/. Embryol. exp. Morph. Vol. 20, 3, pp. 319-27, November 1968
Printed in Great Britain
3] 9
Endocrine influences on growth and pigmentation
of embryonic down feather cells
By LELAND G. JOHNSON 1
From the Department of Biology, Augustana College
Rawles (1960) and Hamilton (1952) have outlined the development of the
down feather. Details of the development and interaction of the epidermal
constituents and melanocytes of the down feather were reported by Watterson
(1942) and Goff (1949) described the development of the mesodermal portions
of the feather.
There have been several reports that normal down feather development is
dependent upon undisturbed endocrine balance. One of the techniques for
alteration of normal endocrine patterns in the chick embryo is the complete
extirpation of the rudiments of the pituitary gland (Fugo, 1940) and many
developmental studies have been based upon this method of 'hypophysectomy'
by partial decapitation (see Hinni & Watterson, 1963). Several of these studies
have cited gross effects of this operation on down feather development. Fugo
(1940) found pronounced differences between operated and control embryos in
length and general pigmentation of down feathers along with a 'tremendous
increase' in the number of pigment granules in individual barbule cells of
decapitated embryos. He reported that injection of 'thyrotropic preparations'
alleviated the melanotic feather condition, but did not affect the general growth
retardation of operated embryos. Thyroxine injections in the concentrations
which he used proved toxic to the operated embryos. Goeringer (1959) reported
that growth in length of down feathers ceased after belatedly reaching the
length of 15- to 16-day control feathers and that the feathers of decapitated
embryos had 'stubby' tips and other anomalies. Both Fugo and Goeringer
mentioned changes in sheath consistency which made sheath splitting difficult
in decapitated embryos even after the time (17 days) when it could be done
readily in control embryos. Yatvin (1966a, b) reported effects of decapitation on
subcellular and molecular processes in feather development. He found that
changes in skin and feather polyribosome size distribution patterns and morphology which normally occurred between 12 and 15 days of incubation were
delayed or possibly prevented in decapitated embryos. This effect was reversed
in decapitated embryos treated with chick pituitary extracts or parabiosed with
unoperated embryos.
1
Author's address: Department of Biology, Augustana College, Sioux Falls, South
Dakota 57102, U.S.A.
320
L. G. JOHNSON
This study was undertaken to obtain basic quantitative data on the cellular
responses of developing down feathers to several kinds of endocrine manipulation. The effects of decapitation and direct chemical manipulation of thyroid
hormone level were examined.
MATERIALS AND METHODS
Fertile New Hampshire Red eggs were incubated in forced draft incubators at
37-5 °C. The embryos were decapitated at approximately the 12- to 15- somite
stages of development by a modification of Fugo's (1940) method. There was
no significant difference in feather cell size between unopened and opened control embryos as thirteen unopened 18-day control embryos had a mean barbule
cell length of 59-12 /^ while seven opened 18-day controls had a mean cell
length of 59-38 JLL. There was no apparent pigmentation difference between the
two groups. Operated eggs were candled frequently for detection and removal
of dead embryos.
Thiourea injections at 10 days of incubation were made through a small hole
punched in the egg over the chorioallantoic membrane. Any eggs which oozed
fluid or bled were discarded. Each egg received 10 mg of thiourea (Romanoff &
Laufer, 1956) dissolved in 0-1 ml of Spratt's unbuffered saline solution (Rugh,
1962). Control injections of equal volumes of saline were made. Some of the
thiourea-injected embryos also received 1 jug of L-thyroxine sodium pentahydrate (Nutritional Biochemicals Corporation) in 0-1 ml of saline injected through
the same hole on the twelfth day of incubation.
