student research day - Case Western Reserve University School of ...

David Yao
Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation
David Yao*, Quanjian Yan*, Lebing Wei, John Lowe, Lan Zhou
Department of Pathology
Case Western Reserve University School of Medicine
Phenotypes resulting from mutations in Notch loci range from notches at the wing margin in Drosophila to T cell acute
lymphoblastic leukemia in human. These pleiotropic effects of Notch signaling stem from the pivotal role of Notch
receptors in controlling the development of multiple biological systems. Because of this critical role, Notch signaling is
subject to tight regulation at multiple levels. One such regulation is the post-translational modification of Notch receptors
by O-linked fucosylation, a reaction that is catalyzed by Protein O-fucosyltransferase-1 (Pofut1). To address the
physiologic function of O-linked fucose moieties on Notch we have previously studied mice engineered to be conditionally
deficient in O-linked fucose. These mice maintain a targeted deficiency in the FX locus which encodes an enzyme required
for the de novo synthesis of GDP-fucose. Homozygosity for the FX-null allele yields a complete deficiency in O-linked
fucosylation of Notch. It also produces phenotypes that include myeloproliferation and faulty T cell development, each of
which is due to loss of Notch signaling in myeloid progenitor cells. In this study, the physiologic function of fucose in
regulating hematopoietic lineage homeostasis was examined. We first demonstrated that FX-null multi-potential
progenitors differentiated into myeloid cells while co-culturing with Notch-ligand-expressing OP9 cells. Bone marrow
transplant of FX-null hematopoietic stem cells yielded an augmented myelopoietic development phenotype. Consistent
with these findings, using an in vitro Notch-dependent T cell differentiation assay, we observed that mouse embryonic
stem cells (ESCs) carrying targeted deficiencies in either Notch1 or Pofut1 failed to produce T lymphocytes while
maintaining their ability to generate granulocytes. Moreover, in vivo hematopoietic reconstitution of CD34+ stem cells
derived from Notch1-null or Pofut1-null mouse ESCs showed enhanced granulopoiesis with depressed lymphoid lineage
development. Together, our results indicate that fucosylation of the Notch receptor is critical in regulating blood cell
lineage homeostasis.
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