Dr. Kelly McNagny, Research Supervisor (Medical Genetics)
Dr. Carolyn Brown (Medical Genetics)
Dr. Pauline Johnson (Microbiology and Immunology)
Dr. Robert Kay (Medical Genetics)
EXAMINING COMMITTEE
Chair: Dr. Donald Brooks (Pathology and Laboratory
Medicine)
Supervisory Committee: Dr. Kelly McNagny, Research
Supervisor (Medical Genetics), Dr. Robert Kay (Medical Genetics)
University Examiners: Dr. Connie Eaves (Medical Genetics),
Dr. Hermann Ziltener (Pathology and Laboratory Medicine)
External Examiner: Dr. Shirish Shenolikar, Pfizer Global
Research and Development. 2800 Plymouth Road. Ann Arbor, MI. USA.
ABSTRACT
Podocalyxin is a sialomucin expressed on kidney podocytes,
vascular endothelia, and hematopoietic progenitors. Although
podocalyxin and its close relative, CD34 have been studied for many years, their
precise functions have remained elusive, and roles in blocking differentiation,
preventing cell adhesion, and establishing cell polarity have all been proposed. Despite
this ambiguity, the perinatal lethality of podocalyxin knockout mice (as a
result of kidney defects) and podocalyxin’s close association with cancer progression
highlight its biological importance. I therefore used several strategies to clarify
podocalyxin’s functions and mechanisms of action. Podocalyxin was overexpressed in
epithelial cells, and I observed a striking decrease in cell adhesion and an
induction of microvillus formation. Microvillus formation was then used as the
endpoint to assess the activity of podocalyxin mutants: the extracellular
domain was essential while most of the cytoplasmic tail could be deleted
without loss of this function. These in vitro studies also demonstrated that
podocalyxin recruits the scaffolding protein, NHERF1, which may have important implications
in the regulation of NHERF-related processes, such as interaction with ion
transporters and signalling molecules. In order to study podocalyxin in vivo,
generation of conditional podocalyxin overexpressing mice was attempted. The
intention was to generate a single floxed podxl transgenic mouse line that
could be crossed with numerous Cre mice in order to induce podocalyxin
expression in selected tissues. For example, podocalyxin overexpression in
mammary tissue was intended to facilitate evaluation of podocalyxin’s role in
breast cancer progression. Similarly, these mice could be used to selectively
rescue defects in podocalyxin-deficient mice. Unfortunately, chromosomal
abnormalities in the parental embryonic stem cells temporarily prevented completion
of this study. As an alternative strategy, we attempted to selectively
rescue the kidney defects observed in podocalyxin-null mice by creation of mice
with a kidney-specific podxl transgene. Surprisingly, although transgenic
podocalyxin was appropriately expressed, and podocyte morphology appeared relatively
normal in contrast to podocalyxin-null mice, transgenic mice still died
perinatally. This suggests the presence of other serious, as yet undetermined,
abnormalities in podocalyxindeficient animals. Continued assessment of these defects and
podocalyxin’s role in cancer progression is underway. In summary, this thesis
reveals a new mechanistic role for podocalyxin in the process of cell morphogenesis
and suggests that in addition to its vital role in kidney development,
podocalyxin may play an essential role in other aspects of mammalian development.
PUBLICATIONS
Nielsen
JS, McCoy ML, Chelliah S, Vogl AW, Roskelley CD, and McNagny
KM. The CD34-related molecule, Podocalyxin, induces microvillus formation.
(PNAS, in revision).
*Doyonnas R, *Nielsen
JS, Chelliah S, Drew E, Hara T, Miyajima A, McNagny KM. Podocalyxin is a CD34-related marker of murine
hematopoietic stem cells and embryonic erythroid cells. Plenary paper, Blood 105:
4170-8 (2005). *co-first authors.
