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/ . Embryol. exp. Morph. Vol. 16, I, pp. 83-9, August 1966
Printed in Great Britain
83
The effect of embryonic partial decapitation on the
developmental sequence of some
proteins in the chicken
By CLYDE MANWELL 1 & T. W. BETZ2
From the Department of Biological Science, The Florida State
University, and the Department of Zoology,
University of Illinois
INTRODUCTION
Hormonal control of differentiation at a biochemical level is exemplified by
studies on amphibian and insect metamorphosis. However, Hinni & Watterson
(1963) have reviewed the literature and presented new data on another developmental system with potential for analysis of hormone action. Chicken embryos
at 33-36 h of incubation can be * hypophysectomized' by partial decapitation,
the prosencephalic"and anterior part of the mesencephalic areas being removed.
Absence of the pituitary primordium prevents the formation of a pituitary gland.
Such embryos that continue to develop are noticeably smaller and show retardation in the development of bones, feathering, and several epithelial structures
by 2 weeks of incubation. These 'hypophysectomized' embryos have an increased mortality, especially in the third week of incubation; the few that
escape this 'phenocritical period' never hatch and remain in ovo days after the
normal time of hatching.
Ontogenetic protein sequences in the chicken have been studied in regard to
haemoglobin, lactate dehydrogenase, malate dehydrogenase (Manwell, Baker
& Betz, 1966), alkaline phosphatase (Moog, 1959), and a number of structural
proteins. Accordingly, we have studied the ontogeny of several proteins to
determine the effect of partial decapitation on the normal developmental
pattern.
MATERIAL AND METHODS
Chicken embryos were partially decapitated in the manner described by
Hinni & Watterson (1963). Two types of controls were used: sham-operated and
unoperated embryos. Individuals were sacrificed at the end of each day over the
1
Author's address: Laboratory of the Marine Biological Association of the U.K., Citadel
Hill, Plymouth, Devon, England.
2
Author's address: Department of Biology, Carleton University, Ottawa 1, Canada.
6-2
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C. MANWELL & T. W. BETZ
period of from 4 to 17 days of incubation. The only pooling of samples was at
4 days of incubation; otherwise all data are on individual embryos. Each
embryo was bled to provide erythrocytes for haemoglobin (Manwell et al. 1966)
and then was homogenized with a volume of 001 M-K3PO4 equal to the weight
of the embryo. As 'hypophysectomized' embryos lack the upper beak, eyes and
the anterior part of the head, these structures were removed from controls and
studied separately. Embryo homogenates were frozen immediately and kept
at — 20 °C until the entire series had been accumulated. A volume of toluene
approximately equal to the weight of embryo and buffer was added prior to
homogenization. The samples were kept under a carbon monoxide atmosphere
to avoid inactivation of enzymes by oxidation. From embryos of 14, 15, 16 and
17 days the following organs were homogenized separately: liver, heart, small
intestine, and breast muscle, the last mentioned also including the ribs and
associated connective tissue. After thawing, homogenates were shaken briefly
with the toluene layer to facilitate separation of lipid-protein complexes and
were centrifuged at 30,000 g for 1 h at 0 °C. The supernatants were immediately
used for electrophoresis in the modified Smithies' vertical starch-gel arrangement (see Manwell et al. 1966, for details and references). All samples were
screened in three different buffer systems: potassium phosphate, pH 7-0, ionic
strength = 0-02; Smithies' borate; and Ferguson & Wallace's (1961) discontinuous buffer. This last buffer gives extremely good resolution of the major
protein components extractable in low ionic strength buffer and many of the
enzymes; accordingly, in this paper designation of 'anodal' or 'cathodal'
migration and relative electrophoretic mobilities is based on the FergusonWallace pH 8-0 buffer. Using standard histochemical methods, the following
enzymes were identified after starch-gel electrophoresis: acid phosphatase,
alkaline phosphatase, lactate dehydrogenase (LDH), malate dehydrogenase
(MDH), a-naphthyl acetate esterases, lipase, and N-benzoyl-arginine-naphthylamide catheptic protease. Total protein of low ionic strength tissue extracts
was stained with nigrosin.
As the observations on partially decapitated chicken embryos suggest that the
anterior pituitary gland becomes active quite early in chicken development, and
as various workers have had success in the use of starch-gel electrophoresis in
resolving pituitary hormones (Barrett, Friesen & Astwood, 1962; Catt & Moffat,
1965; Ferguson & Wallace, 1961), both in purified preparations and in crude
pituitary extracts, it was decided to attempt an evaluation of the degree of
embryonic pituitary differentiation in the chicken by electrophoresis. Extracts
of single anterior pituitary glands from an adult male, a laying female, and a
non-laying female chicken were compared with extracts of 200 pooled 21-day
foetal chick pituitaries and of 60 pooled 4-day chick pituitaries.
