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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 84 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 85 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 86 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. 88 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). REFERENCES K.-E., BECKMAN, L. & LUNDIN, D. (1963). Genetic variations of human serum phosphatase. Ada genet. Statist, med. 13, 89-94. BARRETT, R. J., FRIESEN, H. & ASTWOOD, E. B. (1962). Characterization of pituitary and peptide hormones by electrophoresis in starch gel. /. biol. Chem. 237, 432-9. CATT, K. & MOFFAT, B. (1965). Fractionation of rat pituitary extract by starch-gel electrophoresis and identification of growth hormone and prolactin. Endocrinology 76, 678-85. CONKLIN, J. L. & NEBEL, E. J. (1965). Malate dehydrogenase isozymes of the chick embryo. /. Histochem. Cytochem. 13, 510-14. FERGUSON, K. A. & WALLACE, A. L. C. (1961). Starch gel electrophoresis of anterior pituitary hormones. Nature, Lond. 190, 629-30. HINNI, J. B. & WATTERSON, R. L. (1963). Modified development of the duodenum of chick embryos hypophysectomized by partial decapitation. /. Morph. 113, 381-426. JOHNSON, E. M. (1965). Electrophoretic analysis of abnormal development. Proc. Soc. exp. Biol. Med. 118, 9-11. MANWELL, C. (1966). Metamorphosis and gene action. I. Electrophoresis of dehydrogenases, esterases, phosphatases, hemoglobins and other soluble proteins of tadpole and adult bullfrogs. Comp. Biochem. Physiol. 17, 805-23. MANWELL, C , BAKER, C. M. A. &BETZ, T. W. (1966). Ontogeny of haemoglobin in the chicken. /. Embryol. exp. Morph. 16, 65-81. ARFORS, Embryonic hypophysectomy 89 F. (1959). The adaptations of alkaline and acid phosphatases in development. In Cell, Organism, and Mileu, 17th Symp. Dev. Growth, pp. 121-55. Ed. D. Rudnick. New York: Ronald Press. RENDEL, J. & STORMONT, C. (1964). Variants of ovine alkaline serum phosphatases and their association with the R-O blood groups. Proc. Soc. exp. Biol. Med. 115, 853-6. SHORE, R. E. (1965). An electrophoretic analysis of proteins of cellular sap in normal and hybrid frog embryos. /. Embryol. exp. Morph. 14, 1-14. SIMKISS, K. (1964). Phosphatases as crystal poisons of calcification. Biol. Rev. 39, 487-505. WILCOX, F. H. (1963). Genetic control of serum alkaline phosphatase in the chicken. /. exp. Zool. 152, 195-204. MOOG, {Manuscript received 12 January 1966)