Collected embryos were preserved in 10 % formalin. Feathers were always
selected from one small area on the mid-line of the anterior portion of the
saddle tract (Holmes, 1935). An uncleared whole mount of each feather was
prepared by splitting the sheath, spreading the barbs, and mounting the feather
under a coverslip with a non-resinous mounting medium. In each feather two
of the 10-15 barbs radiating outward and distally from the calamus were
studied. Each barb consisted of a proximal portion (about two-thirds to threefourths of the total length in controls) which gave rise to two rows of barbules
opposite each other and at right angles to the barb. The approximate length of
this portion and the length of the distal portion which lacked barbules were
measured with an ocular micrometer and a scanning objective. Individual barbules were randomly selected midway through the length of the barbule-bearing
portion of the barb. The long, tapering barbule tip cell and the two adjacent
cells were excluded. The next ten cells (cells 4-13 counting from the tip) in the
single row of elongate cells making up the barbule were studied. An oil immersion objective was used for the cell measurements and pigment granule counts
in the cells from decapitated embryos and the associated controls. Although the
pigment granule counts cannot be taken as absolute values because of difficulties
in resolving occasional clusters of granules and in focusing on the various planes
Embryonic feather development
321
through the thickness of the cells, repetition of selected counts showed errors of
less than 5 %. All cell measurements in the thiourea and thyroxine injection experiments were made under a high dry objective and cells were measured two at
a time rather than individually. In all cases five feathers per specimen were used
and barbules from two different barbs of each feather were studied. A single
mean cell length for each barbule was calculated for statistical treatment.
Sexing of specimens was done by dissection and direct observation of the
reproductive systems.
RESULTS
General observations
Several observations on the gross effects of decapitation on feather development can be added to those reported by Goeringer (1959). The consistency of the
medullary portion of the barbs of decapitated embryos appeared to be different
from that of the controls and there was generally more pigmentation associated
with the barbs of operated embryos. Rounded brown clumps which ranged in
size up to 15 ft in diameter were observed frequently in the barbs of decapitated
embryos. Such clumps were rare in control barbs. No quantitative data on the
total length of individual barbs are reported because of the difficulty in making
accurate measurements of the barbs which were usually curved on the slides.
However, the barbs did appear to be shorter in the decapitated embryos as
would be expected on the basis of Goeringer's results. One difference between
the two groups which could be accurately measured was in the length of the
terminal portion of the barb. About two-thirds of the barbs in each down
feather had elongate, tapering tips which did not bear barbules and these tips
were usually quite straight on the slides. Data for decapitated embryos were
taken only from specimens in which the sheath could be opened well enough to
completely expose the tips. The mean tip length (mm) in the control embryos
was 2-691 ± 0049 (S.E.) and in the decapitated embryos it was 0-920 + 0-049 (S.E.).
Barbule cell growth
Measurements of lengths of individual barbule cells were made for decapitated
and control embryos at 18, 19, and 20 days of incubation. These data are
presented in Table 1.
When variation in feather lengths among the control groups became obvious,
nine more 20-day embryos which had received no experimental treatment were
examined for purposes of comparison. Feathers from these embryos had a mean
barbule cell length (/*) of 60-611 + 0-664 (S.E.). This result can be compared with
the control data in Table 1 and with the control group for the thyroid-inhibition
experiments reported below.
Thiourea injection at 10 days of incubation also resulted in deficient barbule
cell growth. The mean length (/*) of cells from 150 barbules of fifteen 18-day
embryos which had been injected with thiourea was 46-402 ±0-331 (S.E.) while
322
L. G. JOHNSON
the mean length (jt) of cells from 100 barbules of ten saline-injected 18-day
embryos was 61-781 ±0-636 (S.E.). Eight 19-day thiourea-injected specimens had
a mean cell length O) of 46-665 ± 0-625 (S.E.).
Some of the embryos which had received the thiourea injection on day 10
were given a thyroxine injection on day 12. The mean length (JLC) of cells from
120 barbules of 12 of these embryos at 18 days of incubation was 52-833 +
0-406 (S.E.). Thus, the effects of thiourea injection were partially alleviated by
thyroxine.
Table 1. Mean length (/*) ofbarbule cells in decapitated
and control embryos
Control embryos
Days of
incubation
18
19
20
No. of
barbules*
*±S.E.
Decapitated embryos
No. of
barbules*
P±S.E.
59-213 ±0-489
200
100
44122 ±0-735
100
60-153 ±0-747
80
47-278 ±0-791
63-211 ±1-077
100
50
40-282 ±0-738
* Ten cells were measured in each barbule in all cases.
