Quantitative radioautographic study of intracellular localization of calmodulin antagonist,W-7, in Chinese-hamster ovary cells

Summary The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 M), 2 (33.4 M), 5 (83.5 M) and 10 Ci/ml (167 M) tritiated W-7. Some cells were preincubated in 10, 50 and 100 M unlabelled W-7 for 30 min and then labelled with 2 or 5 Ci/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 m-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

The induction of chromosome condensation in tsBN2, a temperature-sensitive mutant of BHK21, inhibited by the calmodulin antagonist, W-7

The induction of premature chromosome condensation (PCC) in tsBN2 cells, a temperature-sensitive (ts) mutant of BHK21/13 which shows PCC at the non-permissive temperature, was almost completely inhibited by 40 microM W-7, an antagonist of calmodulin. The mitotic phosphorylation of histone H1 and H3 was also inhibited by W-7. W-5, a chlorine-deficient analogue of W-7 and which interacts weakly with calmodulin, did not inhibit the induction of PCC, even at a dose of 80 microM. The content of calmodulin in tsBN2 cells was increased by a temperature shift to 40.5 degrees C. All these results suggested that calmodulin is required for the chromosome condensation.     

Inhibition of the acrosome reaction of sea urchin spermatozoa by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)

A calmodulin antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), inhibited induction of the acrosome reaction of sea urchin spermatozoa by egg jelly at 20-50 microM, but W-5 did not. The latter, a chlorine-deficient derivative of W-7, has a lower affinity for calmodulin than does W-7. These results suggest that a Ca2+-calmodulin-dependent process plays an important role in the triggering process of the acrosome reaction of sea urchin spermatozoa.     

Influence of W-7, a calmodulin antagonist on phospholipid biosynthesis in Candida albicans

AIM: This study was undertaken to investigate the role of calmodulin in phospholipid biosynthesis in Candida albicans using W-7, a calmodulin antagonist. METHODS: Cells were grown as shake cultures in the absence and presence of W-7 at different concentrations. Changes in cell mass, phospholipid content and incorporation of labelled precursor into phospholipid and activities of respective enzymes have been studied. RESULTS: Decreased incorporation of labelled acetate into total lipids and phospholipids was observed in the presence of 40 microm of W-7 which was not as a consequence of altered growth of Candida in the presence of calmodulin antagonist. Further, a significant decrease in the levels of calmodulin and CaM dependent protein kinase activity was observed in cells grown with different concentrations of W-7. This was accompanied by decreased/increased activity of phosphatidic acid phosphatase and phospholipase A, respectively in W-7 grown cells as compared to controls. CONCLUSIONS: These findings suggest definite involvement of calmodulin in the regulation of phospholipid metabolism in Candida albicans.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.     

Differences in Ca2+-mediation of hypotonic and Na+-nutrient regulatory volume decrease in suspensions of jejunal enterocytes

We determined differences in the Ca2+ signalling of K+ and Cl- conductances required for Regulatory Volume Decrease (RVD) in jejunal villus enterocytes passively swollen (0.5 or 0.95.isotonic) compared with swelling because of the absorption of D-glucose (D-Glc) or L-Alanine (L-Ala). Cell volume was measured using electronic cell sizing. In nominally Ca(2+)-free medium containing EGTA (100 microM) RVD after 0.5 or 0.95.isotonic challenge was prevented. L-Ala swelling and subsequent RVD was influenced in Ca(2+)-free medium. Villus cells were incubated with 10 microM of the acetomethoxy derivative of 1,2.bis (2-aminophenoxy) ethane N,N,N1,N1 tetracetic acid (BAPTA-AM) and RVD after 0.5.isotonic swelling or L-Ala swelling was prevented. Niguldipine (0.1 microM), nifedipine (5 microM), diltiazem (100 microM), Ni2+, and Co2+ (1 mM) all prevented hypotonic RVD but had no effect on RVD after L-Ala addition. Charybdotoxin (25 nM) a potent inhibitor of Ca(2+)-activated K+ channels, had no effect on hypotonic RVD but prevented RVD of villus cells swollen by D-Glc. We used the calmodulin antagonists, naphthalene sulfonamide derivatives W-7 and W-13, to assess calmodulin activation of K+ and Cl- conductance in these two models. L-Ala swelling and subsequent RVD was not influenced by 25 microM W-7; hypotonic RVD was prevented by 25 microM W-7 or 100 microM W-13. The W-13 inhibition of RVD was by-passed with 0.5 microM gramicidin. Our data show that hypotonic RVD requires extracellular Ca2+ and that the K+ conductance activated is not charybdotoxin sensitive but requires calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct interaction of calmodulin antagonists with Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase

Trypsin-treated Ca2+/calmodulin-dependent phosphodiesterase (CA2+-PDE), which had lost its sensitivity to Ca2+-calmodulin, was inhibited by various calmodulin antagonists, trifluoperazine, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and aminoalkyl chain analogues of W-7 (A-3, A-4, A-5, I-240, A-6, A-7). These inhibitory effects were less than those on calmodulin-activated Ca2+-PDE. The ability of these compounds to inhibit trypsin-treated Ca2+-PDE correlated well with the inhibitory effect on calmodulin-activated Ca2+-PDE. W-7 inhibited trypsin-treated Ca2+-PDE in a competitive fashion with respect to cyclic GMP and the Ki value was 300 microM. The inhibition of trypsin-treated Ca2+-PDE by W-7 (300 microM) or A-7 (100 microM) was overcome by the addition of excess calmodulin. Trypsin-treated Ca2+-PDE can bind to W-7-coupled cyanogen bromide-activated Sepharose 4B in the presence of 1 mM EGTA. These results suggest that Ca2+-PDE possesses a binding site for calmodulin antagonists and that the binding site for these antagonists on this enzyme may be structurally similar to the binding site on calmodulin itself.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts

Interleukin 1 (IL-1) stimulates the synthesis of collagenase in human uterine cervical fibroblasts. This inductive effect of IL-1 on collagenase production was augmented by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific inhibitor of calmodulin, in a dose-dependent manner. The apparent collagenase activity observed in the culture medium of the cells treated with IL-1 and 40 microM W-7 was about three times higher than that produced by the cells treated with IL-1 alone. The immunoblotting with the specific antibody against human collagenase showed that the increased collagenase activity resulted from the accelerated biosynthesis of collagenase. Another calmodulin inhibitor, trifluoperazine, enhanced the effect of IL-1 on collagenase production similarly. However, the effect of N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor of calmodulin, was negligible. These results suggest that W-7 enhances the collagenase production by specifically inhibiting calmodulin and that calmodulin may act as a suppressor of the IL-1-induced collagenase production in human uterine cervical fibroblasts.     

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

The proliferation of adrenal medullary cells in newborn and adult mice

Summary The proliferative activity of newborn and adult mouse adrenal medullary cells was determined with light and electron microscopic autoradiography. The H³ thymidine labelling index of 2 weeks old mice adrenal medullary cells was about 9.4 % and declined to less than 1 % in adult mice. In electron microscopic autoradiography labelled norepinephrine as well as epinephrine cells could be seen. Only in 1 and 2 weeks old mice some morphologically undifferentiated cells were visible. In formaldehyde induced fluorescence combined with light microscopic autoradiography the fluorescence intensities of labelled and unlabelled medullary cells were measured. On average the fluorescence intensity of labelled cells was lower than that of unlabelled cells. The differences could be explained by a higher number of autoradiographic silver grains laying on the cytoplasm of labelled cells. These results give evidence that fully differentiated adrenal medullary cells are capable of division.This study was supported by Jubiläumsfonds der Österreichischen Nationalbank grant No. 818     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Loss of alpha 2-macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for alpha 2-macroglobulin, and epidermal growth factor (EGF) in fibroblasts. The loss of alpha 2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for alpha 2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 is removed. Receptor loss was measured as a change in the capacity for binding iodinated ligands; no change in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface beta 2-microglobulin, a membrane protein which is excluded from coated pits and which is not internalized, indicating that the effect of W-7 was specific for membrane receptors and not a result of bulk depletion of plasma membrane. The loss of alpha 2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsible for the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: 1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; 2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rhodamine-labeled alpha 2-macroglobulin, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; 3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/cell) and observed no effect on the receptor-mediated endocytosis of rhodamine-labeled alpha 2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Autoradiographic localization in polychaete embryos of tritiated mesulergine, a selective antagonist of serotonin receptors that inhibits early polychaete development.

Developing embryos of the polychaete Ophryotrochal labronica were exposed to tritiated mesulergine, a selective antagonist of the serotonin receptors 5-HT1c and 5-HT2, that also has significant affinity to dopamine D-2 sites, and the labeling was analyzed by autoradiography. Already at the earliest developmental stages (1-4 cells), numerous silver grains visualizing 3H-mesulergine binding sites and possibly also serotonin receptors were recorded over the cytoplasm, mostly in association with decomposing yolk granules, but few grains were detected over the nuclear region. In advanced pregastrular embryos (3 days) the number of silver grains was greatly increased over nuclei, cell borders and non-yolk cytoplasmic elements, notably in the animal half of the embryos. For newly gastrulated embryos (4 days), more than 90% of the grains appeared over non-yolk cellular structures. Abundant access to serotonin receptors is probably a fundamental condition not only for gastrulation but also for the high mitotic activity of the cleavage period. An indication hereof is the observation that exposure of cleaving polychaete eggs/embryos to unlabeled mesulergine inhibited cytokinesis and chromosome movements, whereas spindle formation and chromosome duplication were unaffected.     

Light microscopic radioautographic study on the DNA synthesis of prenatal and postnatal aging mouse retina after labelled thymidine injection.

The DNA synthesis of mouse retina from the 19th prenatal day through 12 months postnatal has been studied by light microscopic radioautography after the injection of tritiated thymidine. A peak of the labeling index after incorporation of tritiated thymidine was found at fetal day 19. The labelled cells decreased gradually with the developing of the eye from the first postnatal day and were completely disappeared in two weeks after birth. The data also indicated obvious regional differences of the incorporation of tritiated thymidine during the periods of the retina development. The labeling index was the greatest in the anterior region compared to the equator region and the posterior region in the same group of age. The average number of the silver grains in labelled nucleus lead to a decrease with the development of the retina after birth, but there was no significant regional differences found in the same group of age. The data shown from this study suggest that the cell differentiation in mouse retina proceed from posterior to anterior region.