1. Highly ‘kidney-specific’ antiserum-globulins prepared against a particular sub-fraction isolated from the microsomal fraction of adult chicken kidney were conjugated with fluorescein and used as a staining reagent to study the development of mesonephros in the chick embryos. Mesonephric cords from the midabdominal region at different developmental stages from stage 16 to stage 31 were studied in (a) paraffin sections, and (b) smeared preparations of the dissociated cells.

  2. In the differentiated mesonephros at stages 27−30 specific fluorescence of antibody staining appeared in the cells of proximal secretory tubules. First intense fluorescence in the development occurred, coinciding with the first histological organization of epithelium at stage 20−21. Weak specific fluorescence was also detected in the cells of the condensed nephrogenic mesenchyme.

  3. Throughout the development of the tubular epithelium fluorescence appeared in a particular locus of the cells, i.e. only the apical part. In the earlier stages perhaps prior to epithelial formation, a fluorescent dot was sometimes detected apparently inside the nucleus. Diffuse reaction over the cytoplasm has never been observed.

  4. The proportion of the fluorescent positive cells in the whole cell population of the mesonephric cords steeply increased from stage 21 (9 per cent.) to stage 27 (45 per cent.). By combining the antibody staining technique with autoradiography, it was revealed that the percentage of the cells incorporating 3H-thymidine in the fluorescent positive cells was about twice as high as that in the negative cells at stages 22 and 25. At the final stage of the development (stage 28) there was little difference in the percentage of the isotopically labelled cells between positive and negative cells.

  5. The present results indicate that the antibody staining technique can be a potent tool to study acquisition of tissue-specificity in nephrogenesis. It is suggested that the initial synthesis of the molecules with immunological kidney specificity occurs in the nuclei in the early stages of development; in the next step when the proliferation rate of the cells with such molecules becomes particularly high, the synthesis may occur only in the apical part of the cells.

In principle, it can be stated that initiation of histogenesis is preceded or accompanied by synthesis of molecular species, specific for cells belonging to a particular type of tissue. It has long been suggested that such molecular species, if present, must display immunological tissue-specificity, and an immunological approach to describe tissue-differentiation in embryogenesis at the molecular level is also not new (recent reviews by Tyler, 1955; Edds, 1958; Ebert, 1959; Flickinger, 1962). In fact, a number of important contributions have been made along this line, especially on the development of the antigenic constituents of muscle (e.g. Ebert, 1953; Ebert et al., 1955; Holtzer et al., 1957; Holtzer, 1961) and lens (recent works by Flickinger & Stone, 1960; Maisel & Langman, 1961,Takata et al., 1964). As for other tissue cells, however, our knowledge is still scanty, largely owing to the difficulty of characterizing and isolating tissuespecific antigens, even from differentiated adult tissues.

Recently, it has become possible to extract ‘kidney-specific’ antigens in a semipurified form from the microsomes of adult chicken (Okada, 1962; Okada & Sato, 1963a) and in the author’s earlier contribution, some preliminary immunodiffusion studies on the distribution of these antigens in the chick embryos at later stages of development were reported (Okada & Sato, 19636 ; also Okada & Yamamura on embryonic liver, 1964). In this paper the chronology of appearance of these antigens in the earlier development of mesonephros is studied by the fluorescent antibody technique, which the author had expected to be more sensitive and to permit detailed observations on the developing tissues (e.g. Clayton, 1960).

Material

Chick embryos ranging from stage 16 to stage 30 (Hamburger & Hamilton, 1951) were used. For observation of nephrogenesis, the mid-abdominal region of the mesonephric cord over 4−5 somites length was usually investigated.

Antiserum

See the author’s earlier publications (Okada & Sato, 1963a, 1963b>) for the procedures for extracting the sub-fraction called K45 which consists mainly of ‘kidney-specific’ antigens as shown by immunodiffusion tests. In short, the washed microsomes of the adult kidneys were dissolved with 0-5 per cent, sodium deoxycholate (DOC), ultra-centrifuged, and the proteins precipitated in a 20 to 45 per cent, saturation of ammonium sulphate were isolated from the DOC extracts. The sub-fraction K45 thus separated was injected into rabbits to prepare the antiserum (anti-K45).

