The regeneration of the mammalian skeleton requires the action of both intrinsic and extrinsic inductive factors
from multiple cell types which function in a hierarchical and temporal fashion to control skeletal progenitor cell
proliferation and differentiation. Sensory nerves have been shown to be an integral part of the bone fracture
repair process, driving the processes of vascularization, ossification, and mineralization of bone. In contrast to
the bone repair process, regeneration, where new growth replaces both the amputated bone and surrounding
soft tissue varies widely in vertebrates. In mammals, regeneration is restricted to only the distal phalangeal
element. More proximal amputations result in the formation of a hypertrophic callus and failed regeneration.
Significant efforts have been placed on dissecting out the distinguishing signaling pathways differentiating
regenerative versus non-regenerative amputations. Beyond the desire to promote full regeneration, unraveling
these processes could allow us to leverage regenerative mechanisms during repair and tissue-engineering
based bone therapeutic approaches. A handful of prior studies have implicated innervation as an essential
component of regeneration, however they relied on complete sciatic nerve resection, making it impossible to
distinguish nerve-specific regenerative outcomes from mechanical loading-induced effects, and the relationship
between innervation and regeneration remains unclear.
Using transgenic mouse models and pharmacological inhibition, our preliminary results point to a severe delay
in digit regeneration following inhibition of sensory nerve tropomycin receptor kinase A (TrkA). In the context of
previous literature, we propose that sensory nerve TrkA signaling is necessary for proper digit regeneration.
Specifically, we propose that: i) sensory nerves are recruited to the amputation site early in the healing process
through the nerve growth factor (NGF)-TrkA signaling axis established in our lab, ii) sensory nerve-derived
signals play an essential role in promoting blastema formation and maintaining cells in a proliferative,
osteogenically primed state, and thus, iii) disruption of sensory nerve signaling through transgenic and/or
pharmacological inhibition severely impairs digit bone regeneration.
Specific Aim 1: Define the spatiotemporal patterning of sensory innervation and characterize the effects of
sensory nerve TrkA signaling disruption during digit regeneration
Hypothesis: Sensory nerve outgrowth and signaling coincides with wound closure, blastema formation and
proliferation, initiating overall digit regeneration.
Preliminary results using a transgenic knockin mouse model (TrkAF592A), demonstrate a substantial deficit in digit
regeneration. In Aim 1 we will first conduct a comprehensive study on the temporal and spatial patterning of
neurotrophin expression and sensory innervation during early and late stages of digit regeneration. Here, we will
make use of commercial antibodies, as well as transgenic mouse lines (Thy1-YFP, NGF-eGFP) to identify the
spatial distribution of sensory nerves during healing. Cellular sources of NGF will be identified using established
markers of mesenchymal and inflammatory cells. Histological and radiological approaches will then be used to
determine the effects of temporally titrated TrkA signaling inhibition on the inflammatory response (day 3, 7),
formation of the blastema (day 10) and subsequent vascular invasion and tissue mineralization (day 14, 28) to
elucidate the multiple facets through which sensory nerves regulate digit regeneration.
Specific Aim 2: Delineate the molecular mechanisms affiliated with sensory nerve signaling disruption during
Hypothesis: Sensory nerves secrete factors to precisely act at the crossroad of digit regeneration, regulating cell
dedifferentiation, proliferation and osteogenic commitment.
Transcriptional data is typically derived from the whole blastema, while spatial information has only been
determined using immunohistochemical approaches one target at a time. Using the cutting-edge and newly
validated spatial transcriptomics (VISIUM 10X Genomics), we will examine the gene signature in innervated and
non-innervated regenerating digits. Though previously only viable in soft tissues, we have recently optimized a
novel approach for bone tissue. This innovative transcriptomics process will be used to gather transcriptional
data during regeneration after amputation following TrkA inhibition from innervated domains in an unbiased
fashion to determine the nerve-specific factors underlying blastema biology. Results will be validated using
publicly available single-cell RNA-seq data sets, in situ hybridization (RNAscope) and histological staining.
Justification for proposal as an R21: The technology of spatial transcriptomics, though extremely powerful, is
yet to be successfully applied to adult mineralized tissue. Using our newly developed protocol, the results of this
R21 will provide first-in-field insights into the spatially-defined regulation of sensory nerves at various stages
throughout regeneration after amputation, as well as fundamental understandings of their role in the overall
maintenance of cell fate and plasticity.