PROJECT SUMMARY
The notochord is a highly conserved developmental tissue that extends along the anterior-posterior axis of all
chordates, including humans. It is composed of inner vacuolated cells surrounded by an external layer of
sheath cells that secrete a thick extracellular matrix. Inflation of the vacuolated cells within the restrictive
sheath creates a pressurized rod that supports locomotion in chordates and ultimately patterns the spine of
vertebrates. As such, the development of the notochord and spine are intimately linked, and defects in the
formation of notochord cells have been linked to scoliosis and vertebral malformations. The notochord is a
difficult tissue to study in mouse models since it is already replaced by the spine at the time of birth. In
contrast, the external development and optical transparency of zebrafish make them suitable for investigating
processes involved in notochord development and maturation. This proposal will use quantitative image
analysis, zebrafish genetics, and modern proteomics approaches to define the role of the inositol
polyphosphate phosphatase-like 1a (inppl1a) gene in notochord and spine development. Mutations in this gene
cause early notochord defects and thoracic scoliosis in zebrafish. In this fellowship proposal, I will test the
hypothesis that inppl1a regulates notochord vacuole inflation and, ultimately, the mechanical stability of the
notochord with three Specific Aims. In Aim 1, I will determine the role of inppl1a in notochord vacuolation by
quantifying changes in notochord cell size and vacuole inflation (1.1) and internal vacuole membrane dynamics
(1.2). I will also define the temporal and spatial requirement of inppl1a during notochord development using
pharmacological and molecular-genetic approaches (1.3). In Aim 2, I will evaluate the mechanical properties of
inppl1a mutant notochords by manipulating mechanical stress (2.1) and vertebral bone mineralization (2.2-2.3)
during development. Finally, in Aim 3, I will define the protein interactors of Inppl1a in notochord and spine
development. I will use a candidate gene approach (3.1) and a proximity-dependent labeling strategy (3.2) to
identify additional proteins required for Inppl1a-dependent notochord vacuole inflation. To supplement these
approaches, I will also use modern proteomics techniques to build a comprehensive atlas of the notochord
protein interaction network (3.3). In doing so, I will build an invaluable resource for future investigation of
proteins involved in notochord development. Altogether, the work in this proposal will add to the knowledge of
how notochord cells vacuolate and will ultimately benefit our understanding of human skeletal health and
disease. Although the notochord is considered an embryonic tissue, it has been implicated in adult diseases,
including intervertebral disc degeneration and chordoma. Additionally, mutations in INPPL1 cause the rare
endochondral bone disorder, Opsismodysplasia. Therefore, this work in zebrafish will be significant because it
will likely reveal a conserved role for inppl1a/INPPL1 in skeletal development and disease.
