Project Summary/Abstract
At the cornerstone of human bipedal locomotion are the pelvis and knee, two hind limb skeletal structures for
which we know little about their respective development in humans. Indeed, these structures have complex
3-D morphologies whose initial patterns arise during the chondrogenic anlagen stage, when coordinated
cellular differentiation and proliferation establish various tissue types and the spatial relationships between
different structural components (e.g., between the knee’s distal femoral condyles and proximal tibial platform,
or between the pelvis’ ilium, pubis, ischium, and acetabular subdomains). Yet, for developing human skeletal
structures, we understand little about these cellular events and their relationships to tissue morphology and
function. Moreover, while one can envision that these events are mediated by a pleiotropic or common ‘skeletal
growth’ gene set and accompanied regulatory apparatus, how this tool kit is used in developing humans to
build each structure, remains a mystery. As biomedicine move towards regenerative therapies for joint tissues
and mechanistic investigations into developmental disorders of the skeleton, it is crucially important to gain a
better understanding of how cells of the skeleton and joints acquire their functional roles, and it is both timely
and critical to establish this at single cell and spatial resolutions. To date, large functional genomics-based
consortia, such as an ENCODE or the ROADMAP EPIGENOMICS PROJECT, have not focused on the
skeleton due to logistical issues in extracting cartilage cells from developing skeletal elements composed of
hard extracellular matrix. However, recent advances on this front by the grant investigators have allowed them
to isolate and study individual cartilage cells from developing human skeletons. The focus of this proposal,
therefore, is to more deeply investigate how the human knee, pelvis, and hind limb in general form in utero, at
the level of individual cells and in understanding how changes in their biology and behavior drive the respective
development of each hind limb structure. This will be accomplished via two main aims, one focused on the use
of spatial transcriptomics to examine expression dynamics histologically (Aim 1), and another on the use of a
single cell (sc) multiomics approach (Aim 2), consisting of scRNA-sequencing (to detect genes) and
scATAC-sequencing (to detect regulatory regions) on the same cell. By simultaneously profiling gene
expression and regulatory element availability at the individual cell level from many cells of these developing
human structures, and spatially, the necessary resolution will be achieved to define small but important
nuances in the genetic programs that govern anatomical-site-specific cartilage cell biology and how it links to
hind limb morphology. Use of these protocols developed by the grant’s Team will help ensure that an
extraordinary resource is provided to the musculoskeletal biology community, and that crucial information
needed to develop novel pharmaceutical and regenerative medicine-based therapeutics is made public.
