Histochemical and Histofluorescence Tracing of Chelatable Zinc in the Developing Mouse
Departments of Morphology (YBN,WFS) and Physiology (IS), Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer Sheva, Israel
Correspondence to: Dr. William F. Silverman, Dept. of Morphology, Faculty of Health Sciences, PO Box 653, Ben-Gurion University of the Negev, Beer Sheva 84 103, Israel. E-mail: szeev{at}bgumail.bgu.ac.il
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Summary |
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Key Words: embryo gestation metal localization tissue
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Introduction |
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Materials and Methods |
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Selenite Autometallography
For the selenite method, pregnant females and newborn pups were injected IP with sodium selenite (20 mg/kg) and allowed to survive for 1 hr. Specimen collection proceeded as described above. Slides were then immersed in a developer solution as described previously (Danscher 1982). Sections were counterstained with toluidine blue, cressyl violet, and hematoxylin and eosin (H&E) alternatively.
TSQ Histofluorescence
Histofluorescence with TSQ was performed as described previously (Frederickson et al. 1987). Tissue images were captured into a PC workstation with a digital camera (SPOT RT; Diagnostic Instruments, Sterling Heights, MI) using a UV-2A filter block (330380 nm; barrier 420 nm). Tissue autofluorescence was assessed and eliminated using sections treated with a solution not containing TSQ. Previous treatment with sodium selenite resulted in elimination of TSQ fluorescence.
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Results |
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Chelatable zinc was concentrated in a variety of tissues and exhibited distinct patterns of distribution in midsagittal sections at E13.5, 15.5, 17.5, and P0. At E13.5, chelatable zinc was barely detectable by selenite (not shown). Although also very low when assessed with TSQ, some signal was observed in the digestive tract epithelium, epidermis, and cartilage (Figures 1 3). At E15.5, TSQ histofluorescence was also observed in bone (Figure 3). Selenite staining generally overlapped that of TSQ at E15.5, although some labeling of hepatocytes and blood vessels was observed (data not shown). Again, at E17.5 and P0, when most organs are fully developed, the two methods demonstrated similar patterns of zinc distribution, being most prominent in the alimentary tract and skin. The liver parenchyma at this stage was heavily labeled with the selenite method but not by TSQ. Other tissues exhibiting more moderate concentrations of zinc were pancreas, blood, cartilage, choroid plexus, and vertebrae. Tissues that displayed very little or no zinc were brain and spinal cord, striated muscle, lung, thymus, and thyroid gland. Under high-magnification light microscopic analysis, zinc appeared primarily in cell cytoplasm. The reader is referred to Figure 4 and Table 1 for an overall description of the findings at E17.5.
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Liver
In the liver, selenite AMG labeling was primarily restricted to hepatocytes, beginning at E15.5 (data not shown). No specific organizational pattern of zinc-positive hepatocytes could be discerned. In addition, zinc appeared in biliary canaliculi (Figure 5A). Hepatic elements devoid of chelatable zinc were also observed, including hematopoietic cells, e.g., megakaryocytes (Figure 5B), and connective tissue (i.e., Glison's capsule) enveloping the liver. The TSQ method, in contrast, did not detect chelatable zinc in the liver.
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Skin
In skin, both AMG and TSQ protocols demonstrated the highest zinc concentrations in the epidermis, particularly in the stratum granulosum (Figures 1E and 1F). The AMG method, however, did not label skin until E17.5, whereas TSQ demonstrated some labeling at earlier ages (Figure 4). Hair follicles of the mystacial whiskers exhibited chelatable zinc with both methods, beginning at E17.5 (not shown).
Pancreas
The pancreas was moderately positive for zinc with both methods, beginning at E17.5. Both AMG reaction product and TSQ fluorescence were most concentrated in pancreatic islets, where labeling was distributed throughout the islet. Moreover, zinc was present in only a small percentage (i.e., <10%) of islet cells (data not shown). Virtually no labeling was demonstrated over the exocrine pancreas.
Bone and Cartilage
Up to and including E15.5, AMG reaction product was absent in bone, whereas TSQ demonstrated light labeling of an occasional vertebra and cartilage (Figure 3). At E17.5, both AMG and TSQ methods labeled bone, especially vertebrae, as well as skull and the long bones of the limbs (Figure 3). However, this labeling was inconsistent with many instances of even a single bone exhibiting both moderate and low levels of zinc. When present, zinc was most concentrated in periosteum and the bony matrix. In cartilage, zinc was most evident in intervertebral discs, mainly in chondrocytes at the periphery of the disc (Figures 3F and 3G). At P0 almost all bones contained zinc, although the bone marrow was noticeably unlabeled (see above discussion of megakaryocytes in liver). Cartilage on the day of birth exhibited little if any labeling with either selenite AMG or TSQ (not shown).
Central Nervous System
Chelatable zinc maintains a well-known distribution in the postnatal rodent CNS, particularly in the forebrain and hippocampus (Czupryn and SkangielKramska 1997; Slomianka and Geneser 1997
; Nitzan et al. 2002
; Valente et al. 2002
). In contrast, neither AMG nor TSQ demonstrated chelatable zinc in the brain during gestation, with the single exception of ependymal and endothelial cells of the choroid plexus (CP) (Figure 6). Labeling for zinc in CP was first observed at E15.5. Demonstration of zinc in the CP was more robust at E17.5, with no apparent changes on P0. Chelatable zinc was demonstrated, albeit in low abundance, at E17, and was somewhat more concentrated at P0. At birth, labeling for zinc was present at all levels of the cord and hindbrain, particularly in the dorsal part (Figure 7).
