The effect of early prenatal hypercapnia on the vascular network in the chorioallantoic membrane of the chicken embryo

Over the last decade, the poultry sector has sought to develop novel ways to monitor chicken embryonic growth, health, and quality to control and optimize egg incubation conditions, particularly the concentration of dissolved gases (O(2), CO(2)). One of the parameters, which may change under different gas concentrations, is the angiogenesis in the chorioallantoic membrane (CAM), the organ for gas exchange of the chicken embryo. In this study, a newly developed methodology was used to quantify the angiogenesis in the CAM under normal and early hypercapnic conditions (i.e., increased CO(2) concentrations). Two experiments were conducted in which the same CO(2) profile was applied. The development of the vascular system was monitored from embryonic day (ED) 10 until ED 14 in Experiment 1, and until ED 16 in Experiment 2. This development was characterized by two different parameters-the vascular fraction (VF) as a measure for the density of the vascular network and the fractal dimension (FD) as a measure for the degree of branching of the vascular network. Moreover, in Experiment 2, embryo weights were compared between both groups. The proposed methodology showed that differences in the development of the vascular system could be observed across groups but also as function of the ED. Both VF and FD and the embryo weights were shown to be higher in the hypercapnia group compared to the control group.     

Development of a fast, objective, quantitative methodology to monitor angiogenesis in the chicken chorioallantoic membrane during development

Recent research investigates the role of different gas concentrations during incubation, on chicken growth, quality and health post hatch. One of the parameters of chicken development which changes under different gas concentrations is angiogenesis in the chorioallantoic membrane (CAM). To be able to perform large incubation experiments under different conditions, angiogenesis in the whole CAM must be quantified objectively and easily. In this paper, a fast, objective, quantitative methodology to assess changes in the overall vascular development in the CAM of chicken embryos is presented. Samples were taken with minimal disturbance by emptying the egg, so that the CAM stayed attached to the shell, which was then cut in pieces. We employed a commercial digital camera and a macro lens set at 5x magnification to take pictures with sufficient contrast and resolution (2.64 mm/pixel). These were processed with computer algorithms to calculate the vascular fraction (VF) and the fractal dimension (FD) automatically on binary images. The ratio of the repeatability and reproducibility variation compared to the parts variation was 0.32 for VF and 0.21 for FD. In a validation experiment (n=284), one group was incubated under hypoxic conditions and the other under normoxic conditions. It was shown that early hypoxia stimulated angiogenesis, while chronic hypoxia impeded growth with significant differences between both groups, which is in accordance with literature data. Thus, we report here a method to asses overall angiogenesis in the CAM under different incubation conditions.     

The effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

Effects of exogenous heparin on the vasculogenesis of the chorioallantoic membrane

On the basis of the well-known stimulating role played by heparin on blood vessel neoformation in the chorioallantoic membrane (CAM), demonstrated by means of slow-release polymers soaked in this substance and placed on CAMs, a new technique of introducing heparin directly into the allantoic sac is proposed. According to observations carried out in ovo and after fixation, morphological modifications are demonstrated in the developing vasculature of the heparin-treated CAMs which, compared with the control CAMs, show dilated and sinuous arterial and venous branches, denser and irregular capillary networks, and a high number of vascular primordia. The results, confirming that heparin is involved in angiogenesis, indicate the suitability of the technique and suggest that the effects of this substance could be enhanced by growth factors released by CAM tissues rapidly growing up from the 5th to the 7th incubation day.     

RNA-seq analysis of the active chick embryo chorioallantoic membrane reveals genes that encode proteins assigned to ion transport and innate immunity

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology.     

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.     

Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.     

Geographic variation in lizard phenotypes: importance of the incubation environment

Geographic variation in phenotypes can result from proximate environmental effects as well as from underlying genetic factors. Reciprocal transplant experiments, in which organisms are moved from one area to another, offer a powerful technique to partition the effects of these two factors. However, many studies that have utilized this technique have focused on the post-hatching organism only and ignored potential effects of environmental influences acting during embryonic development. We examined the phenotypic responses of hatchling scincid lizards ( Lampropholis guichenoti ) incubated in the laboratory under thermal regimes characteristic of natural nests in two study areas in southeastern Australia. Although the sites were less than 120 km apart, lizards from these two areas differed in thermal regimes of natural nests, and in hatchling phenotypes (morphology, locomotor performance). We incubated eggs from each area under the thermal regimes typical of both sites. Some of the traits we measured (e.g. hatchling mass and snout-vent length) showed little or no phenotypic plasticity in response to differences in incubation conditions, whereas other traits (e.g. incubation period, tail length, inter-limb length, body shape, locomotor performance) were strongly influenced by the thermal regime experienced by the embryo. Thus, a significant proportion of the geographic variation in morphology and locomotor performance of hatchling lizards may be directly induced by differences in nest temperatures rather than by genetic divergence. We suggest that future studies using the reciprocal transplant design should consider environmental influences on all stages of the life-history, including embryonic development as well as post-hatching life.     