Embryonic hypophysectomy
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RESULTS
Major proteins of low ionic strength extracts of control and partially decapitated embryos could be resolved electrophoretically into from 5 to 7 cathodal
and from 17 to 20 anodal protein-staining zones. No differences as a result of
'hypophysectomy' could be seen. Some variation in the presence or absence of
two rapidly moving anodal protein zones occurs in individual embryos and
probably represents a genetically based protein polymorphism. The general
pattern of the major low ionic strength extractable proteins changes only slightly
during the first 2 weeks of incubation; similar results have been reported by
Shore (1965) in early development of Rana pipiens and R. sylvatica, although
some striking changes in these proteins are observed in extracts of homologous
organs when tadpoles and frogs of R. catesbeiana are compared (Manwell, 1966).
Acid phosphatase, lipase, and N-benzoyl-arginine-naphthylamide cathepsin are
distinct single electrophoretic zones which change only quantitatively during
development and are not altered by partial decapitation, at least not by day 17
of incubation.
Lactate dehydrogenase follows a sequence reported by other workers
(references in Manwell et al. 1966) and partial decapitation has no apparent
effect on either the amount of LDH activity or the isozyme changes in development.
Malate dehydrogenase is resolved into two major isozyme regions, both of
which show tendencies to further subdivision. The faster MDH zone occurs in
all tissues of chick embryos studied by us. Extra-embryonic membranes, heart
and, especially, liver, have in addition to the 'fast' MDH a slower anodal MDH
isozyme. Conklin & Nebel (1965) report up to 7 MDH's in chick liver, brain
and spleen, but add that not all of this heterogeneity is intrinsic. 'Hypophysectomy' has no detectable effect on MDH differentiation.
Alkaline phosphatase is resolved into four isozyme bands, two trace rapidly
moving anodal zones, a rather smeared out but very intensely staining zone of
intermediate anodal mobility, and a minor zone that is a slowly moving anodal
component. Embryonic liver extracts have all four of these zones and the
greatest alkaline phosphatase activity. Heart, intestine, and breast muscle and
ribs have predominantly the diffuse zone of intermediate electrophoretic
mobility. This major alkaline phosphatase band also occurs in low levels of
activity in extracts of 4- to 10-day-old embryos. Though alkaline phosphatase
occurs in both 'hypophysectomized' and control embryos, the former have
consistently less activity than the latter, especially when the comparison involves
days 15-17 of development. At that time the major alkaline phosphatase is
reduced to approximately one-tenth the normal level, judging by rate of enzyme
activity after electrophoresis. The electrophoretic mobility of the major alkaline
phosphatase is not shifted by partial decapitation, suggesting that any possible
hormonal effect involves synthesis rather than allosteric modification of the
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C. MANWELL & T. W. BETZ
enzyme. The individual variability in levels of alkaline phosphatase observed
in some populations of chickens (Wilcox, 1963) was not observed in our
specimens.
The ot-naphthyl acetate esterases of whole homogenized chicken embryos can
be resolved into five anodal zones and one cathodal zone, the second and third
anodal zones being resolved into several close enzyme bands. Developmental
variation is predominantly quantitative, although there is a progressive increase
in the number of bands during development and some differences in the esterase
complement of organs. Partial decapitation has no effects on the anodal
esterases but did reduce to trace or undetectable levels the cathodal esterase
observed in heart and, especially, in liver of 15- to 17-day embryos.
The haemoglobin 'switchover' described in the accompanying paper is not
altered by partial decapitation, although some partial decapitates have noticeably less haemoglobin than controls.
Pituitary extracts are resolvable into eighteen anodal and three cathodal protein
zones, most of which do not correspond with protein zones in serum, and thus
represent intrinsic pituitary proteins. There are some quantitative differences
in the amounts of certain of the zones but otherwise the chick pituitary is
similar to that of the adult by the time of hatching, at least so far as its major
soluble proteins are concerned. No evidence has been provided that any of these
zones in electrophoresis represent specific adenohypophyseal hormones in the
chicken. However, that a number of the major protein bands of mammalian
pituitaries are, in fact, known pituitary hormones has been established (Barrett
et al 1962; Catt & Moffat, 1965; Ferguson & Wallace, 1961).
DISCUSSION
It appears that most proteins are unaffected by partial decapitation in the
chick embryo, at least by the 17th day of development, which is after the time
that some partial decapitates die. However, there are striking reductions in the
electrophoretic activity, and thus presumably amounts, of alkaline phosphatase and a cathodal esterase. The reduction in alkaline phosphatase is of
interest in that Hinni & Watterson (1963) found this enzyme was reduced or
absent in duodenal cells of partial decapitates. Our studies show that subnormal
amounts of alkaline phosphatase are not confined to the developing intestine,
but occur in other organs as well. A similar result has been recently reported by
Johnson (1965), who observed that in abnormal rat embryos, caused by a
maternal deficiency in pteroylglutamate, the major broad zone of alkaline
phosphatase detected after electrophoresis is reduced or absent.
