Developmental increase in the inotropic and cyclic AMP response to isoproterenol in embryonic and newly hatched chicks

The cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels of ventricles isolated from 15- to 20-day-old chick embryos and 0- to 3-day-old hatched chicks were compared to clarify the mechanism underlying the change in sensitivity to isoproterenol during perinatal developmental stages when the functional sympathetic innervation has been completely achieved. Isoproterenol produced a positive inotropic effect on ventricles isolated from both embryonic and hatched chicks, but the ventricles from the hatched chicks were more sensitive. At both developmental stages sotalol was an equipotent antagonist of isoproterenol. 3-Isobutyl-1-methylxanthine (IBMX) produced an increment in the contractile force of the ventricles at both stages, but the ventricles from the hatched chicks responded to lower doses of IBMX. The reactivity to isoproterenol in increasing cyclic AMP level was significantly higher in the hatched ventricles than in the embryonic ventricles. The results suggest that the different sensitivities to isoproterenol between embryonic and newly hatched chick ventricles may be due to some changes in the process for cyclic AMP production.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Notes and queries

There is presently great interest in using early embryonic tissues, particularly human tissue, for studies of protein and gene expression. Embryonic human tissue is very fragile, and delays often occur before it can be properly prepared for scientific study. Using chick embryos, we have studied the effects of delaying fixation or biochemical isolation on the preservation of cytological characteristics and biochemical molecules. Our study shows that by 60 min post-harvest, tissue morphology and immunofluorescence staining degrades, but the total mRNA profile remains stable. This study suggests that the time between removal of the tissue and fixation is critical to the results and that the critical time is much shorter for embryonic tissues than for more developed tissues. Our results have implications for all research where embryonic tissues are harvested but not processed immediately.     

Preservation of the morphological and molecular stability of embryonic tissues

There is presently great interest in using early embryonic tissues, particularly human tissue, for studies of protein and gene expression. Embryonic human tissue is very fragile, and delays often occur before it can be properly prepared for scientific study. Using chick embryos, we have studied the effects of delaying fixation or biochemical isolation on the preservation of cytological characteristics and biochemical molecules. Our study shows that by 60 min post-harvest, tissue morphology and immunofluorescence staining degrades, but the total mRNA profile remains stable. This study suggests that the time between removal of the tissue and fixation is critical to the results and that the critical time is much shorter for embryonic tissues than for more developed tissues. Our results have implications for all research where embryonic tissues are harvested but not processed immediately.     

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.     

Dynamic properties of axonal transport of proteins and glycoproteins: A study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.     

Developmental changes in vitamin A level and lack of retinyl palmitate in chick lungs

1. Developmental changes in retinol and retinyl palmitate contents in lungs of chick embryos and posthatch chicks were investigated. 2. Remarkable changes in the lung retinol levels were found during development of chicks. Embryonic lungs 5 days prior to hatching contained the highest content of retinol. The level then declined rapidly and was lowest on 1 day before hatching. 3. Its level then rose substantially within 7 days after hatching. 4. No retinyl palmitate in chick lungs was detectable at any of the developmental stages examined, nor even in adult hen. 5. Serum retinol level changed in parallel with the lung retinol. 6. The patterns of changes in liver retinol and retinyl palmitate were remarkably different from that occurring in the lung retinol. In chick embryonic livers, the levels of them were low, followed by a rapid increase after hatching. 7. The high level and its rapid decrease of lung retinol content during development of chick embryos may be functionally connected with retinol action in embryonic lungs for cellular differentiation and maturation.     

The ossification centers in the lower end of tibiotarsus in domestic fowl.

The morphogenesis of lower end of tibia in chick is studied commonly but the process of ossification of the same has received very little attention so far. The present study is directed to throw some light on the appearance of ossification centers in the lower end of tibiotarsus of chick. The histology of lower end of tibiotarsus was studied by procuring developing tibiotarsi from chick embryos (20) of 6th day incubation till hatching and 3 post hatched chicks. The transparancies of chick embryos at different incubation periods and post hatched chicks were prepared by Dawson's Alizarin staining method. Three cartilage center (tibial, fibulare and intermedium) appeared in 6...9 days of incubation period in the tarsal region. These gradually fused with the lower end of tibia. Three ossification centres developed in the lower end of tibiotarsus. One for intermedium appeared on 16th day and two fotibial and fibulare on 20th day. All these three centres could be located in the transparancies of the chick embryos in tarsal region. The present study proves that the three cartilages centres maintain their individuality during the ossification process even though those fuse completely with the lower end of tibia in chick. The centers for tibial and fibulare are similar to epiphyseal centres of mammals in histological details.     

