The effect of embryonic partial decapitation on the developmental sequence of some proteins in the chicken

0 Author's address: Department of Biology, Carleton University , Ottawa 1 , Canada. 6-2 1 Author's address: Laboratory of the Marine Biological Association of the U.K. , Citadel Hill, Plymouth, Devon, England 2 From the Department of Biological Science, The Florida State University, and the Department of Zoology, University of Illinois 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. - 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 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. 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 componen (...truncated)