Bone Morphology is Regulated Modularly by Global and Regional Genetic Programs

Abstract

During skeletogenesis, a variety of protrusions of different shapes and sizes develop on the surfaces of long bones. These superstructures provide stable anchoring sites for ligaments and tendons during the assembly of the musculoskeletal system. Despite their importance, the mechanism by which superstructures are patterned and ultimately give rise to the unique morphology of each long bone is far from understood. In this work, we provide further evidence that long bones form modularly from Sox9 + cells, which contribute to their substructure, and from Sox9 + /Scx + progenitors that give rise to superstructures. Moreover, we identify components of the genetic program that controls the patterning of Sox9 + /Scx + progenitors 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. Additionally, by combining literature-based evidence and comparative transcriptomic analysis of different Sox9 + /Scx + progenitor populations, we identified genes potentially involved in patterning of bone superstructures. We present evidence indicating that Gli3 is 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 coordinately.Collectively, our results provide strong evidence for genetic regulation of superstructure patterning that further supports the notion that long bone development is a modular process.

Making and shaping endochondral and intramembranous bones

“…Subsequent tuberosity enlargement and endochondral ossification require muscle development, implicating mechanics [249][250][251] . Sesamoid bones are small flat auxiliary bones (their name originates from sesame seed) typically located within tendons.…”

“…The human clinical relevance of mechanics in joint formation is clearly shown by joint abnormalities including talipes in foetuses lacking muscle contraction e.g. foetal akinesia deformation sequence247 and clinically-relevant joint incongruities in foetuses whose movement is physically restricted248 .Tuberosities and sesamoid bones, which arise in association with the perichondrium, may be viewed as two halves of the same coin; in fact, gene inactivation of Gli3 in mice can transform the deltoid tuberosity of the humerus into a sesamoid249 . Molecularly, tuberosities and sesamoid bones initially share characteristics of both chondrocytes (SOX9) and tendons (scleraxis, SCX)236;249;250 .…”

Local retinoic acid directs emergence of the extraocular muscle functional unit

“…In this context, our data suggest that transient SOX9 expression may represent a redeployment of the developmental module for tendon attachment, despite the fact there is no cartilage in the mammalian sclera. Finally, transcriptome analysis revealed the existence of global and regional regulatory modules for superstructure patterning in the limb bones (Eyal et al, 2019), offering a mechanism to induce variations in attachment sites without having to rewrite the entire skeletogenic program (Eyal et al, 2019). As several markers of the POM (Pitx2, Foxc1/2) and retinoic acid signaling modulators (Cyp26a1/b1) were identified as part of specific limb superstructure signatures (Eyal et al, 2019), it is tempting to speculate that those genes also play a conserved role in the generation of the attachment module of the EOMs.…”

Loss of Fgfr1 and Fgfr2 in Scleraxis-lineage cells leads to enlarged bone eminences and attachment cell death

“…These fibrocartilage attachments are often found at bone eminences, which are superstructures such as tuberosities and tubercles, that increase leverage between the line of action of applied mechanical force (e.g., skeletal muscle) and the center of a joint (7)(8)(9). In mice, eminence formation initiates around embryonic day (E)12.5 from a recruited progenitor pool that co-expresses Scleraxis (Scx) and Sox9 and surrounds the cartilage anlage of developing bone (7,10). Eminences form "miniature" growth plates at tendon/ligament attachments and follow similar developmental patterns to the primary growth plate during longitudinal bone growth (1,8,9,(11)(12)(13)(14).…”