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The ribonucleotides in the chorioallantoic membrane of 12-day-old chick embryos have been isolated by ion-exchange chromatography and characterized by their spectrophotometric and paper chromatographic properties. The following nucleotides were identified: adenosine-5' phosphate (AMP), uridine-5' phosphate (UMP), cytidine-5' phosphate (CMP), uridine-5' diphosphate galactose (UDPGal), uridine-5' diphosphate N-acetyl hexosamine (UDPNAHexosamine), guanosine-5' phosphate (GMP), cytidine-5' diphosphate (CDP), uridine-5' diphosphate (UDP), adenosine-51 diphosphate (ADP), guanosine-5' diphosphate (GDP), cytidine-5' triphosphate (CTP), uridine-5' triphosphate (DTP), adenosine-5' triphosphate (ATP), and guanosine-5' triphosphate (GTP). Quantitative determinations of these nucleotides were made on the basis of their ultraviolet absorption at 260 mμ. Similarly, concentrations of these nucleotides were estimated in 12-day-old chorioallantoic membranes after infection with vaccinia virus. Larger amounts of ribonucleoside-5' phosphates were present in the infected tissue at 4 and 12 hours after infection. The amounts of ribonucleoside-5' triphosphates were decreased. In tissues where, it is believed, synchronous infection occurred, the amounts of ribonucleoside-5' diphosphates and triphosphates were markedly lower than in controls after 12 hours of infection. Infection in the presence of tritium₌labelled thymidine showed that the amount of labelled thymidine-5' mono-, di-, and triphosphates had increased after 4 hours and that the amounts of these nucleotides subsequently decreased. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Environmental factors (extracellular macromolecules) possibly operating in somite differentiation were examined by using an in vitro system with myogenesis as the end point. It was found that differentiation depended on the time of removal of the somitic tissue from the host, i.e. between stages 17 and 26 (2½ to 5 days of age), and the question was raised as to the relation of these observations and the appearance of glycosamino-glycans (mucopolysaccharides). A review of the literature revealed that no information was available on this subject so that an examination of the time of appearance, distribution, and nature of the glycosaminoglycans and neutral polysaccharides in the axial region (dermatome, myotome, scleratome, neural tube and notochord) of the developing chick embryo during early somite differentiation was necessary. It became apparent that both histochemical and biochemical analysis were required to identify, quantify, and localize the glycosaminoglycans since histochemical techniques alone limit the interpretations possible because interference from proteins and glycoproteins could not be ruled out. Histochemical analysis indicated that there was very little sulphated anionic glycosaminoglycan present in the early embryonic stages examined. The cytoplasm of cells in all axial areas contained strongly acidic material, but extracellularly, sulphated anionic glycosaminoglycans were almost all confined to the notochordal sheath. The extracellular matrix in all areas contained weakly acidic anionic glycosaminoglycans. With development, the weakly acidic anionic glycosaminoglycans increased in concentration in most areas, but most noticeably in the neural tube and scleratome. The concentration of sulphated anionic glycosaminoglycans also increased and they began to appear in the extracellular matrix in all areas although never attaining the deep staining intensity demonstrated by the weakly acidic anionic glycosamino-glycans. By stage 25, however, the extracellular matrix of the scleratome around the notochord was mostly sulphated anionic glycosaminoglycans. Testicular hyaluronidase digestion suggested that most of the stainable material was either hyaluronic acid or chondroitin 4- and/or 6-sulphate (chon-croitin sulphates A and/or C). A small amount of strongly acidic anionic glycosaminoglycan present in the scleratome, neural tube and notochord was polysulphated. Biochemical analysis confirmed that the weakly acidic anionic glycosaminoglycan was hyaluronic acid and that the sulphated anionic glycosaminoglycan was mainly chondroitin 4- and/or 6-sulphate (chondroitin sulphate A and/or C). Only trace amounts of dermatan sulphate (chondroitin sulphate B) were present. A small amount of heparin could be present since some glucosamine was present in the sulphated fractions. This heparin could account for the polysulphated material observed with histochemical staining. On a quantitative basis, the hyaluronic acid concentration (uronic acid/gm dry wt. of tissue) was at a peak between stages 21 to 25 and was greater than the chondroitin sulphate concentration up until stage 25. After that stage, the chondroitin sulphate concentration began to increase very rapidly, concomitant with the formation of cartilage around the notochord, and the hyaluronic acid concentration began to decline slowly. Thus, whereas the hyaluronic acid content was 2½ times greater than the chondroitin.sulphate content in stage 17 embryos, this ratio was almost completely reversed by stage 28 due to the rapid increase in chondroitin sulphate. Histochemical staining supported these findings. It would seem that the increase in sulphated anionic glycosaminoglycans is directly related to cartilage formation while the high hyaluronic acid content present during stages 21 to 25, a time in development when myotube formation and scleratome cell aggregation and orientation are occurring, may play some more general developmental role in somite differentiation. / Science, Faculty of / Zoology, Department of / Graduate

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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.