Study of lens antigens in the developing newt lens with immunofluorescence

Lens cmfigen in lens development third alternative is blocking 01 the normal dividing cycle of the cells by the tissue applied; release would cause ostensible stimulation. This interpretation presupposes specific blocking of the cells of the liver pnrenchyma and there is no evidence to support it. However, studies to elucidate the point are in progress. Our present view is based on the assumption that factors which stimulate specifically the growth of the same tissue arc originated and released during autolysis. The maintenance of normal balance also in labile or constantly regenerating tissues may be explained according to the same principle: substances which stimuIaLe the proliferation of the same cell type in the manner of a feed-back mechanism are originated in connection with the cell destruction during the physiological elimination. Summary. -hutolytic liver tissue injected into the peritoneal cavity of the mouse after 70 per cent hepatectomy stimulated specifically the DNA synthesis 01 the hepatic parenchymal cells. No stimulation was demonstrated in the mesenchymal cells of the liver and in the cells of the renal tubules, gastric mucosa or epidermis. A

REFERENCES 1.

.J. I%., Proc. Nat!. Acad. Sci. I,‘..$. 40, 335 (1951). i\., Aclu &zfhof. ;%!!icrobiot. Stand. suppl. 150 (1961). MCJC‘WKIS, A. F. and BIUXH~~;S, I-I. C., llrclr. Palhol. 12, 900 (1931). PASCIIKIS, I<. E., Cmeer Res. 18, 981 (1958). PASCIIKIS, K. I-l., CANT~ROW, A. and GOUIMRD, J. W., Fed. Proc. 16, 98 (1957). SAE.rRES, I-I., l?xpt1 Cell Res. t1, 229 (1936). STICII, II. 1’. and FLORIAN, 31, I.., CUR. J. Riochem. Physiol. 36, 8% (1958). TEIH, H., Sot. Sci. Fennica, Commenfafiones Iliol. 13, 1 (1951). ~ Verhnndl. Deuf. Ges. Polhol., 45, 150 (1961). TEIR, 1-I. and L.N*IWARJIJ, A., Expll Cell Res. 24, 424 (19til). TF.IH, H. and I3av~s12, K., Espfl Cell Res. 5, 500 (1953). TIXI~, H. and HYT~HAA, ‘I’., IXLh Con@. of the European Sot. Haemat., Lisboa, 8, 220 (1960). T~~IANIS~VII.~, G. D., .J. J;mbr!Jol. I:‘@ Morpho[. W:Erss, I’., Science 115, 487 (1952). \VII.SON, J. \V. and LI:I.~L.c, I-I. II., Anat. Record 97, 471 (1945). EXERT,

2.

~.~HTII?~I~.~L~,

3. 4. 5. 6. 5. 8. 9. 10. 11. 12. 13. 14. 13.

STUDY

OF LEI\JS ANTIGENS WITH C. T.4KATA,

Riology

Division,

IN THE

DEVELOPING

NEWT

1963.

LEXS

IMMUNOFLUORESCENCE J. E’. ALBHIGHT

Oak Ridge

National Received

and T. YAMADA

Laboratory,’ January

Oak

Ridge,

Tenn.,

U.S.A.

7, 1964

1~ has been reported that lens antigens of the adult frog first appear at a developmental stage when the lens placode is transformed into the lens vesicle [la]. Comparable findings arc available for the chick embryo [7, 8, 131. On the other hand, it _- _....__-1 Operated

by Union

Carbide

Corporation

for the I;nitetl

States

Atomic Experimeniui

Energy

Commission.

Ceil Reseurch

34

C. Takafa, J. F. Albright and T. Yamada

Figs. 1 A.-Scclions through the eye region of T. p,qrrhogns/er at different clcvelopmcntal slages stained with the fluorescein-labeled gamma globulin fraction. Weakly bright patches scattered in non-lens regions do not rq)resent itrimunofluorescencc. This was concludrd by considering their color, observations made with different filter combinations, and data obtained with the fluoresccinlabeled normal gamma globulin fraction. x 115. Fig. l.-Section of the lens placode of a stage 27 embryo. The inimunofloorcsc:er11 st,ain is absent in the whole arca. Fig. 2.-- Section through the early lens vesicle still connected with Lhc c&derm of a stage 32 embryo. Note positive immunofluorescencc in the inner layer of the lens vesicle. Fig. 3.- The lens rudiment at stage 37. Strong irnmunolluorescence in the primary lens fiber cells. Fig. 4.- The lens rudiment at stage 38. Strong inlnlrullofluorcscence of the lens fiber cells. The cytoplasm of lens epithclium cells also indicates irnmunofluorescencc.

has been claimed lhat adult lens antigens are present in areas other than the Icnsforming area of the embryo [I- 6,8j. On Lhc basis of the latler observalions and relaled experiments, it has been suggcstecl that the presence of lens antigens or related substances in cmbrxonic areas is connected with Lhc lens-inducing capacily of lhe corresponding embr\;onic areas 11 A]. As an approach to Lhis complex of problems,

