1. The differentiation of T. torosa somite mesoderm between stage 25 and 15 mm. has been examined with regard to variation in nuclear morphology, mitosis, and the development of intercellular matrix. Along with conventional techniques a fluorescent Schiff reagent has been used to obtain a fluorescent Feulgen for observation of DNA pattern, and a fluorescent PAS for observation of the appearance of intercellular mucopolysaccharide.

  2. The development of three kinds of nuclear morphology (DNA pattern) and of the PAS-positive intercellular material of the differentiating somite is described.

  3. The study indicates that nuclear changes and appearance of ground substance spread laterally from the always more differentiated medial portion of the somite and that mitotic activity is frequently observed in those more superficial areas containing cells with nuclei of the first-stage DNA pattern and is not (or is rarely) observed in those more medial areas containing cells With Other DNA patterns and in which the PAS-positive intercellular material is present.

  4. It is concluded that in the differentiating somite through stage 40 there exists a superficially located population of mitotically active, presumably undifferentiated, mesoderm cells.

In order better to evaluate results obtained in this laboratory concerning the responses of differentiating postneurula somite tissue to other mesoderm tissue placed in its immediate vicinity (Finnegan, unpublished), it was necessary to examine somite differentiation in situ. A qualitative examination of somite interphase nuclei of tail-bud and later stages was performed to note their morphological changes since it was assumed, as suggested by Briggs & King (1955), that such changes indicate cellular differentiation and, conversely, that absence of such changes indicates that the cells are not actively differentiating.

Because of the possible role of the intercellular matrix in histogenesis (see Grobstein, 1954, 1959; and Edds, 1958) a study was made of the development in the somite of that portion of the intercellular matrix which is demonstrable histochemically with the periodic acid-Schiff (PAS) technique. The visual clarity of the results has been materially aided by the fluorescent Schiff reagent of Culling & Vassar (1961) which makes possible a fluorescent Feulgen and a fluorescent PAS reaction.

Taricha torosa embryos from stage 25 up to 15 mm. in length (Twitty & Bodenstein stages; see Rugh, 1948) were fixed in Carnoy’s acetic-alcohol mixture under standard conditions of temperature and time, paraffin processed, and sectioned at 10μ. Sections were dried at 37° C. to avoid nucleic acid changes known to occur at high temperatures (Pearse, 1960). The acetic acid in this fixative precipitates nucleic acid (DNA) within the nuclear membrane. Despite the fact that this may produce a chromatin pattern artificial in appearance as compared to the living condition (Baker, 1950, 1958), a constant difference of results, produced by identical technical procedures, may be taken as indicative of some underlying phenomenon within the tested material (the ‘good artifact’ of Gersh, 1959).

Throughout the developmental stages examined the somite is considered as the block of mesoderm tissue lying lateral to the neural tube and notochord. The mesenchyme which appears in the sectioned material both medial and lateral to this block following stage 30 is not included.

The fluorescent Feulgen reaction was as described and discussed by Culling & Vassar (1961). A short acid hydrolysis (10 min.) was used to give the maximum staining response with the fixative (see Pearse, 1960). The intercellular material was demonstrated with the periodic acid-Schiff (PAS) reaction in which the fluorescent and conventional Schiff reagents were applied to the sections after a 10-minute exposure to the periodic acid and the sulphite rinses of the McManus procedure. Sections similarly stained after the Hotchkiss procedure for the conventional PAS technique, in which an acid-reducing rinse before exposure to periodic acid is substituted for the sulphite rinses, gave results identical with the above.

Both the Feulgen and the PAS reactions were controlled by (1) demonstrating the absence of preformed aldehydes (the omission of hydrochloric or periodic acid producing a negative response with the Schiff reagent); and (2) demonstrating that the aldehyde groups formed with the appropriate acid treatment could be blocked chemically from reacting with the Schiff reagent (acetylation gave a negative response in both reactions in the somite material). It appears, therefore, that the results obtained with these two histochemical reactions were due to the presence of an aldehyde produced by the action of the periodic or the hydrochloric acid rather than to any pre-existing aldehyde or reducing lipid.

Control of the Feulgen reaction was by treatment of sections with deoxyribonuclease and with perchloric acid prior to application of the Feulgen procedure; both of these pretreatments produced negative Feulgen reactions. Since a positive Feulgen reaction within nuclei is now generally accepted to be specific for DNA (Pollister & Ornstein, 1959; Pearse, 1960), the Feulgen-positive material observed in the sections will be so discussed in this report.

