Michael, Lydia

2018-01-31T11:48:28Z

2018-01-31T11:48:28Z

2003

Branching morphogenesis is a common mechanism of mammalian development. Branched epithelia of kidneys, lungs, mammary glands etc develop from an initially unbranched epithelial bud which develops under the control of reciprocal interactions between the growing epithelium and the mesenchyme that surrounds it. To examine how morphogenetic signals change the shape of the epithelium this thesis examines the urinary collecting duct as a model for branching and tests the hypothesis that the tips of the epithelium are specialized for morphogenesis.

Kidney development begins when the ureteric bud, an outgrowth of the Wolffian duct, invades the metanephric mesenchyme. This then induces the bud to grow and bifurcate repeatedly to form the collecting duct system. To better understand how the ureteric bud tips are specialized to produce branches, this thesis compares their cell biology to that of the rest of the ureteric bud using the murine metanephric organ culture as a model system. Specifically, it describes the pattern of cell proliferation, cytoskeletal organization as assessed by immunofluorescence and confocal laser scanning microscopy, glycoconjugate expression in the tips and in the rest of the bud by means of lectin histochemistry and gene expression by differential display PCR.

Increased cell proliferation, measured by bromodeoxyuridine incorporation, is found at the tips of the branching epithelium. This pattern of increased cell proliferation correlates with branching activity and is lost when branching is arrested in kidney rudiments depleted of sulphated glycosaminoglycans. Known mediators of ureteric bud branching in culture such as GDNF and TGF-p were examined for their effect on cell proliferation at the ureteric bud tips; TGF-P reduced proliferation and GDNF either increased it or had no effect, depending on whether it was applied locally or generally.

Microfilaments, detected by fluorescent-tagged phalloidin, are strongly expressed in the apical parts of the ureteric bud tips during branching. Inhibition of branching (either by depletion of sulphated glycosaminoglycans or by a MAP kinase inhibitor) disrupts this localized expression. Cytochalasin D disruption of filamentous actin inhibits branching, results in cell scattering from the ureteric bud tips and alters the cell junctions as revealed by E-cadherin distribution. Actomyosin interactions also regulate branching of the ureteric epithelium as shown by a general myosin inhibitor.

Expression of lectin binding glycoconjugates at the ureteric bud tips differs from the stalks. Dolichos Biflorus Agglutinin (DBA), a known collecting duct marker, is found to selectively binding to stalks. This binding pattern is present during active branching but is lost when branching is blocked. Organ recombination experiments show that the ureteric bud stalk cells are able to generate DBAnegative tip cells upon amputation and regeneration. Finally, genes expressed differentially between ureteric bud tips and stalks provide further evidence to support the hypothesis that the tubule tips are specialized structures.

http://hdl.handle.net/1842/28622

The University of Edinburgh

Annexe Thesis Digitisation Project 2017 Block 16

Morphogenetic specializations at the tip of the branching epithelium of the kidney

Thesis or Dissertation

PhD Doctor of Philosophy