Preparation of fluorescent antibody

A crude globulin fraction was isolated from the antiserum (anti-K45) by precipitation with ammonium sulphate at 45 per cent, saturation. Fluorescein isothiocyanate (FITC) was added (protein:dye ratio being 1:50) at pH 9·0 (0·05 M carbonate-bicarbonate buffer). Purification of the conjugated antibodies was done by the following procedures (Okada, 1964; also Nairn, 1962); (1) Removal of the free dyes by filtration with Sephadex G25, (2) treatment with acetone powders prepared from adult chicken livers, (3) treatment with acetone powders prepared from 16-day chicken embryos devoid of kidneys, (4) reprecipitation of the conjugate with ammonium sulphate at 45 per cent, saturation, and then (5) the precipitated conjugate was dissolved with buffered physiological saline and dialysed against it. For the control, crude globulins isolated from the pre-immunized rabbits sera were conjugated with FITC in exactly the same manner as described for anti-K45.

In order to remove unwanted minor cross-reacting antibodies, the anti-K45-FITC conjugate was absorbed with microsomes of lung and liver of adult chicken (each 6−8 mg. proteins/ml. of the conjugate solution). All the conjugates were centrifuged at 24,000 × g. for 60 min. to clarify and kept in deep freeze until use.

Immunohistology

Two types of preparations, paraffin sections and smeared preparations, were made. For preparing paraffin sections, small tissue blocks (ca. 3 mm.3) were dropped into an acetone-methanol mixture (8:2) at − 70°C., kept for about 2−3 hr. at − 20°C. with frequent changes of the mixture, and were put through xylol for 10 min. at room temperature, and then impregnated with paraffin having a low melting point (46°C.) for 15 min. Sections were made at 5 μ thickness, and deparaffinized by a short immersion in xylol. For smeared preparations a cell suspension obtained by brief trypsinization of the tissue block was mounted directly on glass slides. After drying, the slides were fixed with acetone-methanol mixture at − 20°C. for 30 min., dried in vacuo and washed.

The staining by the conjugated globulins lasted overnight under refrigeration. After washing repeatedly with buffered saline the slides were mounted with buffered glycerine. A Zeiss Osram HBO 200 high pressure mercury lamp was the light source for observation.

Autoradiography

Cells were isotopically labelled by dropping 5·0 μc. of thymidine-3H (spec, act 3·0 curies/mM.) in Tyrode solution (50 μc./ml.) on the vascularized yolk sac of the embryos at the desired stage. After 4 hr. of additional incubation paraffin sections of the isotopically labelled mesonephri were made in the same manner as described above. Fluorescent observations were made and were recorded photographically. The slides were then washed, refixed with Bouin, and then autoradiographs were obtained by standard procedures using FUJI Autoradiographic Stripping Film. After the development and staining with Harris’ haematoxylin, the same fields as taken for the fluorescence photomicrographs were observed to count the cell number with isotopically labelled nuclei in respect to fluorescent positive and negative cells.

Specificity of anti-K45-FITC

To discuss information obtained by immunohistological methods it is of utmost importance to establish that the staining was caused by specific immunological reaction, but not by non-specific adsorption of the conjugate, and that the immunological reaction was really against the desired molecular species. Assurances for these points were as follows :

(a) Immunological specificity of anti-K45-FITC studied in vitro

In the agar double-diffusion technique of Ouchterlony, anti-K45 conjugated with FITC (anti-K45-FITC) gave one heavy and one faint precipitin line against the homologous test antigens, i.e., the sub-fraction K45 and DOC-extract of kidney microsomes, but it was completely negative against the post-microsomal supernatants and ethylenediamine tetra-acetate extract of the microsomes of the kidney, and various preparations from livers and lungs. The immunoprecipitate produced by anti-K45-FITC always fluoresced. There was no loss of the precipitating antibodies after treatment of the conjugate with the DOC-insoluble fraction of the kidney microsomes. Thus, anti-K45-FITC really contained the fluorescent antibodies against the ‘kidney-specific’ antigens (abbreviation; ‘K-antigens’) which were the main components of the sub-fraction K45. No positive reaction in vitro occurred against the kidney preparations of mice and newts, Triturus pyrrhogaster.