The specific determination of the PAS-positive intercellular material of the somites was attempted (1) by treatment with diastase to remove simple polysaccharides from the sections (the fluorescent PAS material remained); (2) by treatment with alcian blue, aldehyde fuchsin, and toluidine blue to demonstrate the presence of acid or sulphated mucopolysaccharides (all these agents gave negative results in the intercellular material under investigation); and (3) by staining with Sudan black B to locate any phospho- or glycolipids not previously removed by the fixing procedure (all sections tested gave negative results).

In view of the elimination of the presence of the potentially PAS-positive materials indicated by these tests it is assumed that the PAS-positive material described in this report is most probably neutral mucopolysaccharide.

The fluorescent stained sections were observed and photographed with the Zeiss Large Fluorescence equipment as described by Vassar & Culling (1959).

Somite nuclear morphology

Stage 25-27 group

All somite nuclei in these embryonic stages demonstrated Feulgen-positive material (DNA) as fluorescent aggregates, rather evenly spread within the nuclear membrane; but those nuclei in cells of the medial area of the somite adjacent to the notochord-ventral neural tube region contained a coarser chromatin pattern and produced a less intense fluorescence than the rest (Plate, fig. F; see also Text-fig. 2). The more intense fluorescence in the lateral and middle somite nuclei was produced by a large number of individually small DNA aggregates within the nuclear membrane, to be referred to henceforth as the first-stage chromatin pattern (Text-fig. 1), while the more medial nuclei demonstrated a reduced number of individually larger DNA aggregates, the second-stage chromatin pattern (Text-fig. 1). Mitotic figures were encountered in the lateral and middle regions of the somite where the first-stage chromatin pattern was present in these developmental stages (Text-fig. 2) and may be observed in these areas in routine haematoxylin and eosin sections. Such figures are not encountered in the medial somite area where the second-stage chromatin pattern is observed (in Text-fig. 2, region of numeral II).

Text-fig. 1.

The three varieties of chromatin (DNA) pattern observed in somite nuclei. For explanation see text. (Figures traced from photographs. Approx. ×500.)

Text-fig. 1.

The three varieties of chromatin (DNA) pattern observed in somite nuclei. For explanation see text. (Figures traced from photographs. Approx. ×500.)

Text-fig. 2.

Outline of a section through an anterior trunk somite of a stage 27 T. torosa embryo. The numeral I indicates the location of the first-stage DNA pattern nuclei and the numeral II indicates the approximate location of second-stage nuclei in the somite. (Traced from a projection.)

Text-fig. 2.

Outline of a section through an anterior trunk somite of a stage 27 T. torosa embryo. The numeral I indicates the location of the first-stage DNA pattern nuclei and the numeral II indicates the approximate location of second-stage nuclei in the somite. (Traced from a projection.)

Stage 28-30 group

The second-stage DNA pattern became visible in nuclei more laterally placed in the somite (Text-fig. 3), and in the oldest stages the most medial somite nuclei initiated a chromatin pattern in which the major portion of the DNA appeared to be coalesced near the nuclear membrane and the nucleoli. The nuclei with first-stage DNA pattern were present in the superficial areas of the somite; that is, in the dorsal tip, the lateral face, and the ventral tip regions (Text-fig. 3, numeral I). Again, mitotic figures were found only in these areas with the first-stage chromatin pattern (Plate, fig. G) as also in haematoxylin and eosin sections. They were not encountered in other areas of the somite (Text-fig. 3, numeral II). The older animals in this group respond with a partial flexion of the trunk to strong stimulation with a hair loop.

Text-fig. 3.

Outline of a section through an anterior trunk somite of a stage 30 T. torosa embryo. Numerals I and II as in Text-fig. 2. Numeral III indicates the approximate location of the third-stage (mature) DNA pattern nuclei. (Traced from a projection.)

Text-fig. 3.

Outline of a section through an anterior trunk somite of a stage 30 T. torosa embryo. Numerals I and II as in Text-fig. 2. Numeral III indicates the approximate location of the third-stage (mature) DNA pattern nuclei. (Traced from a projection.)

Stage 33-36 group

The further coalescing of the Feulgen-positive material near the nuclear membrane and around the two nucleoli characteristic of this species (Costello & Henley, 1949) became more evident in the median somite nuclei of this developmental group. The coarse DNA aggregates of the second-stage pattern were no longer visible in these nuclei so that the nucleoli appeared suspended within the fluorescence-outlined nuclear membrane, the third-stage or mature chromatin pattern (Text-fig. 1). This condensation of the chromatin on the nucleoli and nuclear membrane has been noted in differentiating amphibian myoblasts by Sirlin & Elsdale (1959) and animals in this group independently perform C- and S-flexures. At this time only the most superficial layer of lateral-face nuclei and small groups of nuclei in the ventral and in the dorsal tip regions still showed the first-stage DNA pattern, while throughout the remaining portion of the somite nuclei with the second-stage DNA pattern were visible (Text-fig. 4).