Barbule cell pigmentation
Pigment granule counts were made on 2000 cells of 200 barbules from twenty
18-day control embryos and on 1000 cells of 100 barbules from ten 18-day
decapitated embryos. The general increase in pigment granule number per cell
in decapitated embryos observed by Fugo was confirmed, but it was obvious
that there was little uniformity in the granule numbers in either control or
decapitated embryos. The mean granule number for all of the control barbule
cells was 37-1 and 140-8 was the mean for all of the cells from decapitated
embryos. No statistical treatment of these data is reported because of the error
involved in making granule counts. The overall mean granule number per cell
for individual decapitated embryos ranged from 53-2 to 263-6 and for control
embryos the range of means was from 2-5 to 102-6 granules per cell. However,
there was considerable variation from barbule to barbule even in those embryos
having large or small overall granule means. Therefore, these data from all
specimens are grouped and presented in Fig. 1 as a frequency distribution of
mean granule numbers per cell for individual barbules. Only one barbule from
a decapitated embryo had a mean granule number per cell of less than 20 while
more than half (101) of all control barbules had mean granule numbers per cell
of twenty or less. There was also a marked difference on the higher end of the
distribution as the highest mean granule number per cell for a control barbule
was 161-1 while 37 of the 100 barbules from decapitated embryos had mean
granule numbers per cell above that level.
There were no clear sex-associated differences in pigment granule numbers
Embryonic feather development
323
per cell in the control embryos. A similar result was seen in the data from
decapitated embryos. The four embryos having the lowest and highest overall
mean granule numbers per cell in the control and decapitated groups were all
females.
The feather colors of thiourea-injected embryos were within the range of
variation observed in control embryos and did not resemble the chocolate color
of the feathers of decapitated embryos. Because of these gross observations, no
cellular study of the feather pigmentation of thiourea-injected embryos was
made.
20
100
80
20
40 80 120 160 200
No. of granules per cell
Fig. 1. Mean numbers of pigment granules per cell in down feather barbules.
Number of barbules with mean granule counts falling into each range is indicated.
(A) 18-day decapitated embryos (all means over 200 are lumped). (B) 18-day
control embryos.
DISCUSSION
The 'stubby' appearances of feathers of decapitated embryos reported by
Goeringer (1959) can be explained by the observation that the terminal barbulelacking portions of the barbs were much shorter in decapitated embryos than in
controls.
The brown clumps observed in the barbs were probably degenerating melanocytes which had not been crowded off into the prospective pulp membrane area
and carried away in the normal manner at the time of pulp shrinking (Watterson, 1942; Goff, 1949). Goeringer (1959) did report that a number of abnormal
processes occurred during pulp shrinkage in decapitated embryos.
There was a marked reduction in barbule cell size in decapitated embryos.
324
L. G. JOHNSON
The variation among the groups of control embryos was probably due to chance
factors in selection of specimens for study because previous workers have concluded (Watterson, 1942; Koning & Hamilton, 1954) that the development of
the down feather is essentially complete by 18 days. Results from an additional
group of 20-day control embryos supported this view.
The cell growth response in decapitated embryos might also be considered
with reference to Yatvin's (1966a, b) work. He related his findings to earlier
studies (Humphreys, Penman & Bell, 1964; Bell, Humphreys, Slayter & Hall,
1965) which described subcellular events coincident with the period of active
keratin synthesis in embryonic skin (Bell & Thathachari, 1963). Later reports
(Byers, 1967; Humphreys & Bell, 1967) complicated the interpretation of these
results because the nature of the distribution patterns of polyribosomes extracted
from chick embryo skin was found to be influenced by the temperature at which
the extraction process was begun. However, Yatvin (1966a) did find a polyribosome distribution pattern in decapitated embryos which differed from that
of controls at a comparable age and he demonstrated (1966&) that pituitary
hormone replacement would reverse this effect. On the basis of the cell growth
data reported here it might be suggested that in addition to keratinization, the
subcellular events which Yatvin studied could be associated with the synthesis
of other major structural components during the period of feather cell growth.
Thiourea injections also resulted in marked barbule cell growth reduction.
Thyroxine injections did not restore barbule cell size to the control level, but
the dosage used was lower than that required to reverse the effects of thiourea
injection on chick development in some other studies (e.g. Konigsberg, 1958;
Moog, 1962). Possibly injection on day 12 was not early enough for thyroxine
to be fully effective.