(b) Control staining

Staining of the heterologous tissues such as liver, stomach, lung, intestine and spleen of the newly hatched chicks by anti-K45-FITC was negative in both paraffin sections and smeared preparations. The conjugate stained only kidney. Staining by the control conjugates was completely negative with all tissues including kidney. Microscopic preparations of chicken kidneys were pretreated with non-conjugated globulins of either anti-K45 or preimmunized rabbit sera, washed and stained with anti-K45-FITC. Complete inhibition of the staining occurred only after pretreatment with the non-conjugated anti-K45 globulins.

(c) Persistence of the antigenic specificity after fixation

Isolated kidney microsomes were treated with acetone-methanol mixture (8:2) in test tubes under the same conditions as applied to immunohistological preparations, thawed, briefly immersed in xylol, warmed at 45°C. for 20 min., and washed with saline. Some proteins were extracted at this step, but they did not react with anti-K45. However, when the residue was dissolved with DOC, the proteins showing reactions of identity with the ‘K-antigens’ in reaction with anti-K45 in agar were solubilized, though the reaction was somewhat weaker than in the use of the non-treated samples.

Observations on the fully-differentiated mesonephros

Anti-K45-FITC stained differentiated mesonephros (at stages 27−30) brightly. The result indicates a common distribution of the ‘K-antigens’ in both types of kidney, meta- and mesonephros, but the antibody staining occurred only in the cells of the proximal secretory tubules (Plate 1, Fig. A). Within these cells the staining was confined to the apical cytoplasm. Other cell parts remained negative, although in some preparations very faint fluorescence was detectable around the cell surface. Cells of the Wolffian duct and collecting ducts were negative, except for an inconsistent occurrence of very weak surface staining. Glomeruli, mesenchyme and endothelium were completely negative. In the smeared preparations of the dissociated cells obtained from the mid-abdominal region of the mesonephros, fluorescence occurred in the apical cap which corresponds to the luminal side in the intact tubules (Plate 2, Fig. G). The percentage of the positive cells (‘K-antigens’-containing cells, abbreviation ‘K-cells’) was, in an average of five separately prepared samples, 53 per cent, of the total cells at stage 30 and 51 per cent, at stage 27. The cells with inconsistent surface staining were scored as negative in the present study.

Observations of the developing mesonephros

At stages 24-25 the results were similar to those at stages 27-30, though fluorescence was less intense. In the dissociated cell preparations the fluorescent portion of individual cells is narrower than at stages 27-30 (Plate 2, Fig. H) ; the percentage of the ‘K-cells ‘in the total cell population was 29 per cent. (Text-fig. 1).

TEXT-FIG. 1.

Percentages of the positively fluorescent cells (‘K-cells’) to the total cells contained within the mid-abdominal region of the mesonephric cords at various developmental stages. Each number is an average of more than four differently prepared samples, in each of which counting was made of at least 400 cells. Vertical lines represent two standard errors.

TEXT-FIG. 1.

Percentages of the positively fluorescent cells (‘K-cells’) to the total cells contained within the mid-abdominal region of the mesonephric cords at various developmental stages. Each number is an average of more than four differently prepared samples, in each of which counting was made of at least 400 cells. Vertical lines represent two standard errors.

Stage 20 is an initial stage, when definitive fluorescent ‘K-cells’ were recognizable in the sectioned specimens (Plate 2, Fig. F, see also Fig. E.). At this stage an establishment of the epithelial arrangement has started from the condensation of nephrogenic mesenchyme in antero-posterior order. Fluorescence was observed along the inner border of these early epithelia. (Plate 2, Figs. E & F). Some cells of the condensation of the nephrogenic mesenchyme have a tiny spot with weak fluorescence (Plate 2, Fig. F). The Wolffian duct and its evaginations, the collecting ducts, were generally negative. But, in some preparations, very weak fluorescence occurred around the surface of the cells of the condensed mesenchymal cells, the Wolffian duct, the collecting ducts and the cartilaginous condensation of the limb bud. The cause of such a weak surface reaction will be discussed below. Pronephros was negative throughout its development, except for an inconsistent very feeble surface reaction. The percentage of the ‘K-cells’ in the total cell number of the mid-abdominal mesonephric cords at stage 21 was 9 per cent., when the counting was made using the smeared preparations of the dissociated cells (Text-fig. 1).