It is known that alkaline phosphatase plays a major role in bone formation,
as well as in mucoprotein and fibrous protein synthesis (Simkiss, 1964). The
enzyme has also been implicated in determining the specificity of certain blood
groups (Arfors, Beckman & Ludin, 1963; Rendel & Stormont, 1964), which
Embryonic hypophysectomy
87
indicates that it might alter cellular aggregation mechanisms dependent on the
properties of the cell surface. Besides possible direct pituitary control of the
synthesis of alkaline phosphatase, it is known that the administration of corticosteroids can 'induce' the premature appearance of this enzyme in chick
embryos (Moog, 1959).
Although it is hardly likely that reduction in alkaline phosphatase accounts
for the entire 'foetal' syndrome of partial decapitates, the failure to synthesize
adequate amounts of this enzyme might well influence enough biochemical
processes to retard embryonic development and, ultimately, cause the death
of the embryo. That bone formation is retarded in late partial decapitates is
obvious from inspection and dissection. Homogenization of partial decapitates
is easier at all stages, suggesting some failure in connective-tissue stabilization
or ossification, effects that might arise from the lack of sufficient alkaline
phosphatase.
The morphological defects of partial decapitates can be largely neutralized
by grafting anterior pituitaries into partial decapitates (Betz, unpublished
studies). Thus, it is reasonable to suggest that the biochemical and morphological
peculiarities of partial decapitates are due directly or indirectly to the absence
of the anterior pituitary gland, rather than the absence of other head structures.
In support of the idea that the embryonic pituitary is already well differentiated
by the time of hatching, the similarity of electrophoretic patterns of the major
soluble proteins, presumably including the protein hormones, to those of adults
is of interest.
SUMMARY
1. Partial decapitation of chick embryos at 33-36 h of incubation,' embryonic
hypophysectomy', markedly decreases the amount but not the electrophoretic
mobility of alkaline phosphatase and a cathodal esterase, although several other
esterases, acid phosphatase, lactate dehydrogenase, malate dehydrogenase, a
cathepsin, lipase, haemoglobin and the major protein components extractable
at low ionic strength are essentially unaffected.
2. Electrophoresis of extracts of pituitaries of chicks just prior to hatching,
at 4 days after hatching, and adults, suggests that with regard to the major
proteins, which most likely include many of the protein hormones, the pituitary
becomes well differentiated by hatching in normal embryos.
3. The absence of pituitary hormones is responsible for the 'foetal' syndrome of partial decapitates and the reduction in bone formation may well be
the result of inadequate amounts of alkaline phosphatase.
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C. MANWELL & T. W. BETZ
RESUME
%
Ueffet de la decapitation embryonnaire partielle sur Vapparition de
quelques proteines au cours du developpement, chez le poulet
1. La decapitation partielle d'embryons de poulet a 33-36 heures d'incubation, ou 'hypophysectomie embryonnaire', diminue de facon marquee la
quantite presente, mais non la mobilite electrophoretique de la phosphatase
alcaline et d'une esterase cathodique, bien que plusieurs autres esterases, la
phosphatase acide, la deshydrogenase lactique, la deshydrogenase malique, une
cathepsine, la lipase, l'hemoglobine et les principals proteines extractibles de
faible force ionique restent essentiellement non affectees.
2. L'electrophorese d'extraits d'hypophyses de poulets juste avant l'eclosion,
4 jours apres l'eclosion, et adultes, suggere que, sous le rapport des proteines
principales, qui renferment tres vraisemblablement plusieurs des hormones
proteiques, l'hypophyse est bien differenciee a l'eclosion chez les embryons
normaux.
3. L'absence d'hormones hypophysaires est responsable du syndrome
'foetal' des decapites partiels et la reduction de la formation des os pourrait
bien etre le resultat d'une teneur anormale en phosphatase alcaline.
The authors thank Professor Ray Watterson, Department of Zoology, University of
Illinois, for advice and encouragement in these investigations. Miss C. M. Ann Baker supplied
valuable information concerning electrophoretic and histochemical techniques as applied
to characterization of chicken enzymes. The research was supported by the United States
National Science Foundation (GB-612 and GB-3037). One of us (CM.) thanks the U.S.
Public Health Service for a Special Postdoctoral Fellowship (1-F3-AM-22-232-01).
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{Manuscript received 12 January 1966)