The Development of the Ultimobranchial Body in Xenopus laevis Daudin and its Relation to the Thyroid Gland and Epithelial Bodies

The ultimobranchial body is a derivate of the pharyngeal cavity which has been found in all vertebrates studied at least at some stage of development. Its origin, development, and structure has been studied in many species but nevertheless nothing certain is known about its significance and activity. Earlier authors have used several terms for this organ, e.g. accessory thyroid gland, lateral thyroid, corpus Y, suprapericardial body, telobranchial body, and postbranchial body. The term mostly used at present, ultimobranchial body, was suggested by Greil (1904).

It is not possible to give a consistent picture of the formation of the ultimobranchial body, since it is evidently different in different vertebrates. In fishes it is formed from the sixth rudimentary visceral pouch (van Bemmelen, 1889); in amphibians Maurer (1888) regards it as being formed from the ventral wall of the pharyngeal cavity, while Greil (1904) derives it from the sixth visceral pouch. In reptiles it develops as a common bud together with the fifth and sixth visceral pouch (Johnson, 1922). In birds, according to Rabl (1907), it develops from the fifth and sixth visceral pouches, or else from the sixth pouch only (Sicher, 1921; Dudley, 1942). Opinion in regard to the mammals varies. Tandler (1909) derives it from the fifth visceral pouch, Lewis (1906) and Klapper (1946), in contrast, from the fourth visceral pouch. Kingsbury (1914,1915) considers that the ultimobranchial body does not represent a visceral pouch at all.

The later development of the ultimobranchial body in the lower vertebrates is in general similar. Laterally to the larynx it forms a pair of uni-or multilobar organs, most nearly resembling the thyroid gland (Watzka, 1933, &c.). The size, structure, and localization of the organ vary considerably and in Urodeles, for instances, only the left body is developed (Wilder, 1929). The later development of the ultimobranchial body in mammals is incompletely known. It is evident that it does not form an independent, persistent organ. Most authors agree that during development it fuses with the lateral thyroid lobes, forming there rudimentary cysts (Rabl, 1931; Politzer & Hann, 1935; van Dyke, 1944,1949,1955) or ultimately disappears altogether (Grosser, 1910; Kingsbury, 1914,1935,1939; Klapper, 1946). It is an opinion fairly generally held that the ultimobranchial body forms active thyroid tissue (Badertscher, 1918; Rogers, 1929; Weller, 1933; Massart, 1940) and that the thyroid gland in mammals is thus formed from three anlagen, one median and two lateral ones. This theory, however, is unproved.

The normal development of the ultimobranchial body has been studied on the material which the Hubrecht Laboratory has collected for the ‘Normal table’ of X eno pus laevis Daudin. The stages 41–56 investigated in this study were prepared by Prof. J. Ariëns Kappers, M.D., Groningen, and stages 57–66 by one of us (Toivonen). The former part of the material was sectioned serially at 10 μ and stained with haematoxylin-eosin. The latter part was serially sectioned at 20 μ and stained with ‘Nucplascol’ (Grübler-Hollborn). For the more detailed histological study and for the experimental work embryos reared in our laboratory have been used. Their developmental stages are given as Hubrecht Laboratory stage numbers. In the detailed studies Bouin’s fluid or formol-alcohol was used as fixative. The thickness of the sections was 6 μ and the staining Heidenhain’s azan.

The experiments with I¹³¹ were performed as follows. The experimental animals were kept for 4 days in a solution containing radioactive iodine (200 μC. I¹³¹ in 1,000 ml. water). The temperature of the solution was 22° C. Thereafter the thyroid gland and the larynx, together with the surrounding tissues, were taken out and fixed for 3 hours in Carnoy’s fluid, the pieces being simultaneously stained with eosin. After embedding in paraffin the blocks were serially sectioned at 10 μ, mounted on slides and covered with a thin layer of celloidin (0·4 per cent, celloidin solution). The preparations were then placed on the film (Guilleminot Collodium 4). Exposure lasted 14 days. After the autoradiographs had been made, the preparations were stained in the usual way.