At stages 17-18 we failed to detect any definitive fluorescence in the paraffined sections, except for inconsistent surface staining around the cells of the Wolffian duct and the mesenchymal condensation of the nephrogenic cells. In the preparations of the dissociated cells, however, fluorescence occurred in 6 per cent. of the total cell populations of the mid-abdominal mesonephric cords (Text-fig. 1). The positive staining was usually seen as one tiny dot per single cell (Plate 2, Fig. I). In 33 per cent, of these positive cells, the fluorescent dot was detected not in the apical cytoplasm, but in the central portion of a cell, presumably in the nucleus (Plate 2, Figs. J & K ; see Text-fig. 2). In a limited number of the cells two dots, one in the peripheral part of the cytoplasm and the other apparently in the nucleus, were detected (Plate 2, Fig. K). There appears to be no diffuse localization of the fluorescence over cytoplasm.

TEXT-FIG. 2.

Percentages of cells having a nucleus with a fluorescent dot to the total positively fluorescent cells (‘K-cells’) found in the mid-abdominal region of the mesonephric cords at various developmental stages. Vertical lines represent two standard errors.

TEXT-FIG. 2.

Percentages of cells having a nucleus with a fluorescent dot to the total positively fluorescent cells (‘K-cells’) found in the mid-abdominal region of the mesonephric cords at various developmental stages. Vertical lines represent two standard errors.

At earlier stages than stage 17 no positive fluorescence was detectable in either the smeared preparations or the paraffin sections.

Proliferation of the ‘K-cells’

As shown in Text-fig. 1, the percentages of the ‘K-cells’ in the mid-abdominal region of the mesonephric cords were sharply increased between stage 21 and stage 27. A strict comparison of the percentages in different developmental stages cannot be permitted, since there is no proof to indicate that the region used for the observation of a given stage exactly corresponds to that for the other developmental stages. In spite of these limitations, there seems to be a consistent tendency to an enormous increase in the relative proportion of the ‘K-cells’ during a period of about 48 hr. Such a steep increase could be interpreted by two alternative assumptions; (1) a number of the negative cells constantly gain ‘K-antigens’ over this period, or (2) the proliferation rate of the ‘K-cells’ is higher than that of the negative ones. As an initial effort to answer the question, some preliminary observations were made to decide the percentages of the cells picking up 3H-thymidine, not in the whole cell population but in the ‘K-cells’ and negative cells of the developing mesonephros respectively. This was done by combining the antibody staining with autoradiography, and the results are summarized in Table 1. They indicate that the percentage of the isotopically labelled cells is much higher in the positive cells than in the negative cells at stage 22 and stage 25 (Plate 1, Figs. C & D). The numbers became approximately equal at the later stage of differentiation (Plate 1, Figs. A & B). However, the observation is yet far from enough to exclude the first assumption of the gain of ‘K-antigens’ by the negative cells.

TABLE 1.

Frequencies of the cells incorporating 3H-thymidine in three different tissue types. At least 800 cells were counted in respect to each type at a given developmental stage

Frequencies of the cells incorporating 3H-thymidine in three different tissue types. At least 800 cells were counted in respect to each type at a given developmental stage
Frequencies of the cells incorporating 3H-thymidine in three different tissue types. At least 800 cells were counted in respect to each type at a given developmental stage

Limitations of fluorescent antibody technique

Limitations of the technique were extensively discussed by Holtzer, especially in the case of application to the study of the cell differentiation (Holtzer, 1961 ; also Holtzer et al., 1957). Since the present work is the first contribution of this technique to the study of kidney development, it seems to be necessary to discsus the value of the antibody staining method in this particular case.

