The forming limb skeleton serves as a signaling center for vascular patterning
Our study identified the forming skeleton as a signaling center that patterns the vasculature to segregate from the skeleton and, concurrently, to enrich its vicinity by expressing both pro- and anti-angiogenic signals. As the molecular mechanism that underlies the regulation of limb bud vasculature by the skeleton, we demonstrated that SOX9, a key regulator of skeleton formation, controls the expression of Vegf, a pivotal angiogenic factor. From a broader perspective, this work suggests a paradigm for a mechanism that allows for coordination of organ development and vascular patterning by a shared transcriptional regulation.

Figure 1: Vasculature and skeleton development are coordinated
(A-C): Staining for vascular endothelial cells (green) shows vascular distribution of E10.5 (A), E11.5 (B) and E12.5 (C) whole limbs of control embryos. Red box indicates axial artery, white box indicates interdigital vasculature near the avascular area of future metacarpal. Yellow arrows indicate vascular-rich stems that divide and separate the future metacarpals. (D-F): Transverse sections of E10.5 (D), E11.5 (E) and E12.5 (F) autopods stained for endothelial cells (green) and chondrocytes (red) illustrates vascular patterning and chondrocyte differentiation, respectively. Circled area in E indicates areas of mesenchymal cells that undergo differentiation into chondrocytes.

 

HIF-1α regulates the differentiation of hypoxic prechondrogenic cells during early skeletogenesis
The segregation of the forming skeleton from the limb vasculature induces a localized reduction in oxygen tension at those vessel-free domains, forming hypoxic niches. To tackle the question of the mechanism that enables mesenchymal differentiation into chondrocytes in hypoxic niches, we studied the involvement of HIF-1α, a pivotal mediator of cellular adaptive response to hypoxia, in chondrocyte differentiation. Our study identified HIF-1α as a key regulator of chondrocyte differentiation, as in its absence the hypoxic mesenchymal cells failed to differentiate. Moreover, our finding that HIF-1α regulates Sox9 expression in hypoxic prechondrogenic condensations provides a molecular entry point for the elucidation of this unique regulatory mechanism).

Figure 2: Developmental aberrations in limb skeleton following Hif-1α deletion
  Staining of mouse forelimbs from E18.5 wild type (top) and mutant that lack Hif-1α(bottom) demonstrate severe retardation in limb skeleton formation (A) and joint fusion (B) in the absence of Hif-1α expression. Autopod skeletons (C) show cartilage formation at the periphery of the forming digits and joint loss in the Hif-1α-null limb.

 

Figure 3
  Micro-CT analysis of 21-day-old wild type and Hif-1α-deleted autopods: dorsal view demonstrates severe retardation in the mutant’s skeleton including bone deformation, lack of phalanges (bracket), and joint fusion. H and I show dorsal and lateral views of digit 3, respectively; yellow arrows indicate fused joints and fused sesamoid bones.

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