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
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.