  1. Some observations proving that the fluorescent staining is due to the specific immunological reaction were given in the results. They indicate that the staining is due to the reaction between the ‘K-antigens’ contained in the sub-fraction K45 and their corresponding antibodies, but the possibility of the involvement of some other minor components in the staining cannot be excluded. Since in in vitro studies of the antibody pattern only precipitin tests were adopted, the presence of the trace antibodies against insoluble antigens may be undetected. We tentatively assume that inconsistent weak surface fluorescence detected in the cells of several tissues was due to such minor antibodies, while the major antibodies which were directed against ‘K-antigens’ caused the bright staining of the apical part of the cells of secretory tubules. This assumption seems likely, because concentrated anti-K45-FITC gave more consistent surface staining than was observed in the present study (see Okada, 1964, in which a twice concentrated anti-K45-FITC solution was used).

2. The sensitivity of the antibody staining is not yet quantitated. If an antigen is concentrated in a particular site of the cell, its presence can be easily visualized. On the other hand, a diffusely distributed antigen needs a much larger amount to permit successful detection. Besides, conditions for fixation and sectioning should be kept in mind. It could be speculated that at some stage of development the determinants may be coupled with the protein molecules in such a way that they can be easily extracted or destroyed during the course of preparing the microscopic preparations. Throughout the present observations, fluorescence always appeared as a dot or spot. The fact shows a concentrated localization of ‘K-antigens’ in a definte locus of the cell, but it does not exclude a possible distribution of the antigens in other parts.

Histogenesis of nephric tubules and ‘kidney-specific’ antigens

A number of contributions have appeared on kidney antigens in the field of pathology in relation to glomerulonephritis (Hill & Cruikshank, 1953; Mellors, 1955; Mellors et al., 1955a; Mellors et al., 1955b; Ortega & Mellors 1955). These antigens whose antibodies cause pathological lesions were immuno-histologically localized in the glomerulus basement membrane and are different from ‘K-antigens’ studied in this paper. In past works, ‘kidney-specific’ antigens studied by Weiler (1956a, 1956b, 1959) and Nairn et al. (1962) showed much similarity to the present ‘K-antigens’ in their histological distribution. These authors prepared their antibodies against the microsomal fractions, but no efforts were made towards further purification of the antigens. At any rate, such localizations of the different groups of antigens in the different parts of the kidney well expresses a molecular specialization of the tissue elements in kidney.

In early embryogenesis, the first sign of the appearance of ‘K-antigens’ is detectable in the closely packed cells of the nephrogenic mesenchyme, and their presence became demonstrable coinciding with an initiation of the epithelial architecture of the cell. Is the presence of ‘K-antigens’ a necessary prerequisite for the arrangement of the cells into the epithelial structure ? Is organization of the cells into epithelium a necessary condition for active synthesis or deposition of ‘K-antigens’? These questions which may arise from the present observations will be fully discussed in the author’s next papers which deal with observations on the kidney cells cultured in monolayer conditions (Okada, 1965, and in preparation).

Cellular differentiation of kidney cells

As discussed before, there are a number of difficulties due to the limitation of the antibody staining technique, in interpreting the observations on the intracellular distribution pattern of the antigens. However, such an invariable localization of ‘K-antigens’ in the apical part of ‘K-cells’ strongly suggests the occurrence of an intracellular specialization within the single cell. Some electron microscopical observations also support this view. They reveal that the apical part of the cell is well characterized by the presence of, besides an abundance of microvilli, an ample number of intracellular tubules which are often considered to be continuous with the surface membrane (Sjôstrand & Rhodin, 1953; Pease, 1955). Judging from the procedures of isolation as well as gross chemical analysis, our ‘K-antigens’ were assumed to be lipoproteins probably constituting the membranous elements of the cells (Okada & Sato, 1963). The possibility that the membranes of the intracellular tubules contain ‘K-antigens’ can be surmised. Therefore, localization of the ‘K-antigens’ in the apical part of the cells could be interpreted as an occurrence of differentiation of the molecular constituents of the membranous structures inside a single cell.