Somasiri A, Nielsen
JS, Makretsov N, McCoy ML, Prentice L, Gilks CB, Chia SK, Gelmon KA,
Kershaw DB, Huntsman DG, McNagny KM, Roskelley CD. Overexpression of the
anti-adhesin podocalyxin is an independent predictor of breast cancer progression.
Cancer Research 64: 5068-73 (2004).
Nielsen
JS, Doyonnas R, and McNagny KM. Avian models to study the transcriptional
control of hematopoietic lineage-commitment and to identify lineage specific genes.
Cells Tissues Organs 171: 44-63 (2002).
SELECTED ABSTRACTS AND POSTER PRESENTATIONS
Nielsen JS, McCoy ML, Chelliah S, Vogl AW, Roskelley CD,
and McNagny KM. Ectopic expression of a single protein is sufficient to drive
microvillus formation. American Society for Cell Biology 45th Annual Meeting (San
Francisco 2005).
McCoy ML, Nielsen JS, Somasiri A, Makretsov N, Stingl J,
Vogl AW, Gilks CB, Huntsman DG, Kershaw DB, McNagny KM, and Roskelley CD.
Podocalyxin in
mammary epithelial morphogenesis and breast cancer
progression. American Society for Cell Biology 45th Annual Meeting (San
Francisco 2005).
Nielsen JS, McCoy ML, Chelliah S, Vogl AW, Roskelley CD,
and McNagny KM. The CD34-related stem cell marker, Podocalyxin, induces
microvillus formation. Stem Cell Network AGM (Calgary 2005).
Roskelley CD, McCoy ML, Nielsen JS, and McNagny KM. The
polar route: An alternative first leg on the road to breast cancer metastasis.
Epithelial Mesenchymal Transition (EMT) Conference (Vancouver 2005).
McNagny KM. Hematopoietic stem cell migration is
facilitated by the anti-adhesins CD34 and Podocalyxin. Keystone Symposium: Molecular
Regulation of Stem Cells (Banff 2005).
Nielsen JS, Doyonnas R, Chelliah S, McCoy M, Roskelley C,
McNagny KM. The anti-adhesins CD34 and Podocalyxin facilitate
hematopoietic cell migration and upregulation leads to poor outcome in human breast cancer.
Stem Cell Network AGM (Montreal 2004).
Nielsen JS, Drew E, Tan P, Chelliah S, Merkens H, Doyonnas
R, Roskelley CD, McNagny KM. The CD34 family: Essential modulators of
adhesion and cell-cell contact. 12th International Congress of Immunology
(Montreal 2004).
Nielsen JS, Doyonnas R, Chelliah S, Somasiri A, Roskelley
CD, McNagny KM. The CD34-related molecule, podocalyxin, is a hematopoietic
stem cell marker that functions as an anti-adhesin for stem cell migration. Stem
Cell Network AGM (Vancouver 2003).
Nielsen JS, Doyonnas R, McNagny KM. The CD34-related
protein MEP21/Podocalyxin is a stem cell marker involved in reducing cell
adhesion and can be relocalized upon phosphorylation. Keystone Symposium: From
Stem Cells to Therapy (Steamboat Springs, Colorado 2003).
AWARDS
NSERC Canada Graduate Scholarship, 2003-2005
Stem Cell Network Travel Award, 2005
UBC Graduate Travel Award, 2005
Stem Cell Network Travel Award, 2004
Albert B and Mary Steiner Travel Award, 2003
Heart & Stroke Foundation of BC & Yukon
Traineeship, 2001-2003
NSERC Postgraduate Scholarship, 2001-2003
UBC Graduate Fellowship, 2000-2001
GRADUATE STUDIES
Field of Study: Podocalyxin function in epithelial and
hematopoietic cells
Courses
MEDG 510 Advanced Immunogenetics, Dr. K. McNagny
MEDG 520 Advanced Human Molecular Genetics, Dr. C. Brown /
Dr. W. Robinson
MEDG 530 Advanced Human Genetics Dr. B. Simpson / A. Dircks
