J. Embryol. exp. Morph. Vol. 24, 1, pp. 65-71, 1970
Printed in Great Britain
65
Thyroid hormone effects on chick scale
morphogenesis and keratinization
By NANCY K. LAWRENZ 1 AND LELAND G. JOHNSON 1
From the Department of Biology, Augustana College
SUMMARY
The effects of thiourea injection on scale development were studied in New Hampshire
Red x 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 15- to 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 (1966 a, 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).
1
Authors' address: Department of Biology, Augustana College, Sioux Falls, South
Dakota 57102, U.S.A.
5
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66
N. K. LAWRENZ AND L. G. JOHNSON
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 10 jug 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 /.L 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 Mest (Simpson, Roe & Lewontin, 1960) was
applied to differences between experimentals and controls.
RESULTS
Morphological observations
At 13 days the thiourea-injected specimens showed no significant morphological differences from the control specimens (Fig. 1 A, 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.
Thyroid hormone and morphogenesis
67
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 /-i and four 15-day specimens had a mean scale overlap of 444-9 (i.
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 301 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.
Table 1. Mean lengths (/*) of overlapped portion of the scale in
thiourea-injected and control embryos
Days of
incubation
Controls
(X± S.E.)
Thiourea-injected
(X± S.E.)
13
14
15
16
109-2 ±7-4
264-5 ±10-9
448-0 ±30-8
573-2 ±32-9
1150 ±9-9
1620 ±12-1*
344-2 + 28-1*
464-9 ±26-0*
Significantly different from control (P < 005).
Table 2. Mean length to thickness ratios in scales of
thiourea-injected and control embryos
Days of
incubation
Controls
(X± S.E.)
Thiourea-injected
13
14
15
16
1-46 ±0-141
3-36 ±0129
607 ±0-633
6-01+0-522
1-40 ±0136
200 ±0-129*
3-27 ±0-426*
4-73 ±0-352
(X±S.E.)
Significantly different from control (P < 005).
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
5-2
68
N. K. LAWRENZ AND L. G. JOHNSON
G •
H ' ••
Fig. 1. 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.
Thyroid hormone and morphogenesis
69
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. ID). In doubly injected specimens, the peridermal stain extended
around the entire margin of the scale including the inturned area (Fig. IE).
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. IF). Sixteen-day controls showed a
general increase in intensity of stain all around the scale margin (Fig. II).
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 (19616).
70
N. K. LAWRENZ AND L. G. JOHNSON
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.
RESUME
Effets de Vhormone thyro'idienne sur la morphogenese et la
keratinisation des ecailles du poulet
Les effects des injections de thiouree sur le developpement des ecailles onteteetudieschez les
embryos de poulet du croisement New Hampshire rouge x Leghorn blanche.
La morphogenese normale et la keratinisation des ecailles ont ete retardees d'une maniere
significative dans les embryons traites par la thiouree. Des injections de thyroxine apres
traitement ont pour consequence un developpement normal ou meme precoce des ecailles.
La reaction des ecailles a l'hormone thyro'idienne est comparee a d'autres recherches sur les
effets des hormones sur la differentiation de la peau et de ses derives.
This investigation was supported by grants from Research Corporation and the Augustana
Research and Artist Fund.
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(Manuscript received 13 August 1969)