The first developmental stage of the ultimobranchial body is seen in stage 40. On the caudal wall of the pharyngeal cavity, on the median and caudal side of the fifth visceral pouch, a small pit with a pointed bottom is seen. The epithelium has not yet developed a fold at this site. In the following stage the pit has deepened and forms a finger-like lumen which is encircled by the fold of entoderm. The picture corresponds wholly with that of a young visceral pouch, in this case the sixth. It is unique in being directed medioventrally and thus does not reach the ectoderm as do the other visceral pouches. In stage 42 (Text-fig. 1) the sixth visceral pouch is still seen approximately unchanged in size and form. In the following stage (43) the formation of the ultimobranchial body itself begins. The finger-like lumen of the sixth pouch has narrowed to a slit and the entoderm fold becomes thinner at its base and separates from the pharyngeal wall (Text-fig. 1; Plate 1, fig. 1).

In stage 44 the ultimobranchial body is seen for the first time as an independent organ anlage. The lumen of the sixth visceral pouch is closed and the undifferentiated entodermal cell ball thus formed is connected with the pharyngeal wall only by a thin stalk (Text-fig. 1; Plate 1, fig. 2). In the following stage the connexion is broken and the body is found as a separate cell group laterally to the laryngeal aditus (Text-fig. 1). The islets gradually grow and in stage 47 form solid balls, about 35 μ in diameter. Thereafter the balls are dispersed over a wide space, forming small, solid islets of 8–10 μ in diameter. In stages 48–50 no differentiation of these islets is to be observed. The cells are rather small, the limits of the cells indistinct, and the nuclei large and poor in chromatin.

The differentiation of the ultimobranchial cell islets starts in stage 51 (Text-fig. 2; Plate 1, fig. 3). The cells orientate themselves at the periphery of the ball, forming short strands or segments. The cell limits are still indistinct and one cannot remark any distinct formation of follicles. In stage 52 the cell limits have become clearer and at the same time some nearly complete follicles with a very small lumen can be observed. Both here and in the following stage the majority of the islets are still without a lumen. In stage 54 (Text-fig. 2) the follicles have grown in size and number. The lumen has pronounced limits, but it is still narrow, the diameter always being smaller than the height of the epithelium. In a transverse section the wall shows 8–10 cells. In the lumen one can now and then observe an indistinct content which cannot, however, be considered with certainty to be a secretion. The size and form of the follicles are fairly uniform, but their number in different individuals varies greatly. In stage 56 (Text-fig. 2; Plate 1, fig. 4) the follicles are typical and their lumen has widened, being approximately equal to the height of the epithelium. The wall is composed of columnar epithelium, the large nuclei are generally localized in the proximal part of the cells. The epithelium appears to have a basal membrane. It is not possible to state with certainty that a secretion is present, but in the follicles one may often observe a clear, faintly staining content. In the apex of some epithelial cells there are small balls, which may be apocrine secretion granules. The follicles are surrounded by a rich capillary net which in stages 57–58 is further enlarged.

After the ultimobranchial body has reached the developmental stage described above, the following stages reveal no noteworthy changes. The size of the follicles and the ratio of the epithelium and lumen are rather variable, but a clear increase in size is observable both in the whole follicle and in the lumen (Text-fig. 2; Plate 1, fig. 5; Plate 2, fig. 7). The follicles still show the above-mentioned content, which is faintly stained with methylene blue, but in the epithelial cells there are no definite signs of any secretion. A great variability in the number of follicles is seen, as earlier. This stage of slow growth extends until the last stages of metamorphosis, stages 63–64. Thereafter a phase in the development of the ultimobranchial body is observed which for several reasons may be considered as a decline. The follicles no longer increase in size, the earlier wide lumen becomes narrower and the number of the follicles is reduced. In stage 66 as well as in the young adult individuals studied only 2–5 follicles are found and in their narrow lumen no secretion is seen (Text-fig. 2; Plate 1, fig. 6; Plate 2, fig. 8).

