The causal and temporal aspects of blood tissue specification in the chick embryo were investigated in this study. The main focus was on the role of basic fibroblast growth factor (bFGF) in the determination of the erythropoietic tissue, particularly in context with its representation as a non-axial mesodermal derivative which arises in the posterior domain of the chick embryo. The initial strategy employed in this study was the use of agents that are known to block the activity of bFGF, and to determine their effects on erythropoiesis. Treatment of unincubated chick embryo explants with heparin, which binds specifically to the FGF family, was found to inhibit primitive streak formation and erythropoiesis, and also inhibited the formation of other mesodermal tissues. These initial findings suggested that one or more growth factors had become bound to the heparin, and that their activity is important for the specification of primitive streak formation and mesodermal patteming. The development of the erythropoietic tissue was assayed by a cytochemical test for haemoglobin using 0dianisidine; and by histological examination for blood islands and red blood cells in serial sections of the embryos after 48 hours incubation. Microscopic examination of the embryos at the stages of gastrulation on the first day of incubation revealed that heparin caused holes to appear in the ventral layer; and although a primitive streak did not form, a middle layer of mesenchymal cells were seen to accumulate between the ectodermal and ventral "endodermal" layers. It was significant that heparin's inhibitory effect on erythropoiesis could be reversed after the addition of a recombinant bovine bFGF to the heparin-treated embryos. However, the exogenous bFGF did not neutralize the inhibitory effect of heparin on the primitive streak and other mesodermal derivatives (Chapter Two). The inhibition of erythropoiesis by heparin was also reversed by the addition of a mesodermal-inducing factor extracted from a Xenopus embryonic cell line, namely XTC. The XTC mesodermal-inducing factor (MIF), which belongs to the transforming growth factor-B family and is a homologue of activin, could also reverse the inhibitory effect of heparin on primitive streak formation; but no recognizable axial mesodermal structures subsequently developed. Of consequence, was that both bFGF and XTC-MIF blocked heparin's effect on the ventral layer, preventing the gaps forming. Therefore, it is suggestive that the VI development of an intact ventral layer is important for the determination of the erythropoietic sequence (Chapter Three). By taking a more specific approach using antisera to bFGF (anti-FGF) and the bFGF receptor (anti-FGFR) on whole embryo explants, it was found that anti-FGP and anti-FGFR were able to inhibit erythropoiesis, but not primitive streak formation. However, these antisera caused defects in the posterior region of the embryonic axis. These embryos not only lacked posterior blood tissue, but heart and somites were missing; whereas the anterior head structures were well formed. These results therefore suggest that bFGF signalling is important for the development of the posterior body plan, which includes erythropoiesis (Chapter Four). Further evidence for the role of bFGF in the determination of the blood mesodermal tissue line was reached in an in vitro bioassay. In this part of the investigation, specific pieces of the blastoderm, namely pieces dissected from the posterior marginal zone (PMZ) and inner core of the central disc (lCD) were able to form haemoglobin under particular conditions. The PMZ components were found to have the capacity to form haemoglobin when dissected from blastoderms of stages X to xm when cultured in serum-free medium. This commitment to form haemoglobin could be blocked by treatment with anti-FGP at stages X and XI, but not at the later stages of xn and XIII. The ICD components were found to have a commitment to form haemoglobin only if this component was dissected from embryos at stage XIT and XITI, but not before. These results suggest that a determinative event for the haemoglobin differentiative pathway occurs between stages XI and XII. It was also found that the stage X central disc component could be induced to form haemoglobin if a stage xm hypoblast was added to it in tissue recombination sandwich cultures, or if bFGF (75 - 150 ng/ml) was added to the medium. These results lend further support that bFGF plays an important role in the determination of erythropoiesis; and furthermore, suggest that the hypoblastic tissue is the source of this induction (Chapter Four). Finally, immunocytochemical labelling with a polyclonal antibody to bFGF has revealed that bFGF increases significantly from stage XI in cells within the developing hypoblast layer and in the middle mesodermal layer. These cells are located predominantly in the posterior domain of the embryo. This polarized distribution of bFGF with the high value of bFGF concentration in the posterior area, is presumably responsible for inducing the overlying epiblast to form the posterior horseshoe-shaped region from which blood tissue is seen to arise. An immunocytochemical analysis of the distribution of the FGF receptor was vu assessed, as an indicator of the possible competence of the cells to respond to the bFGF signal. The bFGF receptor was found to be expressed at stage XII in cells that appeared to be in register with those immunoreactive to the bFGF ligand; therefore suggesting an autocrine function. It was interesting that at stage Xli an intense immunostaining with the anti-FGFR developed in the nuclei of cells within the epiblast layer (Chapter Five). In conclusion, this study has demonstrated that the initial determination of the erythropoietic cell lineage in the chick is at the time when the hypoblast is in the process of forming beneath the epiblast, Le. between stages XI and XII. Furthermore, it was found that an induction by an FGF-like signal from the hypoblast layer (or middle mesodermal cells that may be closely associated with the hypoblast) induces "competent" cells (Le. FGFR-positive cells) in the epiblast to form blood tissue in the posterior domain of the chick embryo. / Thesis (Ph.D.)-University of Natal, 1994.