Dimethyl phenyl piperazine iodide (DMPP) induces glioma regression by inhibiting angiogenesis

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis of DMPP's inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis – possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis.     

Pericytes in experimental MDA-MB231 tumor angiogenesis

The role of pericytes (PCs) during embryonic or tumor angiogenesis is a matter of debate. We studied the expression of cytoskeletal, membrane, and matrix markers in experimental tumors of the human mammary ductal adenoma MDA-MB231 cell line that were grown on avian chorioallantoic membranes (CAMs) from incubation day 10 to 18 (chick) or 8 to 15 (quail). The expression patterns of alpha-smooth muscle actin (alphaSMA) and desmin, of adhesion molecules beta1 integrin and neurothelin, and of fibronectin and laminin were analyzed with conventional and confocal laser scanning microscopy. The CAM arterial wall showed strong alphaSMA signal in all smooth muscle cell layers but the innermost layer, which was desmin positive. Ramified alphaSMA-negative cells with delicate desmin staining accompanied most minor vessels and were also seen basal to the capillary plexus indicating the presence of PCs. In the tumor nodules, a diffuse alphaSMA signal without definite relationship to vascular structures was detected. Strongly desmin-positive, alphaSMA-negative cells were frequent in the zone of contact to the CAM in small nodules, and were scattered in larger tumors. In some regions they were associated with microvessels, and in others appeared detaching from endothelial cells (ECs) or as single migrating cells. We conclude that: (a) the CAM tumor angiogenesis assay is useful for studying PC/EC interactions, (b) PCs are recruited from the CAM into experimental tumor nodules, (c) variability of vasculature in MDA-MB231 tumors may be due to variable PC/EC interactions, and (d) alphaSMA should be used with caution as a general PC marker.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

Regional hypoxia elicits regional changes in chorioallantoic membrane vascular density in alligator but not chicken embryos

Hypoxic incubation increases vascularization in the chick chorioallantoic membrane (CAM). The effect of regional hypoxia on the vascular density of American alligator (Alligator mississippiensis) and chicken (Gallus gallus) CAMs was studied to determine if hypoxic proliferation of blood vessels is localized or global across the CAM. Eggs were incubated under normoxic conditions with a portion of the eggshell covered with non-toxic beeswax to induce external regional hypoxia. CAMs were examined under a microscope with a 'bulls eye' coverslip and a vascular density index (VDI) was determined. The hypoxic portions of the alligator CAMs were more vascular than the normoxic portions (VDI = 200.9 versus 157. 8, respectively). Presumably this response is maladaptive by causing increased blood flow to the poorly oxygenated portions, i.e. increased 'shunt'. Thus, we hypothesize increased vascularity due to hypoxic incubation is due to local release and subsequent rapid local breakdown or uptake of angiogenic factors. In contrast, the hypoxic and normoxic portions of the chick CAMs exhibited virtually no difference in VDI (VDI= 211.5 versus 217.9, respectively). We suggest the air cell and air space of the chicken eggs allows for circulation of gas in ovo, eliminating the possibility of regional internal hypoxia.     

Enhanced survival of motoneurons in the chick lateral motor column: effects of embryonic skeletal muscle extracts and myoblast-conditioned medium

Muscle-derived factors have shown neurotrophic effects in culture, but their possible effects on the maintenance of embryonic motoneurons have not been demonstrated in vivo. Soluble extracts derived from embryonic chick muscle, or medium conditioned by chick myoblasts, were instilled onto the chorioallantoic membrane (CAM) between the 5th and 11th days of incubation. Counts of the lumbar lateral motor column (LMC) at embryonic day 12 revealed modest but significant increases (12-15%) in motoneuron number for these experimental groups as compared with control treatments. The results suggest that sustaining effects of muscle-derived factors on motoneurons may be demonstrated on the developing LMC by the simple expedient delivery via the CAM, and that these factors can modify the normal program of cell death occurring during this critical period of development.     

