.

Heavy endosomes isolated from the rat renal cortex show attributes of intermicrovillar clefts

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis. A fraction containing entrapped fluorescein was isolated from a cortical homogenate after differential centrifugation and Percoll density gradient centrifugation. This fraction has been dubbed heavy endosomes. To our surprise, small-particle flow cytometry techniques demonstrated that heavy endosomes are homogeneous for entrapped fluorescein dextran and the presence of H(+)-adenosinetriphosphatase activity. The abundance of heavy endosomes, combined with the findings that true endosomal populations are identifiable in other renal cortical fractions, led us to test whether heavy endosomes had the attributes of intermicrovillar clefts. First, we tested whether heavy endosomes vesiculate in vivo or in vitro. Vesicle-by-vesicle flow cytometry analysis of uptake of fluorescein dextran added to the homogenate demonstrated that virtually all the vesicles form in vitro (99 +/- 2%, n = 4). Second, the fraction contains markers associated with intermicrovillar clefts: clathrin light chains, actin, glycoprotein gp280, and gp330, the "Heymann antigen." The presence of the brush border enzyme markers gamma-glutamyl transpeptidase and leucine aminopeptidase in > 99% of the heavy endosomes confirms that the vesicles are of apical origin. The activity of the enzymes colocalized with entrapped markers but was tenfold less than in brush-border membrane vesicles. Heavy endosomes isolated from the rat renal cortex vesiculate in vitro and contain several intermicrovillar markers.

Trafficking of apical proteins into clathrin-coated vesicles isolated from rat renal cortex.

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Clathrin-coated vesicles (CCV) were isolated from the rat renal cortex utilizing discontinuous sucrose density gradients and negative lectin selection. More than 99 +/- 1% (n = 4) of the isolated vesicles contain entrapped fluorescein dextran when analyzed by small-particle flow cytometry techniques. Similarly, flow cytometry analysis demonstrates brisk H(+)-adenosinetriphosphatase activity in virtually all the vesicles. Western blot analysis of the vesicle proteins with a polyclonal anticlathrin antibody stains bands consistent with clathrin and adaptins. When the isolated vesicles are decoated by exposure to 0.5 M tris(hydroxymethyl)aminomethane, the proteins released match the molecular weights of the proteins identified on Western blot analysis. Flow cytometry demonstration of brush border enzymes in > 99% of the vesicles and Western blot identification of maltase suggests both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Additionally, two glycoproteins detectable in this fraction on Western blot analysis and flow cytometry immunocytochemistry are derived from intermicrovillar clefts traffic into the endosomal pathway. Hence, apical proteins traffic into a population of CCV isolated from the rat renal cortex.

The immediate fate of cells in the epithelial somite of the chick embryo.

The precise origins and fates of cells forming the epithelial ball and those contained within the somitocoele at the centre of the somite in the chick embryo are unknown. In particular, it is not known whether the progeny of the proliferating cells in both the epithelial ball and the somitocoele simply mix with each other, or whether they represent two separate populations that remain isolated during proliferation. We tested whether cells found in both of these locations are the result of cell migration, cell division or both. To do this, we injected single cells in both the epithelial ball and the somitocoele with lysinated fluorescein dextran, a molecule that can only be transferred to other cells through cell division, and observed their development over the next 24 h. Control experiments were also performed to assess the incidence of error associated with the microinjection method and to provide reliable and valid comparative data. Our results showed that the progeny of the cells in the somitocoele, as well as any non-proliferating cells, appear to remain in the somitocoele. In contrast, the progeny of the cells in the epithelial ball either remain in the epithelial ball or migrate into the somitocoele to mix freely with the other cells present. These results suggest that the cells in the epithelial ball and the somitocoele develop as discrete groups, with the possibility of diverse fates accompanying further development.

Early development of dorsal column-medial lemniscal projections in the clawed toad, Xenopus laevis

In Xenopus laevis fluorescent dextran amines were applied to study the development of the dorsal column-medial lemniscal projection: rhodamine dextran amine was applied at the mesodiencephalic border to retrogradely label the cells of origin of the medial lemniscus in the dorsal column nucleus (DCN); fluorescein dextran amine to the spinal cord to anterogradely label the primary afferent projections to the DCN. The first mesodiencephalic projections were found at stage 51, i.e. almost immediately after spinal afferent fibers had reached the DCN.

Macropinosome maturation and fusion with tubular lysosomes in macrophages.

Macropinosomes formed by addition of recombinant macrophage colony-stimulating factor (rM-CSF) to mouse macrophages migrate centripetally and shrink, remaining detectable by phase microscopy for up to 15 min. This longevity allowed us to study how macropinosomes age. Macropinosomes were pulse labeled for 1 min with fixable fluorescein dextran (FDx10f), a probe for fluid phase pinocytosis, and chased for various times. To quantify changes in their antigenic profile, pulse-labeled macropinosomes of different ages were fixed and stained for immunofluorescence with a panel of antibodies specific for the transferrin receptor (TfR), the late endosome-specific, GTP-binding protein rab 7 or lysosomal glycoprotein A (lgp-A), and the percentage of antibody positive, FDx10f-labeled macropinosomes was scored. Some newly formed macropinosomes were positive for TfR, but few were rab 7 or lgp-A-positive. With intermediate chase times (2-4 min), staining for rab 7 and lgp-A increased to > 60%, while TfR staining declined. After a long chase (9-12 min), rab 7 staining returned to low levels while lgp-A staining remained at a high level. Thus, macropinosomes matured by progressive acquisition and loss of characteristic endocytic vesicle markers. However, unlike a maturation process, their merger with the tubular lysosomal compartment more nearly resembled the incorporation of a transient vesicle into a pre-existing, stable compartment. Shortly after their formation, FDx10f-labeled macropinosomes contacted and merged with Texas red dextran (TRDx10)-labeled tubular lysosomes. This occurred in two steps: macropinosomes acquired lgp-A first, and then several minutes later the cation-independent mannose-6-phosphate receptor (CI-MPR) and markers of lysosomal content (cathepsin L or pre-loaded TRDx10), all apparently derived from tubular lysosomes. Thus, macropinosome progress through macrophages showed features of both the maturation and vesicle shuttle models of endocytosis, beginning with a maturation process and ending by merger into a stable, resident lysosomal compartment.