Such an apical staining was already observed from the very initial stage of the epithelium formation. Does this fact indicate that an initial synthesis of the antigens really starts at the particular locus in the cytoplasm? The present observations suggest that the initiation of the synthesis begins in the nuclei, since at the early stages of development there were some cells with a fluorescent spot apparently in the nuclei. So far, our experience has not yet been substantial enough to accept that the observed fluorescence is really due to the antigens or determinant groups localized inside the nucleus. But, if it were accepted as a fact, the following speculations, for example, would be proposed; the nuclei of presumptive nephrogenic cells will respond to an inductive effect so as to initiate the synthesis of molecules with immunological kidney-specificity, and the products may be deposited inside the nuclei for a whole. On the other hand, the inductive effect may cause a regional differentiation in the cytoplasm of the underlying cells so as to deposit the molecules to be transferred from the nuclei at the particular locus of the cells. At the next step of differentiation synthesis of the antigens perhaps occurs only in the cytoplasm, presumably at the apical part of the cell. Some published reports indicate that a synthesis of general proteins revealed by means of isotope incorporation is active only in nuclei at the early stage of embryonic development (e.g. Waddington & Sirlin, 1959; Brachet, 1960). As to the appearance of immunologically specialized molecules inside nuclei, the paper by Urso & Makinodan (1963) can be cited; these authors demonstrated by means of antibody staining that the appearance of the antibody molecules in the spleen cells of the immunized animals starts in the nuclei, presumably in the nucleoli ; in later stages of the antibody producton, the antibody molecules are seen only in the cytoplasm.

Développement d’antigènes rénaux spécifiques: étude immunohistologique

  1. Des globulines d’antisérum ‘spécifiques du rein’ ont été préparées à l’aide d’une sous-fraction particulaire isolée de la fraction microsomique de reins de poulets adultes. On les a conjuguées à la fluorescéine et utilisées comme réactif coloré pour étudier le développement du mésonéphros d’embryons de poulet. Des cordons mésonéphrétiques prélevés dans la région abdominale moyenne à différents stades du développement (du st. 16 au st. 31) ont été étudiés (a) sur coupes à la paraffine, et (b) sur frottis de cellules dissociées.

  2. Dans les mésonéphros différenciés, aux stades 27−30, la fluorescence spécifique de l’anticorps est apparue dans les cellules des tubules sécréteurs proximaux. Une première fluorescence intense coïncide avec la première organisation histologique de l’épithélium aux stades 20−21. Une faible fluorescence spécifique a aussi été décelée dans les cellules du mésenchyme néphrogène condensé.

  3. Tout au long du développement de l’épithélium tubulaire, la fluorescence est apparue en un point particulier des cellules, c’est-à-dire seulement la région apicale. Dans les stades plus jeunes, peut-être antérieurs à la formation de l’épithélium, une tache fluorescente a été parfois décelée, apparemment à l’intérieur du noyau. On n’a jamais observé de réaction diffuse au niceau du cytoplasme.

  4. La proportion de cellules fluorescentes dans la population cellulaire totale des cordons mésonéphrétiques s’est accrue fortement du stade 21 (9%) au stade 27 (45 %). En combinant la technique des anticorps fluorescents à l’autoradiographie on a trouvé que le pourcentage de cellules incorporant la thymidine-3H était environ deux fois plus élevé parmi les cellules positivement fluorescentes que parmi les cellules non fluorescentes, aux stades 22 et 25. Au stade final du développement (stade 28) le pourcentage de cellules marquées à l’isotope était peu différent dans les cellules fluorescentes et non fluorescentes.

  5. Ces résultats indiquent que la technique des anticorps fluorescents peut constituer un outil puissant pour étudier l’acquisition de la spécificité tissulaire dans la néphrogenèse. On suggère que la synthèse initiale de molécules à spécificité immunologique rénale a lieu à partir du noyau aux premiers stades du développement ; dans l’étape suivante, quand le taux de prolifération des cellules pourvues de telles molécules devient particulièrement élevé, la synthèse peut avoir lieu seulement dans la région apicale des cellules.

I am grateful to Professor C. H. Waddington and Dr. I. Campbell for reading through the manuscript before publication. My interest in the antibody staining technique was aroused by my former colleague, Mrs R. M. Clayton, to whom I am deeply indebted. I cordially thank Miss Hiroko Seki for her cheerful and devoted assistance throughout the course of the present work. I also thank Mrs E. Okada for preparing the manuscript. The work was partly supported by the Fund for Scientific Research of the Japan Ministry of Education to Professor T. Fujii.

