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Multipotent mesenchymal stromal cells (MSCs) are required for skeletal formation, maintenance, and repair throughout life; however, current models posit that postnatally arising long-lived adult MSCs replace transient embryonic progenitor populations. We previously reported exclusive expression and function of the embryonic patterning transcription factor, Hoxa11, in adult skeletal progenitor-enriched MSCs. Here, using a newly generated Hoxa11-CreERT2 lineage-tracing system, we show Hoxa11-lineage marked cells give rise to all skeletal lineages throughout the life of the animal and persist as MSCs. Hoxa11 lineage-positive cells give rise to previously described progenitor-enriched MSC populations marked by LepR-Cre and Osx-CreER, placing them upstream of these populations. Our studies establish that Hox-expressing cells are skeletal stem cells that arise from the earliest stages of skeletal development and self-renew throughout the life of the animal., Prior evidence suggested mesenchymal stromal cells (MSCs) required for skeletal formation, maintenance, and repair arise postnatally. Here, the authors show that Hoxa11 lineage-marked cells give rise to all skeletal lineages from embryogenesis through adulthood and are upstream progenitors of LepR- and Osx-lineage MSCs

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα + /CD51 + /LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton.This article is protected by copyright. All rights reserved

The limb musculoskeletal system provides a primary means for locomotion, manipulation of objects and protection for most vertebrate organisms. Intricate integration of the bone, tendon and muscle tissues are required for function. These three tissues arise largely independent of one another, but the connections formed during later development are maintained throughout life and are re-established following injury. Each of these tissues also have mesenchymal stem/progenitor cells that function in maintenance and repair. Here in, we will review the major events in the development of limb skeleton, tendon, and muscle tissues, their response to injury, and discuss current knowledge regarding resident progenitor/stem cells within each tissue that participate in development, repair, and regeneration in vivo.

The limb musculoskeletal system provides a primary means for locomotion, manipulation of objects and protection for most vertebrate organisms. Intricate integration of the bone, tendon and muscle tissues are required for function. These three tissues arise largely independent of one another, but the connections formed during later development are maintained throughout life and are re-established following injury. Each of these tissues also have mesenchymal stem/progenitor cells that function in maintenance and repair. Here in, we will review the major events in the development of limb skeleton, tendon, and muscle tissues, their response to injury, and discuss current knowledge regarding resident progenitor/stem cells within each tissue that participate in development, repair, and regeneration in vivo.

Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11EGFP reporter mice, we demonstrate expression exclusively in multipotent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, and exhibit colony-forming unit fibroblast activity and tri-lineage differentiation in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments, and these defects are zeugopod specific. In the stylopod (humerus and femur) and sternum, bone marrow MSCs express other regionally restricted Hox genes, and femur fractures heal normally in Hox11 mutants. Together, our data support regional Hox expression and function in skeletal MSCs.

The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species1. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous2. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb1–6, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish3–7, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process. This article has an associated ‘The people behind the papers’ interview.

Hox genes are critical regulators of skeletal development and Hox9-13 paralogs, specifically, are necessary for appendicular development along the proximal to distal axis. Loss of function of both Hoxa11 and Hoxd11 results in severe malformation of the forelimb zeugopod. In the radius and ulna of these mutants, chondrocyte development is perturbed, growth plates are not established, and skeletal growth and maturation fails. In compound mutants in which one of the four Hox11 alleles remains wild-type, establishment of a growth plate is preserved and embryos develop normally through newborn stages, however, skeletal phenotypes become evident postnatally. During postnatal development, the radial and ulnar growth rate slows compared to wild-type controls and terminal bone length is reduced. Growth plate height is decreased in mutants and premature growth plate senescence occurs along with abnormally high levels of chondrocyte proliferation in the reserve and proliferative zones. Compound mutants additionally develop an abnormal curvature of the radius, which causes significant distortion of the carpal elements. The progressive bowing of the radius appears to result from physical constraint caused by the disproportionately slower growth of the ulna than the radius. Collectively, these data are consistent with premature depletion of forelimb zeugopod progenitor cells in the growth plate of Hox11 compound mutants, and demonstrate a continued function for Hox genes in postnatal bone growth and patterning., Summary: Hox genes are required for postnatal skeletal growth. Hox11 genes function in the zeugopod skeletal elements and regulate longitudinal growth through controlling chondrocyte maturation within the growth plate.

In the musculoskeletal system, muscle, tendon, and bone tissues develop in a spatially and temporally coordinated manner, and integrate into a cohesive functional unit by forming specific connections unique to each region of the musculoskeletal system. The mechanisms of these patterning and integration events are an area of great interest in musculoskeletal biology. Hox genes are a family of important developmental regulators and play critical roles in skeletal patterning throughout the axial and appendicular skeleton. Unexpectedly, Hox genes are not expressed in the differentiated cartilage or other skeletal cells, but rather are highly expressed in the tightly associated stromal connective tissues as well as regionally expressed in tendons and muscle connective tissue. Recent work has revealed a previously unappreciated role for Hox in patterning all the musculoskeletal tissues of the limb. These observations suggest that integration of the musculoskeletal system is regulated, at least in part, by Hox function in the stromal connective tissue. This review will outline our current understanding of Hox function in patterning and integrating the musculoskeletal tissues.