ABSTRACT
The effects of thiourea injection on scale development were studied in New Hampshire Red × White Leghorn Cross chick embryos.
Normal morphogenesis and keratinization of scales were significantly delayed in thioureatreated specimens. Subsequent thyroxine injection resulted in normal or even precocious scale development.
The thyroid hormone responsiveness of scale development is discussed in relation to other studies of endocrine effects on differentiation of skin and skin derivatives.
INTRODUCTION
The normal differentiation of skin and skin derivatives in the chick embryo is endocrine dependent. Fugo (1940) reported that ‘hypophysectomy’ by partial decapitation caused growth deficiencies and pigmentation changes in the down feather. Bartels (1944) found that thyroxine injections accelerated epidermal differentiation. Goeringer (1959, 1968) showed that in decapitated embryos epidermal differentiation was significantly delayed. Down feathers of decapitated embryos had structural anomalies and stopped growing after belatedly reaching the length of 15to 16-day control feathers. Johnson (1968) reported that individual down feather barbule cells of decapitated embryos were shorter than those of controls. A comparable barbule cell growth deficiency was induced by thiourea injection. This deficiency was partially corrected by a subsequent thyroxine treatment.
In addition to the endocrine dependence of normal morphogenesis, another endocrine effect is suggested by Yatvin’s (1966a, b) finding that some subcellular aspects of keratin synthesis in embryonic chick skin are endocrine dependent.
This study was undertaken to examine directly the effects of manipulation of thyroid hormone level on morphogenesis and keratinization in the chick scale. Normal scale developmental patterns have been described by Thomson (1964), Bartels (1944) and Wessells (1961a).
MATERIALS AND METHODS
New Hampshire Red (♂) x White Leghorn (♀) Cross embryos were incubated at 38°C in a forced-draft incubator. Thiourea was injected on to the chorioallantoic membrane through a small hole punched in the shell. Doses of 5 mg of thiourea dissolved in 0·1 ml Howard Ringer (DeHaan, 1967) were injected at 10 days of incubation. Control embryos received 0·1 ml of Howard Ringer. Some of the thiourea-injected embryos were given 1·0μg of L-thyroxine sodium pentahydrate (Nutritional Biochemicals Corporation) a day later. Henceforth, these specimens will be called ‘doubly-injected’.
Chicks were collected daily from 13 days through 16 days of incubation and legs were removed and fixed for 24 h in absolute alcohol containing 5% glacial acetic acid. They were trimmed, mounted in paraplast, and sectioned at 7 μ, thickness. This study concentrated on scales in the lower half of the anterior side of the leg. This is the area of most rapid scale development (Thomson, 1964).
Some specimens were stained with hematoxylin and eosin. The mercurybromphenol blue method of protein staining (Pearse, 1960) was used with schedule modifications. Keratin staining was by the method of Barrnett & Seligman (1952).
Morphological changes in scale development were examined in the most highly developed scale from each specimen. Ocular micrometer measurements were made of the length of the overlapped portion of the scale. Thickness was measured at the midpoint of the overlapped portion. The length and the ratio of length to scale thickness were used as indices of scale maturation. In order to eliminate variability in sectioning angles, only those sections which met certain morphological criteria including position of the first toe were used for measurement. Even this selection process could not completely eradicate error due to variable section angles. Student’s t-test (Simpson, Roe & Lewontin, 1960) was applied to differences between experimentáis and controls.
RESULTS
Morphological observations
At 13 days the thiourea-injected specimens showed no significant morphological differences from the control specimens (Fig. 1A, B). At 14 days, however, some morphological differences could be seen. The thickness of the periderm and the amount of scale overlap were greater in the controls. In 14-day controls the peridermal cells in the flat part of the scale were slightly more elongated and flattened than those in the thiourea-injected specimens. The peridermal thickness in controls was much greater than in scales of thiourea-treated specimens at 15 days. The periderm of controls was also more strongly eosinophilic. At 16 days the differences were less marked, but 16-day controls showed a flattening of peridermal cells in the inturned area of the scale which was lacking in thiourea-injected embyros.
Sections of chick embryo scales at various stages of development. A and B stained with hematoxylin and eosin. C-I stained for protein by the mercurybromphenol blue method. Magnification is the same for all pictures (see scale line in A). (A) 13-day control, (B) 13-day thiourea-injected, (C) 14-day control, (D) 14-day thiourea-injected, (E) 14-day doubly injected (thiourea plus thyroxine), (F) 15day doubly injected, (G) 15-day control, (H) 15-day thiourea-injected, (I) 16-day control, (J) 16-day thiourea-injected.
Sections of chick embryo scales at various stages of development. A and B stained with hematoxylin and eosin. C-I stained for protein by the mercurybromphenol blue method. Magnification is the same for all pictures (see scale line in A). (A) 13-day control, (B) 13-day thiourea-injected, (C) 14-day control, (D) 14-day thiourea-injected, (E) 14-day doubly injected (thiourea plus thyroxine), (F) 15day doubly injected, (G) 15-day control, (H) 15-day thiourea-injected, (I) 16-day control, (J) 16-day thiourea-injected.