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All fluorescence photomicrographs were taken under the same photographic conditions.

Plate 1

FIGS. A & B. Fluorescence photomicrograph (A) and autoradiograph (B) of one and the same field in a paraffin section of the mesonephros of stage-28 embryo. Apical parts of the cells of the secretory tubules (T1, T2, T3 and T4) are brightly fluorescent, whereas the collecting ducts (t1 and t2) are completely negative. Several nuclei incorporating 3H-thymidine are indicated by arrows, × 800.

FIGS. C & D. Fluorescence photomicrograph (C) and autoradiograph (D) of one and the same field in a paraffin section of the mesonephros of stage-22 embryo. Many nuclei of the fluorescent tubule, T, incorporated 3H-thymidine. × 800.

Plate 1

FIGS. A & B. Fluorescence photomicrograph (A) and autoradiograph (B) of one and the same field in a paraffin section of the mesonephros of stage-28 embryo. Apical parts of the cells of the secretory tubules (T1, T2, T3 and T4) are brightly fluorescent, whereas the collecting ducts (t1 and t2) are completely negative. Several nuclei incorporating 3H-thymidine are indicated by arrows, × 800.

FIGS. C & D. Fluorescence photomicrograph (C) and autoradiograph (D) of one and the same field in a paraffin section of the mesonephros of stage-22 embryo. Many nuclei of the fluorescent tubule, T, incorporated 3H-thymidine. × 800.

Plate 2

FIGS. E & F. Fluorescence photomicrographs of the paraffin sections of the mid-abdominal region of the mesonephric cords of stage 21 (E) and stage 20 (F) embryos, showing an initial stage of the intense fluorescence along the inner border of newly established epithelia. Some positive cells in the mesenchymal condensations are indicated by arrows. ( × 1200).

FIGS. G, H & 1. Fluorescence photomicrographs of the cells in smeared preparations of the dissociated cells of the mesonephric cords of stage 30 (G), stage 24 (H) and stage 18 (I) embryos. In Fig. G two ‘K-cells’ (K), in Fig. H one ‘K-cell’ and two negative cells (N) and in Fig. I two ‘K-cells’ are shown. (× 1400).

FIG. J. Fluorescence photomicrograph of a cell having a nucleus with a fluorescent dot in the smeared preparation of the mesonephric cords of stage 18 embryo. (× 1500).

FIG. K. Fluorescence photomicrograph of a small cell-cluster which contains three cells (n1 n2 and n3) having fluorescence in the nuclei and one cell (c) having fluorescence in the cytoplasm. Note n1 and n2 have fluorescent dots in the peripheral part of the cytoplasm (indicated by arrows) besides in the nuclei. (× 1500).

Plate 2

FIGS. E & F. Fluorescence photomicrographs of the paraffin sections of the mid-abdominal region of the mesonephric cords of stage 21 (E) and stage 20 (F) embryos, showing an initial stage of the intense fluorescence along the inner border of newly established epithelia. Some positive cells in the mesenchymal condensations are indicated by arrows. ( × 1200).

FIGS. G, H & 1. Fluorescence photomicrographs of the cells in smeared preparations of the dissociated cells of the mesonephric cords of stage 30 (G), stage 24 (H) and stage 18 (I) embryos. In Fig. G two ‘K-cells’ (K), in Fig. H one ‘K-cell’ and two negative cells (N) and in Fig. I two ‘K-cells’ are shown. (× 1400).

FIG. J. Fluorescence photomicrograph of a cell having a nucleus with a fluorescent dot in the smeared preparation of the mesonephric cords of stage 18 embryo. (× 1500).

FIG. K. Fluorescence photomicrograph of a small cell-cluster which contains three cells (n1 n2 and n3) having fluorescence in the nuclei and one cell (c) having fluorescence in the cytoplasm. Note n1 and n2 have fluorescent dots in the peripheral part of the cytoplasm (indicated by arrows) besides in the nuclei. (× 1500).