Before any inference relating the cell growth effects of decapitation and
thiourea injection can be drawn, data on thyroxine effects on cell growth in
decapitated embryos are needed. However, injection of thyroxine into decapitated embryos so increases mortality that it seems inappropriate to regard
results from the few survivors as reliable indices of physiological response. It
can only be concluded that barbule cell growth is reduced by either decapitation
or thiourea injection. The partial recovery in thiourea-injected embryos treated
with thyroxine provides a departure point for future studies of feather cell
growth. It also suggests a new approach for studies of subcellular events during
differentiation in this system.
The pigmentation results in decapitated embryos could be caused by an increase in the number of active melanocytes, an increase in the number of granules
transferred by each of a normal number of cells through increased synthetic
activity, or by transfer of more pigment granules to each cell as a consequence
of the slower growth rate of the feather. Possibly there was a change in the
relative proportions of the two types of melanocytes present in New Hampshire
embryonic skin (Hamilton 1940, 1941).
Embryonic feather development
325
Fugo's conclusion that thyroid deficiency was the cause of feather darkening
in decapitated New Hampshire embryos must be questioned because of observations in this study and because several other workers (Trinkaus, 1950;
Markert, 1948) have reported that embryonic pigment cells were unresponsive
to altered thyroid hormone levels. Hamilton's report that desoxycorticosterone
acetate inhibited melanogenesis in explanted New Hampshire embryonic back
skin suggests another hypothesis. Decapitation influences embryonic adrenal
cortical function (Case, 1952) and may result in removal of a normal inhibition
to melanocyte activity. Hamilton (1941) found that gonadal hormones also
influenced New Hampshire melanocyte differentiation in vitro, but the significance of these results was challenged by Trinkaus (1948). Recently, GroenendijkHuijbers (1966, 1967) has supported Hamilton's viewpoint. In light of these
disagreements concerning endocrine regulation of embryonic melanocytes,
further experimentation will be needed in order to determine the means by
which decapitation affects pigmentation.
It can be concluded that the embryonic down feather does show several
obvious cellular responses to endocrine manipulation and that it could become
a very useful analysis system for future studies of some aspects of endocrine
regulation of vertebrate ontogeny.
SUMMARY
1. The effects of endocrine deprivation following partial decapitation or
thiourea injection were studied in New Hampshire Red embryonic down
feathers.
2. Growth of barbule cells was significantly reduced by either decapitation or
thiourea injection. Thyroxine injections partially alleviated the feather cell
growth deficiency in thiourea-treated embryos.
3. The melanotic feather condition in decapitated embryos results from a
general increase in the number of pigment granules in the barbule cells, but the
developmental modifications leading to this increased pigmentation level remain
to be studied.
4. It is concluded that the embryonic down feather shows clear cellular
responses to several types of endocrine manipulation and appears to hold considerable promise for future studies of endocrine regulation of development at
both the cellular and subcellular levels.
RESUME
Influences endocrines sur la croissance et la pigmentation
de cellules plumaires du duvet embryonnaire
1. On a etudie les effets de la privation d'elements endocrines consecutive
a la decapitation partielle ou a l'injection de thiouree, sur du duvet embryonnaire
de New Hampshire Red.
326
L. G. JOHNSON
2. La croissance des cellules des barbules a ete reduite de maniere significative
soit par decapitation, soit par injection de thiouree. Des injections de thyroxine
ont partiellement compense les defauts de croissance plumaire chez les embryons
traites a la thiouree.
3. L'etat melanise de la plume chez les embryons decapites provient d'un
accroissement general du nombre des granules pigmentaires dans les cellules
des barbules, mais les modifications du developpement conduisant a cette
pigmentation accrue restent a etudier.
4. On conclut que la plume de duvet embryonnaire presente des reactions
cellulaires nettes et tranchees a l'egard de plusieurs types d'action endocrinienne
et qu'elle presente un interet considerable pour des recherches ulterieures sur
la regulation endocrine du developpement aux niveaux cellulaire et sub-cellulaire.
This investigation was supported by a grant from Research Corporation.
REFERENCES
BELL, E., HUMPHREYS, T., SLAYTER, H. S. & HALL, C. E. (1965). Configuration of inactive
and active polysomes of the developing down feather. Science 148, 1739-41.
BELL, E. & THATHACHARI, Y. T. (1963). Development of feather keratin during embryogenesis
of the chick. / . cell Biol. 16, 215-23.