In trying to elucidate the significance of the ultimobranchial body and especially its relation to the thyroid gland and epithelial bodies, it is important to compare its development with that of these endocrine glands, whose function is known.

In stage 40, where the sixth visceral pouch is found for the first time, the anlage of the thyroid gland is seen as a paired cell strand on both sides of the hyoid crest. The connexion with the thyroglossal duct is lost but at the cranial part the duct itself is still present. The cell strands show no cell differentiation. Not until stage 48 do the cells form 4–5 ill-defined lobes. In the following stage the beginning of follicle formation is seen in the same way as in the ultimobranchial body in stage 51. In stage 50 the follicles are already well defined and contain faintly staining material, but only in stage 51 is there any hint of a secretion. At this time the wall of the follicles is complete, the lumen contains strongly staining colloid and there are resorption vacuoles at the site of the epithelial cells. Measurements reveal that the organ continues to increase in size until stages 62–63. At the end of metamorphosis the thyroid gland is continuously regressing in size and in stage 66 its size is only about 70 per cent, of what it was in stage 63.

Experiments with radioactive iodine show that the thyroid gland is already active in stage 51. The storage of I¹³¹ is proved by the distinct autoradiograph.

It should further be stated that throughout development the situation of the thyroid gland is much more cranial than that of the ultimobranchial body. Even though their distance apart decreases at the end of metamorphosis, no sign of a connexion between them can be observed.

The first phase in the development of the epithelial bodies (parathyroid glands) is observed in stage 43, when a small thickening is seen in the ventral epithelium of the third and fourth visceral pouches. The thickening grows to form club-like anlagen remaining in connexion with the epithelium of the visceral pouches. They begin to differentiate in stages 52–53. Their cells then orientate themselves to give the characteristic concentric formation and a thin membrane composed of squamous epithelial cells is formed around the gland. Except for the gradually increasing size of the glands there are no noteworthy changes until the end of metamorphosis. In stages 59–60 the stalk connecting the epithelial bodies with the epithelium of the pouches is broken and their club-like form is rounded off. Thereafter the glands are nearly spherical. Some of the older tadpoles show 2–3 accessory bodies, which are always located in the direct neighbourhood of the main gland and inside the same capsule.

The question of the relation between the ultimobranchial body and the thyroid gland, which has constantly engaged the attention of investigators, has been a source of controversy which has remained unsettled. As Kingsbury (1939) points out, the thyroid-like structure of the gland and the observation of a colloidal secretion in the follicles are not sufficient to prove that the gland has a thyroid-like activity or is actually a separate thyroid gland. He continues: ‘The final test would be whether the cell metabolism is of such a character that substances binding iodine are produced.’ Such an experiment has been published by Gorbman (1947). He studied the thyroid gland of a young rat, using the autoradiograph technique and stated that here and there the thyroid gland tissue was not able to store iodine. These iodine-negative regions, which also differed morphologically from the thyroid gland proper, are, according to Gorbman’s hypothesis, formed by the ultimobranchial tissue. Since, however, the origin of these iodine-negative regions is not known with certainty, and, further, the experiment was performed on one individual only, it needs confirmation. The species we have investigated has an ultimobranchial body which is distinctly separate from the thyroid gland and it is therefore well suited to such a study. Since the histological studies showed that the ultimobranchial body is most active during metamorphosis, tadpoles of different stages were investigated.

The experiments were performed using the method already described. As a control an autoradiograph of the thyroid gland of each animal was also taken. The number of animals investigated was six, including two individuals each from stages 51 and 60 and two young adult animals. The result was without exception negative. While the thyroid gland of each individual gave a distinct autoradiograph, the ultimobranchial body never stored any iodine (Plate 2, figs. 9–12).