Text-fig. 4.

Outline of a section through an anterior trunk somite of a Stage 36 T. torosa embryo. Numerals I, II, and III as in Text-fig. 3. (Traced from a projection.)

Text-fig. 4.

Outline of a section through an anterior trunk somite of a Stage 36 T. torosa embryo. Numerals I, II, and III as in Text-fig. 3. (Traced from a projection.)

Stage 40 group

The second-stage DNA pattern now appeared in the nuclei of the lateral face and the first-stage pattern remained only in the nuclei of the superficial dorsal and ventral tip area, the number of such nuclei (maximum of six per section) being greater in the ventral tip than in the dorsal tip. Elsewhere in the somite the third-stage DNA pattern was present. It would seem, then, that in the torosa somite at stage 40 there exist two cell populations (growth centres) in which the first-stage or mitotically active DNA pattern is present, a small dorsal-tip group, and a larger ventral-tip group (Text-fig. 5). It is in these two areas that the occasional mitotic figure observed in somite tissue of these older animals is to be found.

Text-fig. 5.

Outline of a section through an anterior trunk somite of a stage 40 T. torosa embryo. Numerals I, II, and III as in Text-fig. 3. The dotted area overlying each of the dorsal somite tips represents the grouped melanophores which produce the dorsal band pigment pattern typical of this species. (Traced from a projection.)

Text-fig. 5.

Outline of a section through an anterior trunk somite of a stage 40 T. torosa embryo. Numerals I, II, and III as in Text-fig. 3. The dotted area overlying each of the dorsal somite tips represents the grouped melanophores which produce the dorsal band pigment pattern typical of this species. (Traced from a projection.)

14 mm. larva group

The third-stage DNA pattern was present in nearly all somite nuclei at this time. The first-stage DNA pattern was no longer visible and the second-stage pattern appeared in a small group of nuclei (less than six per section) in the ventral tip. In the area immediately medial to the dorsal somite tip a small group of nuclei with the second-stage DNA pattern were present. This group may constitute a growth centre in T. torosa similar to the dorsal growth centre described by Holtzer & Detwiler (1953) for somites of Ambystoma punctatum though it must be noted that, as in the earlier developmental stages, mitotic figures were not observed in these regions occupied by cells with the second-stage chromatin pattern present.

Somite intercellular material

Stage 25-27 group

The intercellular PAS-positive material was discernible as a discontinuous granular system throughout the major portion of the somite. Adjacent to the notochord and neural tube the PAS-positive material was present as a continuous entity which appeared to be physically continuous with the PAS-positive intercellular substance in the medial area of the somite. In some cases the reactive intercellular material of the medial area seems to be aligned with the intercellular material of the neural tube (Plate, fig. D). During these stages of development the superficial regions of the somite were free of, or contained small amounts of, the discontinuous granular PAS-positive intercellular material (Plate, fig. A).

Stage 28–30 group

The continuous PAS-positive substance now extended further peripherally from the medial area, the discontinuous granular material was evident in the dorsal tip and lateral face areas, and only the superficial ventral tip of the somite appeared to be free of the PAS-positive material.

Stage 33–36 group

The PAS-positive material became continuous throughout the somite during these stages except for the lateral face, the dorsal tip, and the ventral tip areas where the discontinuous granular phase was found (Plate, fig. C).

Stage 40 group

The now thicker continuous PAS-positive material covered the entire somite from the medial border to the lateral surface and appeared in the ventral and dorsal tip areas. However, the discontinuous granular stage constituted most of the PAS-positive substance present in the somite tip areas and the lateral surface region (Plate, fig. E).

14-mm. larva group

The entire somite section now demonstrated the presence of the continuous PAS-positive material. Accompanying it in the dorsal tip and ventral tip areas was a large amount of the discontinuous granular PAS-positive substance.

Somite reticulum

The basement membrane is considered to consist of a meshwork of argyrophilic reticular fibrils plus a homogeneous component or ground substance (Edds, 1958; Selby, 1959), the PAS-positive material being associated with the homogeneous ground substance. The appearance of this reticulum in the somite was examined with Gomori’s silver impregnation method.

