Abstract
The genetic and developmental mechanisms involved in limb formation are relatively well documented, but how these mechanisms are modulated by changes in chondrocyte physiology to produce differences in limb bone length remains unclear. Here, we used high throughput RNA sequencing (RNAseq) to probe the developmental genetic basis of variation in limb bone length in Longshanks, a mouse model of experimental evolution. We find that increased tibia length in Longshanks is associated with altered expression of a few key endochondral ossification genes such as Npr3, Dlk1, Sox9, and Sfrp1, as well reduced expression of Fxyd2, a facultative subunit of the cell membrane-bound Na+/K+ ATPase pump (NKA). Next, using murine tibia and cell cultures, we show a dynamic role for NKA in chondrocyte differentiation and in bone length regulation. Specifically, we show that pharmacological inhibition of NKA disrupts chondrocyte differentiation, by upregulating expression of mesenchymal stem cell markers (Prrx1, Serpina3n), downregulation of chondrogenesis marker Sox9, and altered expression of extracellular matrix genes (e.g., collagens) associated with proliferative and hypertrophic chondrocytes. Together, Longshanks and in vitro data suggest a broader developmental and evolutionary role of NKA in regulating limb length diversity.
Highlights
The size and shape of skeletal elements, of the limbs, is a key driver of adaptive radiation in mammals (Smith and Savage, 1956)
Previous work has shown that Na+/K+ ATPase pump (NKA) pump subunits are highly expressed in chondrocytes (Mobasheri et al, 2012), indicating a potentially important role in chondrocyte physiology and/or differentiation, but to date that role has not been well characterized
One role of NKA in chondrocytes could be to regulate the process of cell swelling associated with hypertrophy, by regulating osmotic changes across the cell membrane
Summary
The size and shape of skeletal elements, of the limbs, is a key driver of adaptive radiation in mammals (Smith and Savage, 1956). Mesenchymal stem cells (MSCs) initially form condensations in which the cells eventually differentiate into chondrocytes (Karsenty et al, 2009). These cartilaginous condensations (anlagen) are subsequently replaced by bone through the process of endochondral ossification (Mackie et al, 2008). Longitudinal growth of the skeletal anlagen occurs within the growth plate, a highly organized structure that contains a stem cell niche (resting zone) (Abad et al, 2002), a transit-amplifying zone (proliferative zone), and a differentiation zone in which cells undergo hypertrophy, producing and remodeling extracellular matrix (hypertrophic zone) Heritable length variation within and among bones is thought to be a product of variation in the initial anlagen size (Sears et al, 2006; Sanger et al, 2011), in the cellularity and mitotic activity of the proliferative zone (Kember, 1993; Rolian, 2008; Marchini and Rolian, 2018), and/or in the rate and extent of hypertrophy in individual hypertrophic cells (Cooper et al, 2013; Rolian, 2020)
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