BYERS, B. (1967). Structure and formation of ribosome crystals in hypothermic chick embryo
cells. / . molec. Biol. 26, 155-67.
CASE, J. F. (1952). Adrenal cortical-anterior pituitary relationships during embryonic life.
Ann. N.Y. Acad. Sci. 55, 147-58.
FUGO, N. W. (1940). Effects of hypophysectomy in the chick embryo. / . exp. Zool. 85,271-97.
GOERINGER, G. C. (1959). Modified development of the integument of hypophysectomized
chick embryos. I. The epidermis. II. The feather germs. Ph.D. Dissertation, Northwestern
University.
GOFF, R. A. (1949). Development of the mesodermal constituents of feather germs of chick
embryos. / . Morph. 85, 443-81.
GROENENDIJK-HUIJBERS, M. M. (1966). Sex-linked feathering of female hybrid chick embryos
hormonally conditioned. Experientia 22, 302-3.
GROENENDIJK-HUIJBERS, M. M. (1967). Hormone dependency of sex-linked feathering in
female hybrid chick embryos (cross New Hampshire <? x Light Sussex $). Experientia 23,
46-9.
HAMILTON, H. L. (1940). A study of the physiological properties of melanophores with
special reference to their role in feather coloration. Anat. Rec. 78, 525-47.
HAMILTON, H. L. (1941). Influence of adrenal and sex hormones on the differentiation of
melanophores in the chick. / . exp. Zool. 88, 275-305.
HAMILTON, H. L. (1952). IAllie's Development of the Chick. New York: Henry Holt.
HINNI, J. B. & WATTERSON, R. L. (1963). Modified development of the duodenum of chick
embryos hypophysectomized by partial decapitation. / . Morph. 113, 381-425.
HOLMES, A. (1935). The pattern and symmetry of adult plumage units in relation to the order
and locus of origin of the embryonic feather papillae. Am. J. Anat. 56, 513-37.
HUMPHREYS, T. & BELL, E. (1967). The in vivo aggregation of chick embryo ribosomes in
response to low temperature. Biochem. Biophys. Res. Commun. 27, 443-7.
HUMPHREYS, T., PENMAN, S. & BELL, E. (1964). The appearance of stable polysomes during
the development of chick down feathers. Biochem. biophys. Res. Commun. 17, 618-23.
KONIGSBERG, I. R. (1958). Thyroid regulation of protein and nucleic acid accumulation in
developing skeletal muscle of the chick embryo. / . cell. comp. Physiol. 52, 13-41.
Embryonic feather development
327
A. L. & HAMILTON, H. L. (1954). Localization of enzyme systems, nucleic acids and
polysaccarides during morphogenesis in the down feather of the chick. Am. J. Anat. 95,
75-107.
MARKERT, C. L. (1948). The effects of thyroxine and antithyroid compounds on the synthesis
of pigment granules in chick melanoblasts cultured in vitro. Physiol. Zool. 21, 309-27.
MOOG, F. (1962). Developmental adaptations of alkaline phosphatases in the small intestine.
Fed. Proc. 21, 51-6.
RAWLES, M. E. (1960). The integumentary system. In Biology and Comparative Physiology of
Birds, vol. 1, ed. Marshall, A. J., pp. 189-240. New York: Academic Press.
ROMANOFF, A. L. & LAUFER, H. (1956). The effect of injected thiourea on the development
of some organs of the chick embryo. Endocrinology 59, 611-19.
RUGH, R. (1962). Experimental Embryology, 3rd ed. Minneapolis: Burgess.
TRINKAUS, J. P. (1948). Factors concerned in the response of melanoblasts to estrogen in the
Brown Leghorn fowl. J. exp. Zool. 109, 135-69.
TRFNKAUS, J. P. (1950). The role of thyroid hormone in melanoblast differentiation in the
Brown Leghorn. /. Exp. Zool. 113, 149-77.
WATTERSON, R. L. (1942). The morphogenesis of down feathers with special reference to the
developmental history of melanophores. Physiol. Zool. 15, 234-59.
YATVIN, M. B. (1966a). Polysome morphology: Evidence for endocrine control during chick
embryogenesis. Science 151, 1001-3.
YATVIN, M. B. (19666). Hypophyseal control of genetic expression during chick feather and
skin differentiation. Science 153, 184-5.
KONING,
{Manuscript received 22 March 1968)