HIF1α regulation ofSox9is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis

Abstract

During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1␣ (HIF1␣) in mouse limb bud mesenchyme. Developmental analysis of Hif1␣-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1␣ is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1␣-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1␣ in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1␣-depleted limbs raised the possibility that HIF1␣ regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1␣ expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1␣-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1␣ to the Sox9 promoter, thus supporting direct regulation of HIF1␣ on Sox9 expression. This work establishes for the first time HIF1␣ as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.

Hypoxia-driven pathways in bone development, regeneration and disease

“…46,47 Conditional knockout of HIF-1α in the limb bud mesenchyme of mice indicated that HIF-1α expression is probably not required for the initiation of mesenchyme condensation. 46,47 However, HIF-1α is critically involved in the timely differentiation of the mesenchymal cells into chondrocytes, via mechanisms that are still largely unknown, although regulation of SOX-9 (a principal transcription factor involved in chondrogenesis) might be involved. 48,46 In addition, lack of HIF-1α delays the terminal differentiation of chondrocytes, which is probably a downstream effect of delays in the initiation of chondrogenesis.…”

“…46,47 However, HIF-1α is critically involved in the timely differentiation of the mesenchymal cells into chondrocytes, via mechanisms that are still largely unknown, although regulation of SOX-9 (a principal transcription factor involved in chondrogenesis) might be involved. 48,46 In addition, lack of HIF-1α delays the terminal differentiation of chondrocytes, which is probably a downstream effect of delays in the initiation of chondrogenesis. 46,47 Notably, conditional HIF-1α -knockout mice also showed a striking impairment of joint development, 46,47 a defect that could also be related to SOX-9 because (at least in vitro) hypoxia increases matrix accumulation by chondrocytes of the articular surface, in a manner dependent on this transcription factor.…”

“…48,46 In addition, lack of HIF-1α delays the terminal differentiation of chondrocytes, which is probably a downstream effect of delays in the initiation of chondrogenesis. 46,47 Notably, conditional HIF-1α -knockout mice also showed a striking impairment of joint development, 46,47 a defect that could also be related to SOX-9 because (at least in vitro) hypoxia increases matrix accumulation by chondrocytes of the articular surface, in a manner dependent on this transcription factor. 49 Finally, consistent with the notion that hypoxia leads to arrest of growth, 50 HIFs seem to negatively modulate chondrocyte proliferation.…”

“…36 Altogether, conditional knockout of HIF-1α in chondrocytes or limb bud mesenchyme led to dwarfism and marked shortening of the limbs. 36,47,46 In contrast to HIF-1α -knockout, HIF-2α -knockout causes only a modest and transient delay in endochondral bone development. 51 , 21 Embryos heterozygous for a null allele at the locus encoding HIF-2α (Epas1 +/-) were mildly dwarfed, but this phenotype was no longer detectable by 2 weeks after birth in comparison with wild-type (Epas1 +/+ )…”

Expression analysis of limb element markers during mouse embryonic development

“…Here, we show nerve innervation patterns throughout limb outgrowth. Similarly, cartilage and bone analysis is usually assessed in mouse limb studies at specific time points to study specific gene function and action (for example, Amarilio et al, 2007; Collinson et al, 2018; Firulli et al, 2005; Nowlan et al, 2010; Ray et al, 2015; Tavella et al, 2004). Here, we show in detail the progression of cartilage and bone development throughout fore‐ and hindlimb patterning.…”