Lineage restriction, cellular heterogeneity, and patterning during mammalian digit tip regeneration

Abstract

Regeneration can be broadly defined as the renewal of biological structures. This occurs over the lifetimes of all multicellular organisms as structures like epithelia are replaced by cell division and growth. Some vertebrates, however, are capable of regenerating entire appendages, such as limbs, after amputation. This type of regeneration goes beyond the maintenance of tissues and requires re-growth of entire appendages. One striking example is the axolotl, which can regenerate lost limbs and tails, among other complex tissues. Regenerative ability is more restricted within mammals, but mice and human children have the innate ability to regenerate digit tips. The digit tip is an anatomical structure that includes bone, connective tissue, nerves, blood vessels, the nail, and the nail epithelium. After amputation there is first a wound healing response that leads to closure of the wound epithelium. The blastema, a heterogeneous mass of proliferating cells, then forms and regenerates the non-epithelial structures of the digit tip. Mouse digit tip regeneration provides a model to study regeneration in mammals to determine principles of regeneration that will lead to advances in regenerative medicine in humans. This dissertation addresses two aspects of digit tip regeneration that advance our understanding of the mouse digit tip blastema: what is the cellular composition of the blastema, and does patterning of the blastema require the same genes that pattern the limb bud during embryonic limb development. Classically, the blastema has been described simply as a collection of proliferating cells. In the mouse digit tip, the blastema is made up of heterogeneous and broadly lineage restricted cells, but the full extent of heterogeneity is unknown. In Chapter 2 we use single-cell RNA-sequencing to define the cell types present in the unamputated and regenerating digit tip. We find that the blastema is largely made up of heterogeneous fibroblast populations with distinct gene expression programs. Other cell types such as immune cells, Schwann cells, osteoblasts, vascular cells, and more are also found in the blastema, some of which have been described before using histology. These broad cell types are also present in the unamputated digit tip. A computational lineage trajectory of the vascular, monocytic, and fibroblast populations confirms broad lineage restriction in the mouse digit tip blastema but suggests that subtler trans differentiation relationships may exist. We also find that certain fibroblast clusters expand during regeneration and express regeneration specific genes such as Mest. Patterning during limb regeneration has long been thought to be mediated by the same genes that pattern developing limbs. In Chapter 3 we explore the patterning of the mouse digit tip blastema in the dorsal-ventral axis using the well-known set of transcription factors that define the dorsal-ventral axis during limb development, En1 and Lmx1b. We show that En1 and Lmx1b are expressed during regeneration but not with dorsal-ventral polarity. Further, we develop a computational method to show that loss of En1 or Lmx1b does not perturb the dorsal-ventral morphology of the regenerated digit tip bone, indicating that the limb development dorsal-ventral patterning genes are not re-used during digit tip regeneration. Together, these chapters advance our knowledge of the digit tip blastema and provide a wealth of data and methods that can be used to further investigate the mechanisms of regeneration in a mammalian system.