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Discussion |
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It is noteworthy that the distribution of AMG reaction we observed in the liver is reminiscent of the staining patterns reported there previously for metallothioneins (MTs) I/II (Nishimura et al. 1989; Penkowa et al. 1999
), including the strikingly "blank" megakayocytes shown in Figure 7. It is possible that the AMG reaction noted in the liver is due to zinc that is dynamically competed for by selenite from the pool of protein-bound zinc (e.g., MT-bound).
To address the issue of a possible false-positive reaction with selenite AMG, we employed a negative control, i.e., application of developer to sections derived from animals not previously exposed to selenite. Gold, silver, and mercury are the three metal species most likely to produce a spurious reaction (Danscher 1996) and will do so with or without previous treatment of tissue with sulfide or selenite. Kristiansen et al. (2001)
note that this approach is the best-controlled method to ascertain specificity and, in fact, the specificity of the selenite method has been assessed previously in brain (Danscher 1982
). In the present work, control animals did not exhibit autometallographic deposits.
A major consideration in employing both protocols is the dependence of the selenite method on various biological factors (e.g., Slomianka et al. 1990; Valente et al. 2002
). In addition, gestation is a complex process that no doubt contributes additional factors that can affect selenite pharmacokinetics (Danielsson et al. 1990
). It was therefore necessary to confirm the selenite findings by another method, preferably a direct method, e.g., TSQ. Finally, differences, albeit minor, that were obtained using the TSQ and AMG methods have been noted previously (Frederickson et al. 1992
). In general, AMG permits mapping of the metal with reference to surrounding histology although, as mentioned above, it is subject to a variety of physiological influences such as transport mechanisms and tissue permeability (for review see Frederickson et al. 2000
).
Although the present work assessed only chelatable zinc, our findings largely correlate with published concentrations of total tissue zinc (Table 1). This suggests that "free" or chelatable zinc, which exists as a fraction of total zinc, is maintained in balance with protein-bound intracellular zinc, possibly being stored and released as needed. Recent studies using fluorescent resonance energy transfer (FRET) have provided important support for this idea, demonstrating, for example, that nitric oxide induces release of zinc bound to metallothionein (Pearce et al. 2000).
Functional Aspects of Chelatable Zinc
The detection of zinc in bile canniliculi and throughout the digestive tract, including the intestinal lumen and the liver, illustrates a possible zinc enterohepatic circulation in utero. The interplay between secretion and absorption of zinc throughout the gastrointestinal (GI) tract has been described previously (Methfessel and Spencer 1973; Krebs 2000
). It appears that zinc is essential for normal digestive tract physiology, although its precise contribution is not well understood (Semrad 1999
). Our demonstration here of significant quantities of heterogeneously distributed zinc in the GI tract is consistent with the idea that zinc, and more specifically chelatable zinc, is involved in development and/or function of the fetal GI tract as well as in postnatal and adult animals.
Another area in which zinc distribution can readily be related to function is bone, where it is known to be stored (Jackson 1989; King et al. 2000
). Zinc participates in bone formation and growth (Ma et al. 2001
; Ma and Yamaguchi 2001
; Ovesen et al. 2001
). Zinc deficiency leads to a variety of bone pathologies, among which are impaired fracture healing and osteoporosis (Lowe et al. 2002
). The localization of free zinc in bone suggests that it is secreted by local cells to exert effects in a paracrine, autocrine, and possibly an endocrine manner.
For decades zinc has been regarded as an essential element for the normal physiology of skin (PerafanRiveros et al. 2002), possibly via its role as an antioxidant (Rostan et al. 2002
). The present study shows that free zinc is abundant in skin, which is consistent with the thesis that a readily releasable pool of zinc is involved in skin development and function. Hershfinkel et al. (2001)
have recently described an extracellular zinc-sensing receptor on skin cells, which mediates the intracellular release of calcium and, in turn, cell proliferation in a model of wound healing (Hershfinkel and Sekler, unpublished observations). The presence of significant quantities of chelatable zinc in epidermis may therefore suggest that this layer serves as a "trip wire" for initiating cell mechanisms in the dermis to respond to skin injury.
The CNS is one area of the developing mouse in which chelatable zinc, with the exception of the choroid plexus and spinal cord, is conspicuously absent. Areas of the mouse brain that contain high concentrations of free zinc postnatally, e.g., the hippocampus and amygdala, exhibit virtually no chelatable zinc before birth. It is interesting in this regard that, postnatally, the cerebellum contains among the highest concentrations of (total) zinc in the brain (Takeda et al. 2001), although little of it is the chelatable variety. In fact, the lack of chelatable zinc in the fetal brain does not indicate the absence of zinc, because many enzymes and transcription factors containing zinc are present even in early stages of mammalian development. It is therefore, interesting to speculate about the nature of the change that occurs during the days after birth leading to the establishment of a pool of free zinc from existing stores. This comes to be concentrated principally in synaptic vesicles in forebrain excitatory neurons (Frederickson et al. 2000
).
Our study shows that, with a few outstanding exceptions such as brain, "free" or loosely bound zinc is present and is heterogeneously distributed throughout the fetal mouse. Furthermore, we have observed that chelatable zinc is particularly abundant in those organs known to possess high concentrations of (total) zinc. This suggests, first of all, a well-regulated system of zinc homeostasis, even at this early age. Second, it suggests a role for zinc in a wide variety of biological processes, especially because this zinc is present in its most readily usable form. Further research is needed to elucidate the specific functions subserved zinc in the various organs in which it is concentrated.
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Acknowledgments |
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We wish to thank Dr Gershon Perach for assistance in data analysis.
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Footnotes |
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