Metabolism of cholesterol in the tissues and blood of the chick embryo

Three artificially inseminated laying White Leghorn hens were given 35-50 micro c of cholesterol-4-(14)C intravenously. Their subsequently produced eggs contained cholesterol-(14)C-labeled yolks. Some of the fertilized eggs were analyzed for cholesterol content and radioactivity. Other eggs were incubated until hatching. The specific activity of the cholesterol contained in the serum and tissues of newly hatched chicks was determined and compared with that of yolk sac, which was taken as representative of egg yolk cholesterol before its metabolic transfer into the chick embryo. The specific activities of cholesterol in intestine, liver, serum, heart, and skeletal muscle and the whole chick were 95-98% of that in yolk sac, but that of brain cholesterol was only 11% of this value. These results indicate that whereas most of the cholesterol in the chick originated from the egg yolk, cholesterol biosynthesis was active in the brain and provided about 90% of its cholestero content. Newly hatched chicks were found to be hyperlipemic compared with older chicks and had fatty livers with a high cholesterol content. Desmosterol was found in 9- and 15-day old chick embryos but not in the newly hatched chicks, in which the only sterol was cholesterol.     

The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick

Trypsin inhibitor and proteolytic activities were studied in incubated eggs, embryos, and newly hatched chicks. After rupture of the secondary seroamniotic suture at 11 days, the trypsin inhibitor content of the albumen gradually passes into the amniotic cavity; from there it is taken up orally by the chick embryo. It is supposed that between 11 and 18 days of embryonic development the trypsin inhibitor passes from the gut to the yolk sac through the vitellointestinal duct. The thin yolk contained only traces of trypsin inhibitor, and the allantoic fluid was entirely free from it. The amylase activity demonstrable in the liquid intestinal contents of the chick embryo indicates the presence of pancreatic secretion. The trypsin inhibitor probably suppresses the proteases not only directly, but also through prevention of the activation of zymogens. Enterocytes of chick embryos showed no morphological indication of the absorption of undigested proteins on histological examination. The cloacal membrane of the newly hatched chick ruptures shortly after the bird has dried up, and the trypsin inhibitor is subsequently eliminated along with the intestinal contents. The intestinal proteolytic enzymes appear immediately afterward. The proteolytic activity appeared regardless of whether the birds were or were not fed. Maximum proteolytic activity was measured in the small intestine of chicks that were fasted for 2 days after hatching. The pattern of proteolytic enzymes as well as their sensitivity to protease inhibitors did not notably differ from that of mammals.     

Hormonal regulation of fatty acid synthetase in chick embryo liver.

Fatty acid synthetase activity in chick embryonic liver is negligible compared to that in newly hatched, fed chicks. The enzyme activity is prematurely induced 5–50-fold in 20-day-old embryos and in newly hatched chicks by the administration of insulin, hydrocortisone, growth hormone, glucagon or dibutyryl cyclic AMP. The induction of the enzyme activity is blocked by the administration of cycloheximide, indicating that new protein synthesis is required. Immunochemical titrations of different enzyme preparations from 5-day-old chicks, adult chicken and various inducer-treated embryos gave an identical equivalence point, indicating that the changes in synthetase activity after hormonal induction in embryos are related entirely to changes in content of enzyme. The increase in liver synthetase content after administration of insulin, glucagon or dibutyryl cyclic AMP is directly related to an increase in the rate of synthetase synthesis. The induction of the synthetase activity by suboptimal doses of glucagon or cyclic AMP is potentiated by the phosphodiesterase inhibitory theophylline. There is a very rapid decay of synthetase activity, with a half-life of about 4 h after elevation to higher levels following administration of insulin, glucagon or dibutyryl cyclic AMP. Glucagon and dibutyryl cyclic AMP induction of the synthetase activity is observed early in the embryonic development, whereas insulin induction is noted 2 days before hatching. Insulin, glucagon and cyclic AMP are potentially capable of altering the levels of glycolytic intermediates which may be involved in the induction of synthetase.     

Synthesis of Retinal Gangliosides During Chick Embryonic Development

Abstract: Embryonic retina cells incorporated radioactivity from D-[6-³H]glucosamine into gangliosides in vitro. The incorporation was higher in retinas from younger embryos. The pattern of labeling of individual gangliosides of the retina changed gradually from a predominant labeling of gangliosides running chromatographically as GD₃ (nomenclature of Svennerholm) and GM₃ in retinas from 8-day-old embryos to a predominant labeling of those running as GD_Ia and GT, in retinas from 13–18-day-old embryos and newly hatched chicks. The shift in the pattern of labeling correlated with a temporary increase of about sixfold of the activity of UDP-N-acetyl-galactosamine:GM₃ N-acetylgalactosaminyltransferase occurring between days 8 and 14 of embryonic development and with a regular increase of the activity of the UDP-galactose:GM₂ galactosyltransferase occurring from day 8 until hatching. The activities of the CMP-NeuAc:lactosylceramide-, CMP-NeuAc:GM_3-, and CMP-NeuAc:GM₁-sialosyltransferases in the retinas of newly hatched chicks were 40, 20, and 40%(in comparison with the corresponding activities determined in retinas of the 8-day-old embryo.     

