This study examines the association of tissue non-specific alkaline phosphatase (AP) (E.C. 18.104.22.168) with the cellular events of gastrulation of the African clawed toad, Xenopus laevis. In particular, the temporal and spatial localisation of AP was established during this key developmental period by using a highly sensitive histochemical stain, 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to detect AP activity in situ in whole mount and dissected embryos and in cryo-sections of embryos. Activity was first detected at the onset of gastrulation at Nieuwkoop-stage 10.25 within the dorsal marginal zone above the early dorsal blastopore groove. In sagittal section, AP was localised within the area of the prospective chordamesodermal mantle, extending from the dorsal lip towards the anterior margin of the blastocoele. Subsequently as gastrulation progressed, this activity was expressed entirely by the converging and extending dorsal mesendoderm which actively invaginates within the embryo. Eventually, expression of AP was later associated with the invagination of the ventral mesoderm.
In ex vivo experiments using so-called sandwich and open-faced explants, it was found that the isolated prospective dorsal mesendoderm, which retains the ability to generate the movements of convergent-extension, similarly expressed AP activity. Furthermore, when these explants were treated in culture with levamisole, a specific AP inhibitor, disaggregation of the explants took place.
In order to assess whether and how AP might influence gastrulation in intact embryos, living embryos were cultured with inhibitors of the enzyme. Levamisole affected dorso-anterior development in varying degrees, ranging from normal morphology to forms with a bend in the head-tail axis or by producing embryos with even more shortened axes. The exposure of prospective mesodermal cells to levamisole by way of making a hole in the blastocoele roof affected the morphology and directional movement of these cells: scanning electron microscopy showed a disruption of the cell streaming process central to the ingression of the dorsal mesoderm and in addition, the dorsal mesodermal cells appeared rounded and lacked the processes typical of migrating mesodermal cells. The use of brefeldin A (BFA), which disrupts intracellular processing of AP, prevented gastrulation from occurring. In these embryos an AP positive group of cells became localised to the presumptive dorsal marginal zone.
Further to determining the possible developmental role of AP, it was queried whether dorsally positioned cells might still express AP, either in the case of abnormal gastrulation or in the absence of gastrulation. During exogastrulation, an aberrant state of gastrulation, AP was associated with the evaginated prospective mesodermal cells. When gastrulation was prevented from taking place (after the blastocoelic microinjection of trypan blue) and as with BFA treatment, an AP positive group of cells was found to be localised to the presumptive dorsal marginal zone.
Finally, it was questioned whether there was a link between mesoderm induction and AP expression by converging mesodermal cells. By using the established animal cap assay, this was found to be the case. These naive ectodermal cells are induced to form mesoderm and to extend in culture after treatment with the peptide factor, activin, and AP was expressed by the extending cells of these explants. In controls, in the absence of activin and where there was an absence of mesoderm induction, no AP was detected. The in vivo treatment of these cultures with either levamisole or brefeldin following on the activin signal, prevented the cellular extension of the animal caps and no AP activity was seen. It is concluded therefore that AP is required for the induction of the dorsal mesoderm and furthermore is part of the morphogenetic process of convergent extension.
|Degree Type||Doctoral degree|
|Degree Description||PhD (Science)|