Data from measurements of the amount of overlap for thiourea-treated and control embryos at 13, 14, 15 and 16 days of incubation are presented in Table 1. Although only a few doubly injected specimens met the criteria for measurement, scale overlap in those specimens was similar to that of controls. Five 14-day specimens injected with both thiourea and thyroxine had a mean scale overlap of 218·7 μ and four 15-day specimens had a mean scale overlap of 444·9 μ. These results can be compared with the data in Table 1. Means of the ratios of the length of overlap to scale thickness are given in Table 2. Data from a few doubly-injected specimens showed mean length to thickness ratios of 3·01 for 14-day scales (five embryos), 5·36 for 15-day scales (three embryos), and 5·34 for 16-day scales (two embryos). These data should be compared with the data in Table 2.
Histochemical observations
Two main sites of intense protein staining were the periderm of the scale and the basement membrane area. In both injected and control embryos at all ages examined, the basement membrane stain was a thin line which extended throughout the flat part of the scale.
Thiourea-injected and control specimens showed only slight peridermal protein staining at 13 days. In control specimens at 14 days, protein stain was quite concentrated in the periderm of the flat part of the scale and around the apex. The inturned area of the scale showed very little peridermal protein concentration (Fig. 1C). The very weak peridermal stain in the thioureatreated specimens at 14 days was localized in the flat part of the scale close to the apex (Fig. 1D). In doubly injected specimens, the peridermal stain extended around the entire margin of the scale including the inturned area (Fig. 1E). Keratinization in most of these specimens was more advanced than in controls of the same age. At 15 days the peridermal stain of the controls extended all around the scale margin (Fig. 1G). The stain was more dense in the flat part of the scale than in the inturned area. The 15-day thiourea-injected specimens showed a dense peridermal stain only in the flat part of the scale (Fig. 1H). Protein distribution in doubly injected specimens was comparable to the controls but the stain was generally heavier (Fig. 1F). Sixteen-day controls showed a general increase in intensity of stain all around the scale margin (Fig. 1I). At 16 days the thiourea-injected specimens were also stained all around the scale marign (Fig. 1J), but the stain in the inturned areas of the thioureainjected specimens was much less dense than that of the controls. Doubly injected embryos were generally stained even more heavily than the controls at this age.
The distribution of positive keratin staining by the Barrnett-Seligman method was virtually identical to that of dense protein staining.
Clearly, thiourea treatment retarded certain aspects of morphogenesis and keratinization in the chick scale. Scale differentiation in 16-day thioureainjected specimens had overtaken the controls to some extent, but limited numbers of older specimens showed that this apparently progressive recovery was still not completed even at 17 days.
DISCUSSION
Gross morphological development and histochemical differentiation of chick scales were delayed by thiourea injection. The definite reversal of this retardation by thyroxine therapy demonstrated that the observed effects were due to thyroid deficiencies in the injected embryos and that chick scale development is thyroid hormone dependent.
The apparent recovery of thiourea-treated embryos at 16 or 17 days of incubation could be due to the passing of the hormone-dependent phase of scale development. It seems likely, however, that the effective inhibitory action of this thiourea dosage may have lapsed by this time. Future experiments with repeated injections of thiourea to test the effect of continuing inhibition are needed. Some aspects of scale differentiation in doubly injected embryos appeared to be more advanced than scales of control embryos of comparable ages. Possibly the thyroxine level in the doubly injected embryos was actually higher than that in controls during early stages of scale development. If this were the case, acceleration would be expected in light of the results of Bartels (1944) and Wessels (1961 b).
This thyroid hormone responsivenesss of scale morphogenesis and keratinization is consistent with results of studies which indicate endocrine relationships of other aspects of differentiation of skin and skin derivatives (Goeringer, 1959, 1968; Bartels, 1944; Fugo, 1940; Johnson, 1968). Special emphasis should be placed on the thyroid hormone influence on keratin synthesis reported here because it is of interest in relation to other studies of protein synthesis in chick skin. Several reports (Humphreys, Penman & Bell, 1964; Bell, Humphreys, Slayter & Hall, 1965) have described subcellular events during the period when keratin synthesis is occurring in embryonic skin (Bell & Thathachari, 1963). Yatvin (1966a, b) found that the polyribosome size distribution pattern and morphology changes which normally occur between 12 and 15 days of incubation were delayed or even prevented in decapitated embryos. This effect was reversed by treatment with chick pituitary extracts or by parabiosis with unoperated embryos. Unfortunately, later reports (Byers, 1967; Humphreys & Bell, 1967) of regularly reproducible temperature artifacts in chick-skin polyribosome distribution patterns have complicated the interpretation of these results. However, it must be emphasized that Yatvin did find changes in polyribosome distribution patterns which appear to be endocrine dependent.
On the basis of the findings in this study, it seems appropriate to propose that endocrine control of keratin synthesis be re-examined at the subcellular level. Such work might also yield useful general information on the nature of thyroid hormone effects on differentiation. Preliminary studies on the effects of alteration of thyroid hormone level on skin polyribosome distribution profiles are underway in this laboratory.
RÉSUMÉ
Effets de Phormone thyro ïdienne sur la morphogenèse et la kératinisation des écailles du poulet
Les effects des injections de thiourée sur le développement des écailles ont été étudiés chez les embryos de poulet du croisement New Hampshire rouge × Leghorn blanche.
La morphogenèse normale et la kératinisation des écailles ont été retardées d’une manière significative dans les embryons traités par la thiourée. Des injections de thyroxine après traitement ont pour conséquence un développement normal ou même précoce des écailles.
La réaction des écailles à l’hormone thyro ïdienne est comparée à d’autres recherches sur les effets des hormones sur la différentiation de la peau et de ses dérivés.
Acknowledgements
This investigation was supported by grants from Research Corporation and the Augustana Research and Artist Fund.