Ultrastructural observations on the six-day chick embryo lens reveal that fibre cell differentiation, which involves extensive cell elongation, is characterized by the presence of numerous oriented microtubules and by marked changes in intercellular relationships which are felt to be important for cell extension. Increased Golgi activity in cells initiating elongation appears to be related to the formation of intercellular junctions and the elaboration of new surface membrane. Studies concerning the differentiation in vitro of anterior lens epithelia demonstrate that their cells elongate either to a great extent or to a limited extent, depending on factors deriving from the epithelial conformation.The conformation of an explanted anterior lens epithelium can be manipulated so that its cells elongate consistently to either a limited or a great extent. These cells, whether elongated to a limited or to a great extent, are ultrastructurally similar with respect to features reflecting cytoplasmic differentiation, including those important for morphogenesis. Their fine structure closely resembles that of fibre cells differentiating in vivo. The cells of anterior lens epithelia differentiating in vitro, whether elongating to a limited or to a great extent, are the same in terms of the nature and relative proportions of their soluble proteins as indicated by polyacrylamide gel electrophoresis. There is a change in the relative proportions of the soluble proteins accumulated by the anterior lens epithelia when they differentiate in vitro that is similar to that which occurs in cells undertaking fibre cell differentiation in vivo. It is concluded that those factors deriving from the conformation of the anterior lens epithelium that affect the degree of elongation of its cells differentiating in vitro are physical in nature. Since such physical factors are important with regard to restraining or encouraging the expression of morphogenetic potential in vitro, it is suggested that similar physical forces are important in lens morphogenesis in vivo. Observations concerning anterior lens epithelia elongating to a great extent in vitro demonstrate that their cells progressively elongate for only about three days. Well differentiated explants cultured for longer periods do not show greater elongation or further cytoplasmic differentiation. Their cells, at the ultrastructural level, resemble differentiating fibre cells in vivo rather than differentiated ones. It is concluded that the anterior lens epithelium has some capacity for differentiation independent of the ocular environment, but that the latter is essential for the expression of its full developmental potential. / Science, Faculty of / Zoology, Department of / Graduate

In order to determine the feasibility of using the laying hen as a source of molybdenum containing eggs, a dose response experiment was conducted which involved the feeding of graded levels of molybdenum to White Rock laying hens. The feeding of 2000 ppm of molybdenum resulted in decreases in egg production and hen weight. Only egg production was decreased at 1000 ppm of molybdenum. The 500 ppm level of supplementation appeared to be most applicable to further studies in that little effect on either hen weight or egg production was noted. It was also observed that at this level the egg concentration of molybdenum plateaued at 16.4 ppm after 14 days of feeding the experimental ration. An experiment designed to assess the toxicity of the molybdenum included in the egg by the laying hen to the developing chick embryo permitted the estimation of the L.D.₁₀₀ to be about 17 ppm of molybdenum. This value agrees quite closely to the L.D.₁₀₀ of yolk sac injected molybdenum. The embryonic death response observed in this experiment did not occur during any specific period of development. Succinoxidase assays conducted to study the death response indicated that there was no difference in succinoxidase. It was further noted that there was a decline in lethal response in embryos from hens fed the molybdenum containing ration for periods in excess of three months. This decline was mediated by a decreased concentration of molybdenum in the eggs from these hens. The mode of inclusion of molybdenum in the egg, by the laying hen was studied. It appeared that the majority of the molybdenum is included during the maturation of the ova in the ovary. The high concentration of molybdenum observed in the uterus suggests that the shell be considered in future studies. A chemical fractionation of the eggs from the hens fed the molybdenum containing diet indicated that very little of the molybdenum was present in the lipid fraction. Twenty-five percent of the total egg molybdenum was found to be associated with the protein fraction. A rat assay coupled with further chemical procedures indicated that the molybdenum to protein bonding was not of a covalent nature. The observation of n growth depression in chicks which had been injected with molybdenum during their embryonic development was not corroborated by a more extensive study. Although this study was confounded by the possible existence of a copper deficiency, some evidence was obtained for the involvement of molybdenum in the copper metabolism of the chicks. It was surprising that no increase in the blood and liver molybdenum concentrations was observed when the diets containing either 500 or 1000 ppm of molybdenum were compared. The blood and liver copper concentrations were little affected by molybdenum supplementation. / Ph. D.

No description available.

No description available.