While argyrophilic fibres were demonstrable in the areas immediately external to the somite prior to stage 35 they did not appear intrasomitically until around stage 40, at which stage they were found only in the more medial portion and seemed to be continuous with the extrasomitic fibres of the axial area. Between stages 40 and 14 mm. reticular fibres became demonstrable throughout the somite, though no argyrophilic material was found in the dorsal tip or in a small area in the ventral tip.

It would appear therefore that the reticular fibre component of the somite intercellular matrix is not demonstrable until some time after the thick continuous PAS-positive material is present between the somite cells. The fibres were initially demonstrable in the same medial region of the somite as, earlier in development, were the initial nuclear changes and the initial appearance of continuous PAS-positive material. Further, it would seem that the intercellular reticulum spreads laterally across the somite as did the changes in nuclear morphology and the PAS-positive intercellular material. At the oldest stages studied in this investigation (15 mm.) both the homogeneous ground substance and the reticular material were present in the intercellular spaces of the somite though it may be that, as suggested by Edds (1958), the ground substance is gradually replaced by the reticulum.

The results of the present investigation indicate that, as the T. torosa somite differentiates in situ following stage 25, there is present a population of mitotically active, presumably less differentiated, cells residing in the more superficial region of the somite, since up to stage 36 the entire superficial somite area demonstrates the presence of mitotic figures and the first-stage DNA pattern in the nuclei and the discontinuous PAS-positive material in the intercellular area (see Text-figs. 2-4). This population becomes more restricted spatially as the second- and third-stage DNA patterns with their apparent absence of mitotic figures and the continuous PAS-positive material spread peripherally until, at stage 40, there remain two small groups of cells in which the first-stage DNA pattern and the discontinuous PAS-positive material are present, in the dorsal tip and in the ventral tip of the somite (Text-fig. 5).

As measured by the techniques used, the medial area of the somite adjacent to the ventral neural tube-notochord area is further differentiated at any of the developmental stages examined than is the more superficial area, and phenomena associated with somite histogenesis (namely, nuclear chromatin pattern changes and the appearance of intercellular matrix) are initiated in this medial area of the somite appearing later further peripherally.

It has been suggested by Muchmore (1957, 1958) that neural, chordal, and other adjacent tissues influence myogenesis by confining somite tissue in a mass. Possibly this activity of neural and notochord tissue could take the form of initiating the production of somite intercellular ground substance, thereby producing a tissue mass within which histogenesis may occur. A further possible role of the PAS-positive material is indicated by the work of Heilbrunn (1952) and Heilbrunn et al. (1954) in which heparin-like substances appeared to have an inhibitory effect on cell-division. Thus, as the PAS-positive material appears further laterad in the somite it may be inhibiting mitotic activity, thereby assisting differentiation. Finally, as demonstrated by Grobstein & Holtzer (1955; see also Holtzer, 1959) in cartilage production by mouse somite tissue, material of this axial intercellular matrix may be operative in an inductive manner.

Étude des noyaux et de la substance fondamentale intercellulaire au cours de la différenciation des somites in situ chez le Triton Taricha torosa

  1. La différenciation du mésoderme somitique de Taricha torosa, entre le stade 25 et le stade 15 mm., a été étudiée sous le rapport des variations morphologiques des noyaux, de la mitose et de la formation de la matrice intercellulaire. Outre les techniques usuelles, on a utilisé un réactif de Schiff fluorescent afin d’obtenir une coloration de Feulgen fluorescente, pour observer la répartition de l’ADN, et un PAS fluorescent, pour observer l’apparition des mucopolysaccharides intercellulaires.

  2. On décrit le développement de 3 types de morphologie nucléaire (répartition de l’ADN) et du matériel intercellulaire PAS-positif, dans le somite en cours de différenciation.

  3. Ces recherches indiquent que les modifications nucléaires, et l’apparition de la substance fondamentale, s’étendent latéralement à partir de la partie médiane du somite, toujours plus différenciée; une activité mitotique s’observe fréquemment dans les zones plus superficielles, contenant des cellules à noyaux présentant la répartition d’ADN caractéristique du premier stade, mais ne s’observe pas (ou seulement rarement) dans les zones plus centrales contenant des cellules dont les structures nucléaires sont différentes et dans lesquelles se trouve le matériel intercellulaire PAS-positif.

  4. On conclut qu’il existe, dans le somite en différenciation jusqu’au stade 40, une population de cellules mésodermiques localisées superficiellement, à mitoses actives, et probablement indifférenciées.