Ectoderm promotes differentiation of the presumptive ventral mesoderm in early gastrulae of Bufo arenarum

Cultures of both isolated and conjugated explants from early gastrulae of Bufo arenarum were prepared for a study of the development of ventral mesoderm. Only combinations including components of the deep ventral marginal zone and the animal pole successfully differentiated into blood cells (erythrocytes). Histological studies indicated that, while prospective mesodermal cells constituted the only source of such cells, prospective ectodermal cells provided the necessary stimulus for this kind of differentiation. Differentiated cultures, in which the tracer of cell-lineage fluorescein dextran amine was used to label these components, confirmed the above conclusions. These findings are discussed in the context of current concepts about the formation of mesoderm.

PHOTODYNAMIC KILLING OF Dictyostelium discoideum AMOEBAE MEDIATED BY 4′,5′‐DIIODOFLUORESCEIN ISOTHIOCYANATE DEXTRAN. A STRATEGY FOR THE ISOLATION OF THERMOCONDITIONAL ENDOCYTOSIS MUTANTS

Abstract4′, 5′‐Diiodofluorescein dextran has been synthesized by iodination of fluorescein dextran and it was used as a sensitizer in photodynamic killing of amoebae of the cellular slime mold Dictyostelium discoideum. Amoebae from axenic strain AX2 incubated in the presence of 4′,5′‐diiodofluorescein dextran efficiently internalized the molecule by fluid‐phase pinocytosis. Subsequently, illumination of cells showed dose‐ and time‐dependent loss of viability. The tertiary amine, 2,4‐ diaza[2,2,2]bicyclooctane, significantly decreased phototoxicity, whereas the presence of D2O increased phototoxicity. These effects were fully consistent with a role for excited‐state singlet oxygen generated inside the endosomal compartments in the mechanism of photoinjury. A strategy was developed to enrich for temperature‐sensitive Dictyostelium endocytosis mutants in a nitrosoguanidine mutagenized population. Incubation with 4′,5′‐diiodofluorescein dextran at 27°C to load endosomal compartments was followed by light treatment to kill parent‐type cells selectively. In contrast, mutants unable to internalize the fluid‐phase marker would survive the treatment. Six temperature‐sensitive clones were selected that grew normally at 21°C but did not grow at 27°C. They all showed markedly decreased fluid‐phase pinocytosis capacity at nonpermissive temperature as compared to their activity at 21 °C. These mutant strains should be useful in studies designed to characterize endocytosis mechanisms in Dictyostelium.

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes.

An approximately 29-kD protein was purified from the membrane fraction of wheat (Triticum aestivum cv Dganit) mitochondria by the utilization of standard liquid chromatography techniques. The protein, designated MmP29 for mitochondrial membrane protein having a molecular mass of approximately 29 kD, exhibited cationic properties in a buffering solution, adjusted to pH 7.5. This positive charge enabled its passage through a diethylaminoethyl column, without interaction with the positively charged matrix. Subsequently, this protein was separated from the remaining polypeptides by a preferential elution from a hydroxylapatite/celite mixed column. Reconstituted liposomes containing this protein were characterized as being permeable to 8-amino-naphthalene 1,3,6-trisulfonic acid disodium salt (Mr 445) but non-permeable to dextran fluorescein (Mr 40,000). Additionally, MmP29 was inserted into planar phospholipid membranes, and anion-selective, voltage-dependent channels were demonstrated. All of the MmP29 properties mentioned highly resemble voltagedependent, anion-selective channel (VDAC) proteins, suggesting that MmP29 is the mitochondrial outer membrane VDAC protein of wheat.

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.

Potential errors in measuring aqueous humor flow

Breakdown and re-establishment of the blood-aqueous barrier (BAB) was studied following laser irradiation to the rabbit iris by quantitating leakage of protein, fluorescein, and fluorescein-labeled dextran (FLD-70, MW 70,000) into the aqueous humor. Breakdown of the BAB was more prolonged in pigmented than albino animals, possibly due to the increased laser energy absorbed by the pigmented iris. Fluorophotometry appeared to be a more sensitive method for evaluating BAB breakdown than aqueous protein measurements, and small molecule leakage (fluorescein) appeared to be more sensitive than macromolecule leakage (FLD-70). The noninvasive method of evaluating the BAB proved equally as effective as measuring aqueous protein content in assessing the sensitivity of laser-induced trauma to the ameliorating effect of the nonsteroidal anti-inflammatory agent indomethacin.