Hematocrit and hematocrit regulation have the potential to affect developing embryos. To examine the ability of chicken embryos at day 15 to regulate hematocrit, they were subjected to either repeated saline injections (5% of total blood volume) or repeated blood removal (5% of total blood volume). Embryos showed an ability to maintain hematocrit (~20%) despite blood volume increases up to 115% of initial blood volume. Embryos were not able to maintain hematocrit in the face of dramatic blood volume loss. Oxygen consumption of embryos could be affected by their level of hematocrit. To examine this, chicken embryos at day 15, 16, and 17 of incubation were given a high hematocrit (~50-60%) sample of blood (400 μl) to artificially increase the hematocrit of the embryos (~10-12%). Despite the increase in oxygen availability, when monitored over a period of six hours, embryos showed no difference (0.36 ± 0.01 (ml O2 - min-1- egg-1) in metabolism from baseline measurements at day 15, 16 and 17.

Fetal programming results from stressors during fetal development and may influence the occurrence of disease later in life. Maternal nutritional status and/or environment can affect renal development by inducing limited nephron endowment at birth, which results in diseases such as hypertension and coronary heart disease in mammals. Birds are likely to be effective models for this process because, like mammals, they have high pressure cardiovascular systems, mammalian-type nephrons and are homeothermic. This project uses the chicken embryo to explore physiological responses of disrupted hydration state thereby providing insights into renal fetal programming. Under normal conditions the chorioallantoic membrane (CAM) and developing avian kidney work in unison to ensure a proper balance of ions and water within the egg. White leghorn chicken eggs were incubated at 37.5oC±0.5oC and either <35%, 55-60% (normal) or >85% relative humidity. Amniotic fluid serves as the drinking source for the embryo late in development; its composition is important to salt and water homeostasis. High amniotic fluid osmolality increased the blood osmolality for embryos exposed to low humidity incubation thereby indirectly influencing the renal developmental program of the embryos from this group. Indeed estimated filtering capacity was doubled in the low humidity group (6.77 ± 0.43 mm3) compared to normal (4.80 ± 0.33 mm3) and high (3.97 ± 0.30 mm3) humidity groups. The increased filtering capacity seen for those embryos from low humidity may indicate the ability for more efficient recovery of water if similarly stressed as an adult bird. All embryo populations maintained similar oxygen consumption (0.075 ml/min - 0.37 ml/min), hematocrit (15 % - 32 %) and hemoglobin values (4 g/dl - 9 g/dl), thus displaying control over these aspects of the internal environment despite the obvious environmental insult of extreme incubation humidity. These results signify the embryo's immature kidney, along with lower gastrointestinal tract, functions much like the adult form maintaining homeostasis, although the mechanisms may differ. The overall benefits of this research included better understanding of the role the kidney during embryonic development and determining whether environmental factors, such as humidity, leave an imprint on morphological and physiological aspects of the urinary system of the embryo and water compartments of the egg.

How temperature affects physiological development in the chicken embryo is unknown. Embryos incubated at 38°C or 35°C showed no difference in growth or survival. The time to hatching was longer in 35°C than 38°C embryos (23.7 vs. 20.6 days), but unaffected was the relative timing of appearance of developmental landmarks (internal, external pipping). At stage 43-44, 38°C embryos maintained oxygen consumption around 1 mL/g/h despite acute temperature reduction (suggesting thermoregulatory maturation), unlike 35°C embryos. In 35°C embryos the lower oxygen-carrying capacity and temperature insensitive blood O2 affinity (P50 about 30 mmHg) may restrict O2 delivery to tissues, limiting metabolism during decreased ambient temperature. Reduced incubation temperature retards normal hematological and thermoregulatory development.

No description available.

The objective was to study the enzymatic production of triiodothyronine (T₃) in the intestine of chickens during perinatal intestinal maturation in two lines of chickens selected for high (HW) or low (LW) body weight at eight weeks of age. Valid assay conditions (proportionality of enzyme activity with enzyme concentration and assay time) were established and the intestinal 5'-deiodinase (5'D) activity was characterized for comparison with other tissues. The characterization studies showed that intestinal 5'D is like the Type I 5'D in liver of birds and mammals previously studied. Specific activity of adult intestinal 5'D is significantly higher in the HW than in the LW line. In both lines intestinal 5'D increases significantly between embryos that have not pipped into the air cell (NP) and embryos that have pipped into the air cell (AC) and 5'D activity peaks in embryos that have pipped through the shell (TS). In contrast to the line differences in adults, LW embryos have much higher 5'D specific activity than HW embryos until 1d posthatch. Plasma thyroxine (T₄) and T₃ also increased between consecutive stages and peaked in embryos pipped through the shell, then decreased abruptly at 1d posthatch. Both plasma hormones were higher at each perinatal stage in the LW line than in the HW line and the LW line hatched earlier than HW. Intestinal alkaline phosphatase (a marker of differentiation) showed a significant increase in activity at each of the stages of development in both lines. Alkaline phosphatase activity was significantly higher in the LW line than the HW line at the NP, AC and TS stages but not at 1d posthatch. Previous work in other laboratories indicates that T₃ plays a role in triggering intestinal differentiation and maturation of intestinal function for posthatching life. The results of this study indicate that T₃ for this signal originates at least partially from 5'deiodination of T₄ within the intestinal tissues as well as from T₃ available in the plasma. / Master of Science