Hematological toxicology following embryonic exposure to aflatoxin-B1

The influence of embryonic exposure to aflatoxin-B1 (AF-B1) upon the erythroid system of the maturing chicken was examined using a variety of assays. Since the chick embryo is known to possess mixed-function oxidase activity, this animal serves as an excellent model system for studies of human fetal toxicology. AF-B1 (0.1 microgram) was administered to either 6- or 12-day embryos by the air sac method. This level of AF-B1 was highly mutagenic and was found to induce an average of 10.6 sister chromatid exchanges (SCEs) per cell compared with 1.8 SCEs per cell for the acetone control solvent. Despite selection against treated embryos through acute and chronic embryonic toxicity, hatched chicks from AF-B1 treatment groups exhibited erythroid anemia when compared to the acetone controls. Cell count, hematocrit, and hemoglobin concentration were all significantly reduced in the 12-day AF-B1 treatment groups compared with controls. Both sexes were equally affected. While the number of peripheral erythrocytes was reduced following exposure to AF-B1, the differentiation status of erythrocytes was apparently unaltered. Mean cell volume, percentage of circulating reticulocytes, and incidence of an erythroid differentiation marker, chicken fetal antigen, were parameters in which no treatment effects were observed. An apparent maturation effect was noted since adult hematocrits were similar between control and treatment groups. Possible explanations for this age effect are discussed. The ability to detect significant posthatch erythroid toxicity following embryonic exposure to mutagenic levels of AF-B1 suggests the importance of this general approach to perinatal carcinogenic evaluation.     

Development of cardiac rhythms in birds

Heart rate (HR) in avian embryos developing inside an eggshell has been measured by various means while maintaining adequate gas exchange through the eggshell. This is an important requirement in order to avoid adverse effects of impeding gas exchange on the cardiac rhythms of developing embryos. The present report is a review of our ontogenetic study on embryonic HR, which was measured with fulfillment of the above requirement and also hatchling HR measured non-invasively. Firstly, we reviewed measurements of daily changes (developmental patterns) in embryonic mean heart rate (MHR), which were determined from a short-term measurement of HR once a day, in 34 species of altricial and precocial birds. The allometric relationship between the MHR during pipping in altricial birds and their fresh egg masses was the same as that between the MHR at 80% of incubation duration and fresh egg masses in pre-cocial birds. Secondly, we presented the developmental patterns of MHR in chick embryos and hatchlings, which were determined from long-term, continuous measurement of HR before, during and after hatching. The ultradian and circadian rhythms of HR were clearly shown in embryos and hatchlings, respectively. Thirdly, we summarized instantaneous HR fluctuations: HR variability and HR irregularities, in chick embryos and hatchlings. The distinctive patterns were shown in pre-pipped and pipped embryos and newly hatched chicks, individually, which were partly related to autonomic nervous functions and physiological functions.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409]     

Establishment of pluripotent cell lines from porcine preimplantation embryos

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Reconstitution of muscarinic cholinergic inhibition of adenylate cyclase activity in homogenates of embryonic chick hearts by membranes of adult chick hearts

We have previously demonstrated that during embryonic development of the chick heart between days 2 1/2 and 10 days in ovo, muscarinic cholinergic inhibition of isoproterenol-stimulated adenylate cyclase activity increased 4-fold, and the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine increased 26-fold. Although the number of muscarinic receptors remained constant between days 2 1/2 and 10 in ovo, the levels of a 39- and 41-kDa pertussis toxin substrate increased in parallel with the ability of muscarinic agonist to inhibit adenylate cyclase activity (Liang. B.T., Hellmich, M. R., Neer, E. J., and Galper, J. B. (1986) J. Biol. Chem. 261, 9011-9021). These data are consistent with the hypothesis that between days 2 1/2 and 10 in ovo muscarinic receptors were uncoupled from inhibition of adenylate cyclase activity because of limiting levels of pertussis toxin substrates. In the current studies, in order to test this hypothesis homogenates of embryonic chick hearts 3 1/2 days in ovo were reconstituted with membranes from hearts of hatched chicks. In order to rule out reconstitution by factors from hatched chick hearts other than pertussis toxin substrates, muscarinic receptors from hatched chick hearts were inactivated by covalent binding of benzilycholine mustard and adenylate cyclase inactivated by N-ethylmaleimide prior to reconstitution. Reconstitution of benzilylcholine mustard/N-ethylmaleimide treated hatched chick heart membranes with homogenates of embryonic chick hearts 3 1/2 days in ovo resulted in a 2 1/2-fold increase in the ability of carbamylcholine to inhibit adenylate cyclase activity and reconstitution of hatched chick heart membranes with homogenates of hearts 2 1/2 days in ovo resulted in an approximately 10-fold increase in the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine. Membranes from hearts of hatched chicks which had been injected with pertussis toxin were incapable of reconstituting muscarinic inhibition of adenylate cyclase activity in homogenates of hearts 3 1/2 days in ovo. These data support the conclusion that early in embryonic development coupling of muscarinic receptors to inhibition of adenylate cyclase activity is limited by the availability of a pertussis toxin substrate.