The chick embryo chorioallantoic membrane as an in vivo experimental model to study human neuroblastoma

The chick embryo chorioallantoic membrane (CAM) has long been a favored system for the study of tumor angiogenesis because at the stage of development when generally tumor grafts are placed (6–10 days of incubation), the chick’s immunocompetent system is not fully developed and the conditions for rejection have not yet been established. All studies for mammalian neoplasms, including neuroblastoma, have used tumor cell lines, tumor bioptic specimens, cell suspensions derived from tumors, and mouse tumor xenografts bioptic specimens. CAM can also be used to study the effects of antiangiogenic molecules on tumor cell suspensions of tumor bioptic specimens. This review article summarizes and discusses the literature data on the use of the CAM as an in vivo experimental model to study human neuroblastoma.     

Irisin attenuates oxidized low-density lipoprotein impaired angiogenesis through AKT/mTOR/S6K1/Nrf2 pathway

It is known that irisin increases total body energy expenditure, decreases body weight, and enhances insulin sensitivity. Although previous studies have demonstrated that irisin induces vascular endothelial cell (EC) angiogenesis, the molecular mechanisms underlying irisin-induced angiogenesis under conditions reflecting atherosclerosis are not known. The aim of the present study is to investigate whether irisin could inhibit oxidized low-density lipoprotein (oxLDL) impaired angiogenesis. We investigated the effect of irisin on angiogenesis in vitro by evaluating cell viability, cell migration, and the capacity to form capillary-like tubes using human umbilical vein endothelial cells and human microvascular endothelial cells (HUVECs and HMEC-1) that were treated with oxLDL. We also evaluated the effects of irisin on angiogenesis in vivo by Matrigel plug angiogenesis assay and in a chicken embryo membrane (CAM) model. Our results demonstrated that irisin increased oxLDL-treated EC viability as well as migration and tube formation. Moreover, oxLDL inhibited angiogenic response in vivo, both in the Matrigel plug angiogenesis assay and in the CAM model, and was attenuated by irisin. Furthermore, irisin decreased apoptosis, inflammatory cytokines, and intracellular reactive oxygen species (ROS) levels in oxLDL-treated EC. In addition, we found that irisin upregulated pAkt/mTOR/Nrf2 in oxLDL-treated EC. Both mTOR/Nrf2 shRNA and LY294002 could inhibit the protective effect of irisin. Taken together these results, they suggested that irisin attenuates oxLDL-induced vascular injury by activating the Akt/mTOR/Nrf2 pathway. Our findings suggest that irisin attenuates oxLDL-induced blood vessel injury.     

Human natural killer cell adhesion molecules. Differential expression after activation and participation in cytolysis.

Cell adhesion molecules (CAM) participate in interactions between lymphocytes, accessory cells, and target cells that are critical in the generation of effective immune responses. To characterize the involvement of CAM in NK and lymphokine activated killer (LAK) activities, we examined the expression of several CAM by freshly isolated human NK cells and by NK cells activated in vitro with IL-2, and compared this to CAM expression by T lymphocytes under similar conditions. Freshly isolated human NK cells were uniformly LFA-3 (CD58)+ and expressed two to three-fold higher surface levels of LFA-1 (CD11a/CD18) than resting T lymphocytes. More NK cells than T cells also expressed phenotypically detectable levels of intercellular adhesion molecule-1 (CD54). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD11a/CD18, CD2, CD54, CD58, and the NK cell-associated Ag NKH-1 (CD56). Furthermore, essentially all NK cells became CD54+ within 3 days of exposure to IL-2. T cells did not demonstrate comparable up-regulation of CAM after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of E:T cell conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity). The LAK activity induced by exogenous IL-2 could be partially inhibited by anti-CD2, anti-CD11a, or anti-CD54 antibodies and almost completely abrogated by anti-CD2 and anti-CD11a in combination. These studies suggest that CAM play a central role in the regulation of NK cytolysis, and that changes in CAM expression may alter the target cell specificity of activated NK effectors.     

Time course of the age-related alterations in stored blood

The extent and time course of the impairments occurring in whole blood and erythrocyte cells stored under blood bank conditions were studied by monitoring the reduction of MAL-6 spin label added to the media containing whole blood or erythrocyte cells using electron spin resonance (ESR) technique. Impairments forming in the erythrocyte cells incubated for various times at 37 degrees C were also studied. Erythrocyte cells were found to undergo changes during the storage or incubation, leading to fast decay of MAL-6 spin labels signal height. The extent of the changes depends on storage or incubation time. However, the reduction in incubated or artificially aged erythrocyte (AAE) cells was faster than the reduction in whole blood (WB) and aged erythrocyte (AE) cells stored under blood bank conditions. Two exponential curves attributed to the liquid and cellular parts of a given samples were found to be described best in the reduction of MAL-6 spin label in WB, AE and AAE.     