In the present study an attempt has been made to throw light on two questions concerning the ultimobranchial body: what is the function of this organ and what is its relationship to the endocrine derivates of the pharyngeal cavity, the thyroid gland, and the epithelial bodies?

On the basis of histological studies Terni (1927) considers the ultimobranchial body to be an active gland. Watzka (1933) has performed extensive comparative investigations and considers that the structure of the ultimobranchial body in lower vertebrates clearly indicates the internal secretory activity of the gland. Dudley (1942), on the basis of her experiments on birds, formed the opposite opinion and suggests that the organ has no secretory function. The results presented in this paper, however, support the idea that the ultimo-branchial body may have its own specific function. The very development of the organ is in conflict with the concept of a purely rudimentary remnant of a visceral pouch. After becoming separated from the epithelium of the pharyngeal cavity, the ‘remnant’ continues to grow and differentiates into a separate organ which displays a regular structure and which is observed in all individuals studied. Further, it cannot be contested that the structure of the ultimobranchial body, as described, is clearly glandular. The follicles with a relatively wide lumen, the columnar epithelium, and the secretion granules observed in its cells, as well as the wide capillary net surrounding the follicles, go to prove, in our opinion, a secretory function. The histological investigation shows, however, that activity during development varies. It seems not to begin until the emergence of the hind limbs, attaining its maximum near the end of metamorphosis after the emergence of the fore limbs, and weakening thereafter simultaneously with the regression of the tail. Compared with that of the embryo, the ultimobranchial body of the adult is decidedly atrophied, and the typical signs of activity, secretion granules, wide lumen, and secretion, are lacking. Thus the functional significance of the ultimobranchial body after metamorphosis remains an open question, but it is evidently in any case reduced.

The other proposed question is the relation of the thyroid gland and the epithelial bodies to the ultimobranchial body. The relation was of especial interest to the investigators mentioned in the Introduction who studied the development of the ultimobranchial body in mammals. Special attention must be given here to the theory according to which the ultimobranchial body may form active thyroid tissue (Badertscher, 1918; Rogers, 1929; Weller, 1933; Massart, 1940). On the basis of his experimental results, van Dyke (1944, 1949, 1955) has stated that during development the ultimobranchial body may form thyroid tissue and that after a partial destruction of the thyroid gland it acts as a thyroid growth centre, forming new thyroid tissue. After birth, however, the ultimobranchial body atrophies and it is found in the thyroid gland as atrophied follicle-like cysts. This ultimobranchial tissue in the thyroid gland may be the source of some thyroid tumours (van Dyke, 1955). The present results on the frog, where the ultimobranchial body is formed as a separate organ, however, give no hint of a connexion between this organ and the thyroid gland. The ultimobranchial body, according to some authors a ‘lateral thyroid’ (Massart, 1940), is in no developmental stage in contact with the thyroid gland proper, neither does it ever store iodine like the thyroid gland. Although its structure closely resembles that of the thyroid gland, there is never in its follicles any substance staining like the thyroid colloid. Further, the ultimobranchial body is clearly formed later than the thyroid gland. These observations may be sufficient to prove that in the species studied the ultimobranchial body does not form thyroid tissue nor does it function like the thyroid gland.

Of course, these results cannot be generalized with certainty, but it is improbable that this organ has an entirely different activity in different vertebrates. It is another question whether the ultimobranchial body of different authors is indeed the same organ. As mentioned already, its formation in different vertebrates is very variable (from the fourth to the sixth visceral pouch), and it is not certain whether the organs described have anything in common apart from a similar structure and the same name.

Dudley (1942), in birds, has stated that during development the ultimobranchial body forms accessory parathyroid glands. Although the species we have studied also has accessory epithelial bodies, they are probably not derived from the ultimobranchial body. The distance between the two organs throughout development is relatively great and the accessory epithelial bodies are in addition located inside the same capsule as the main gland. A more natural explanation in this case is that a part of the epithelial gland rudiment has isolated itself and developed separately.