1 wish to acknowledge with gratitude the assistance of Mr. Charles Culling, of the Department of Pathology, Faculty of Medicine, University of British Columbia. An expression of appreciation is also extended to Professor H. E. Taylor, Head of the Department of Pathology, for his permission to use the fluorescence equipment of that department. This research was supported in part by a grant (R-T-6178) from the U.S. National Institutes of Health.

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Plate 1

Fluorescent-Feulgen nuclei and Fluorescent-PAS-positive intercellular material during somite differentiation.

Fig. A. Stage 27. Section through an anterior trunk somite. The PAS-positive material of the neural tube (NT) and that of the medial face of the somite appear to be continuous in the area immediately dorsal to the notochord (NO). The discontinuous granular intercellular material is visible within the somite. Approx. ×500.

Fig. B. Stage 28. The discontinuous granular PAS-positive intercellular material is apparent only-in some regions of the lateral face of the somite. The overlying ectoderm (EC) is to the upper left. Compare with fig. E. Approx. × 500.

Fig. C. Stage 36. A section through an anterior trunk somite. The continuous PAS-positive material,, now apparent more laterad than at earlier stages, illustrates its appearance in older somites. The superficial lateral area, the dorsal tip, and the ventral area of the somite show the discontinuous-granular PAS-positive stage in the original sections. Approx. × 100.

Fig. D. Stage 27. A section through an anterior trunk somite. The PAS-positive material of the neural tube (NT) appears aligned with that of the medial part of the somite. The two tissues can be observed as separate entities in the original sections. Approx. ×120.

Fig. E. Stage 40. The discontinuous granular PAS-positive material is now visible along the entire lateral face of the somite. The somite cells (to the right) show the intracellular glycogen displacement characteristic of fixed tissues and the ectodermal epithelium (to the left) demonstrates a thick basement membrane internally. Approx. ×500.

Fig. F. Stage 25. A section through an anterior trunk somite with the medial face to the left side and the dorsal tip at the top of the photograph. The nuclei in the medial area (near notochord-ventral neural tube) are less fluorescent (second-stage DNA pattern) than are the more superficial nuclei (first-stage DNA pattern). Approx. ×120.

Fig. G. Stage 28. A section through an anterior trunk somite with the medial face to the right side and the dorsal tip uppermost in the photograph. The second-stage DNA pattern nuclei in the medial area of the somite are less fluorescent than are the first-stage DNA pattern nuclei in the superficial (to the left) region of the somite. Note the mitotic figures in the areas of first-stage DNA pattern nuclei. Approx. ×120.

Plate 1

Fluorescent-Feulgen nuclei and Fluorescent-PAS-positive intercellular material during somite differentiation.

Fig. A. Stage 27. Section through an anterior trunk somite. The PAS-positive material of the neural tube (NT) and that of the medial face of the somite appear to be continuous in the area immediately dorsal to the notochord (NO). The discontinuous granular intercellular material is visible within the somite. Approx. ×500.

Fig. B. Stage 28. The discontinuous granular PAS-positive intercellular material is apparent only-in some regions of the lateral face of the somite. The overlying ectoderm (EC) is to the upper left. Compare with fig. E. Approx. × 500.

Fig. C. Stage 36. A section through an anterior trunk somite. The continuous PAS-positive material,, now apparent more laterad than at earlier stages, illustrates its appearance in older somites. The superficial lateral area, the dorsal tip, and the ventral area of the somite show the discontinuous-granular PAS-positive stage in the original sections. Approx. × 100.

Fig. D. Stage 27. A section through an anterior trunk somite. The PAS-positive material of the neural tube (NT) appears aligned with that of the medial part of the somite. The two tissues can be observed as separate entities in the original sections. Approx. ×120.

Fig. E. Stage 40. The discontinuous granular PAS-positive material is now visible along the entire lateral face of the somite. The somite cells (to the right) show the intracellular glycogen displacement characteristic of fixed tissues and the ectodermal epithelium (to the left) demonstrates a thick basement membrane internally. Approx. ×500.

Fig. F. Stage 25. A section through an anterior trunk somite with the medial face to the left side and the dorsal tip at the top of the photograph. The nuclei in the medial area (near notochord-ventral neural tube) are less fluorescent (second-stage DNA pattern) than are the more superficial nuclei (first-stage DNA pattern). Approx. ×120.

Fig. G. Stage 28. A section through an anterior trunk somite with the medial face to the right side and the dorsal tip uppermost in the photograph. The second-stage DNA pattern nuclei in the medial area of the somite are less fluorescent than are the first-stage DNA pattern nuclei in the superficial (to the left) region of the somite. Note the mitotic figures in the areas of first-stage DNA pattern nuclei. Approx. ×120.