A mineralized endoskeleton supports the mammalian body. Bone also serves as a vital mineral repository and nurtures non-skeletal cell types, notably stem cells of the hematopoietic system. Skeletal anomalies are amongst the most common birth defects, problems in fracture repair are observed in non-union fractures and bone disorders are increasingly prevalent in an ageing population. Two key biosynthetic cell types generate the specific extracellular matrix of the mammalian skeleton: cartilage-secreting chondrocytes and bone secreting osteoblasts. These cells have diverse origins (neural crest, somitic and lateral plate mesoderm), and generate bone by distinct processes: directly from a mesenchymal stem cell (membranous bone) or via a cartilage template (endochondral bone). Mouse and human genetics have identified a number of critical signaling pathways, and three key transcription factors that are master regulators of chondrocyte (Sox9) and osteoblast (Runx2 and Osx) programs. Haploin sufficiency for Sox9 and Runx2 associates with human skeletal deficiencies. While the importance of these factors is well documented, their regulatory actions are not. To this end, we will determine the gene regulatory networks that underpin the regulatory actions of each of these critical skeletal determinants in chondrocyte and osteoblast development.
Specific Aim 1 : We will use gene-targeting in mouse embryo stem cells to create mice whose endogenous Sox9, Runx2 and Osx proteins are modified by appending a small peptide at their C-terminus. This epitope will enable subsequent analysis of the expression and activity of each factor through common approaches.
Specific Aim 2 : ChlP-seq analysis will be performed with each tagged protein to directly identify their DNA targets in primary chondrocytes or osteoblasts.
Specific Aim 3 : Data from microarray-based transcriptional profiling of normal chondrocytes and osteoblasts, or these same populations following knock-down of Sox9, Runx2 and Osx, will be intersected with ChlP-seq data to predict the targets of each regulatory factors actions. Selected regulatory models will be examined in transgenic mouse studies. These data will provide the first genome scale insight into the gene regulatory networks driving mammalian skeletogenesis.

Public Health Relevance

Defective skeletal programs are a common birth defect, and an increasing problem in aging populations. Our skeleton is synthesized by two cells type, cartilage from chondrocytes and bone from osteoblasts, under the control of three master-regulatory genes. New mouse models will identify the regulatory programs governed by each factor providing the first genome scale insights into mammalian skeletal development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK056246-12
Application #
8564863
Study Section
Special Emphasis Panel (ZDK1-GRB-7 (J1))
Project Start
2012-06-01
Project End
2016-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
12
Fiscal Year
2012
Total Cost
$275,177
Indirect Cost
$72,075
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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