HOXA5 protein expression and genetic fate mapping show lineage restriction in the developing musculoskeletal system

Abstract

HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression are unknown. A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

HOXA5 Participates in Brown Adipose Tissue and Epaxial Skeletal Muscle Patterning and in Brown Adipocyte Differentiation

“…This broad spatial domain suggested that expression of both proteins includes but is not limited to prospective brown adipocytes. In closer view, neither protein is expressed in skeletal muscle (Figures 2E-H; red outline marks the epaxial muscle mass), consistent with our previous reports for HOXA5 (Landry-Truchon et al, 2017;Holzman et al, 2018). However, EBF2 and HOXA5 are co-expressed in muscle connective tissue (MCT) fibroblasts interspersed within the muscle ( Figure 2H, red arrowheads).…”

“…Immunostaining was performed following previously described protocols (Holzman et al, 2018;McGlinn et al, 2019). Briefly, embryos were fixed in 4% paraformaldehyde at 4 • C and embedded in OCT.…”

“…lineage tracing labels both PPARγ-positive brown adipocytes and PDGFRα-positive BAT fibroblasts (Holzman et al, 2018).…”

“…However, EBF2 and HOXA5 are co-expressed in muscle connective tissue (MCT) fibroblasts interspersed within the muscle ( Figure 2H, red arrowheads). Their identity as MCT was based on their co-expression of HOXA5 with MCT markers PDGFRα and TCF4 (Holzman et al, 2018).…”

“…The HOXA5 protein expression domain extends from the third cervical through the second thoracic segments (C3-T2), which includes the somites located at the same axial level as the major BAT depots. Indeed, Cre-mediated fate mapping revealed that Hoxa5 expressing cells contribute extensively to BAT including preadipocytes, adipocytes and connective tissue fibroblasts (Holzman et al, 2018). Together, these findings raise the question of whether Hoxa5 regulates embryonic BAT development.…”