Lens antigen

in lens developmenf

209

the time of appearance and localization of lens anligens in the developing embryo of the newt, Trifurus pyrrhognstm, were studied hy an immunofluorescent technique. The gamma globulin fraction separated from the strum of a rabbit immunized with adult Triturus uiridesccns lens homogenate was conjugated with fluorescein isothiocyanate 191. Serial sections of 3 ,U to 5,~ thickness through 7’. pyrrhvguster embryos were prepared and stained with fluorescent globulin according to the method of Sainle-Marie Ill]. Control experiments were done with fluorescent globulin of the normal rabbit serum. The slides were observed under a large Zeiss fluorescence microscope with a high mercury burner 1lHO-200, using exciter filters, l3G 12, I:G .5, and I!G 1, coupled wilh the barrier filter 47. To make sure that Lhe antiserum against 1’. oiridescens lens is able to react with T. pyrrhogasler lens antigens, agar diffusion tests mere conducted, in which antiserum againsl 2’. viridescens lens was tested against the adull lens homogenate of 1’. uiridescens and 3’. pyrrhogasler. The patterns of lens antigens of two specieswere very similar. The data were interpreted to justify using anti-viridescens lens serum to sludy T. pyrrhognster lens antigens. The lens placode of early Lail-bud embryos (stages 27 and 28, according to Okada and Ichikawa [lo]) did not show fluorescence following Lrcatment with fluoresccnl antibody (Fig. 1). In the later tail-bud stage (stage,32), when the lens placode was transforming into the lens vesicle, immunofluorcscencc was found localized in the cytoplasm of the cells in the inner layer of the lens vesicle, facing the optic cup (Fig. 2). These cells with positive reaction correspond to the primary lens fiber cells. The immunofluorescence staining increased its inlensity with the development of definitive lens Cihcr cells (stage 37-38, Figs. 3, 4). In earlier slages lens epithelium cells were negative (Figs. 2, 3). However, in later stages they indicated immunofluorescence in their cytoplasm (Fig. 4). In all stages examined, the optic cup and non-lens ectoderm were negative. In control seclions treated with fluorescent globulin of normal serum no fluorescence was ohserved. The present results demonstrate that organ-specific lens antigens appear in the cytoplasm of primary lens fiber cells when the lens placode is transformed into the lens vesicle and later increase in amount with tliffercntiation of lens fibers. In the lens epithelium, lens antigens become detectable later, when its regular epithelial arrangement has been attained. The stage of first appearance of lens anligens dcmonstrated in the present experimenl approximately coincides with that determined by Ten Gate and Yan Doorcnmaalen [la] with the precipitin technique in the frog embryo. Our results suggest that occurrence of lens antigens deteclahle with our method is correlated with dilfercntiative events of lens-forming ectoderrn cells, but not with lens-inducing activities of Lhe optic cup or with lens potency of the cctoderm cells. REFERENCES

Y. Yasuda and l’. IIirai J., J. Emhryol. ISxptl Morphol. 7, 193 (1959). H. and IANG~~?\s, .J., J. Emtqol. Expfl Morphol. 9, 191 (1961). 9. .J. J.)., ~ZVELASD, W. C., and SMITH, (1. W., Proc. Sot. Hxptl Mol. Med. 98, 898 (195X). Morphol. (Tokyo) 3, 1 (1947). 10. OKADA, Y. K. and ICIIJKAW~, M., .7. Exptl 11. SAJNTE-!&4RIE, (;., J. Ilistochem. Cytochem. 10, 250 (1962). 12. 'I& CATE, G. and VAX DOOIWSMAALEN, W’. J., Proc. Koninkl. Xed. Akad. Wetenschap. Ser. C 53, X49 (19.X). 13. WOEJ~~.MAX, RI. W., Proc. Koninkl. Xed. nkud. Wetenschnp. Ser. C 65, 145 (1961). 7. 8.

LANDMAN, XAISEL, ~IARSHALL,

INCORPORATION

OF 3H-URACIL

INFECTED

WITH Y.

Plant

Pathology

Laboratory,

YASUDA

Faculty

INTO TOBACCO and

of Agriculture,

Received

January

T.

TOBACCO

LEAF

MOSAIC

VIRUS1

EPIDERMIS

HIRAI Nagoya

University,

Anjo,

Aichi,

Japan

10, 1964

ALTHOUGH intracellular

sites of viral RNA synthesis have been extensively studied, no definite conclusion has been reached. Rald and Solberg [l] and Cadman [3] suggested that the multiplication of tobacco mosaic virus (TMV) and tobacco rattle virus, respectively, has an intimate association with host cell nuclei. ltecently, autoradiographic techniques have been much advanced and a number of evidences on nucleic acid synthesis in plant tissues have been accumulated, especially by using tritium compounds 14, 5, 71. We examined the incorporation of 3H-uracil into TMVinfected tobacco leaf epidermis by means of microautoradiography. I,eaves of tobacco plants (Nicoliana tabacum, “Hright Yellow”), 15.-20 cm long, were detached and cut along the midrib. The upper surface of one half was inoculated with TMV, while the other half served as control. After having been washed with water, both leaves wcrc incubated separately on water for 20 hr in a Petri dish incubated at 25% under continuous illumination from fluorescent lamps, in order to eliminate the physiological disturbance of leaves owing to detachment and to facilitate virus movement to the lower surface of leaves (opposite to inoculated surface) which served for further observations. Then each lower surface of 15 leaf-pieces (2 x 2 cm, lotal fresh weight ca. 3 g), taken away from the half-leaves, was floated on an isotope solution containing 100 ,UC of 311-5,6-uracil (The Radiochemical Centrc, Amersham, England, specific activity 0.87 c/m&f). After definite periods the leaf-pieces were washed with running water and the lower epidermis was peeled away and fixed with Nnvashin solution. hutoradiographs of the epidermis were prepared by the stripping method [2] using Fuji autoradiographic plates (ET-2E type). After 2 weeks of exposure the emulsion was developed using Fuji Rendol. The number of blackened silver grains was counted under a phase microscope. ln a preliminary cxperimcnt using 321’-orLhophosphate, it was found that the 1 Contribution Experimental

No.

77, Plant

Cell Heseorch

34

Pathology

I.aboratory,

Nagoya

1:niversity.