Modulation of angiogenesis by topical application of leptin and high and low molecular heparin using the Japanese quail chorioallantoic membrane model

Pathological angiogenesis characterized by uncontrollable vessel growth is an accompanying feature of many diseases. The avian embryo chorioallantoic membrane (CAM) is an excellent model for angiogenesis research. In our study we used a less common Japanese quail CAM model for the testing of angiogenic potential of leptin, high-molecular (heparin sodium) andlow-molecular (nadroparin calcium) heparins. Heparins play a significant role in vascular endothelial cell function, and they are able to modulate the activities of angiogenic growth factors. On embryonic day 7 leptin (5 μg per CAM), heparin sodium (75 IU per CAM) and nadroparin calcium (47.5 IU per CAM) in 500 μl PBS were applied on the CAM surface. After 24 h the fractal dimension (Df) of the vasculature was evaluated. Samples from each group were histologically analyzed and VEGF-A and Quek1 expression were detected by qPCR. Df was significantly increased in the leptin group. A moderate stimulatory effect of heparin sodium and an inhibitory effect of nadroparin calcium were observed. Both leptin and heparin sodium caused a noticeable increase in the CAM thickness compared to the control and nadroparin calcium groups. We observed an increased number of blood vessels and accumulation of fibroblasts. There was no significant impact on gene expression of VEGF-A and Quek1 24 h after treatment, however, trends similar to the changes in Df and CAM thickness were present. The resulting effect of nadroparin administration on Quek1 levels was exactly the opposite to that of leptin (p < 0.05).     

Inhibitory effects of a gradient static magnetic field on normal angiogenesis

Angiogenesis, the formation of new blood vessels, is critical in many normal and pathological processes such as development, reproduction, tumor growth, and metastasis. Recently, exposure to moderate‐intensity static magnetic fields (1 mT to 1 T) has attracted much attention for its potential therapeutic value as a noninvasive intervening method. Nevertheless, the effects of moderate‐intensity and spatial gradient static magnetic fields (GSMF) on angiogenesis have not received enough attention. In this study, the effects of GSMF (0.2–0.4 T, 2.09 T/m, 1–11 days) on angiogenesis were investigated both in vitro and in vivo. An MTT assay was used as an in vitro method to detect the proliferation ability of human umbilical veins endothelial cells (HUVECs). Two kinds of in vivo models, a chick chorioallantoic membrane (CAM) and a matrigel plug, were used to detect the effects of GSMF on angiogenesis. The results showed that the proliferation ability of HUVECs was significantly inhibited 24 h after the onset of exposure. With regard to the CAM model, vascular numbers in the CAM that was continuously exposed to the GSMF were all less than those in normal condition. In accordance with the gross appearance, the contents of hemoglobin in the models exposed to GSMF for 7–9 days were also less. In addition, similar to the CAM model, the results of vascular density and hemoglobin contents in the matrigel plug also demonstrated that the GSMF exposure for 7 or 11 days inhibited vascularization. These findings indicate that GSMF might inhibit or prevent new blood vessels formation and could be helpful for the treatment of some diseases relevant to pathological angiogenesis. Bioelectromagnetics 30:446–453, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of photoperiod and ageing on the carbon isotope composition of Bryophyllum daigremontianum Berger

Abstract. Changes in crassulacean acid metabolism (CAM) in the leaves of Bryophyllum daigremontianum were studied comparatively under closely controlled (phytotron) and semi-natural (greenhouse) conditions utilizing measurements of natural carbon isotope composition (δ¹³C) of the total leaf matter. Abrupt transfer of plants from long days to short days resulted in an overall increase of CAM in mature leaves (no. 3 from the apex and older) and thus in a higher CAM level for the whole plant. Study during the course of a year in semi-natural conditions showed that a progressive increase in length of day and day night temperature differences (February ← June) activated CAM but only a passage to short days (June ← October) produced the maximum CAM. Under the experimental conditions employed, the maximum shift from the C₃-type to the C₄-type of metabolism was observed in plants subjected to semi-natural progressive variations in the environment, i.e. the δ¹³C values indicated that for plants in the greenhouse the total carbon flow entering the cells was mediated by phosphoenolpyruvate carboxylase activity.