Loss of HIF-1α in the Notochord Results in Cell Death and Complete Disappearance of the Nucleus Pulposus.

The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans

Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements

Quantitative analysis of IVD thickness assessed by X rays at 2 months.

<p>Measurements of IVD and VB thicknesses of control (white bars) and mutant (black bars) mice at 2 months. Statistical analysis was performed using the Student's t test. Differences with a p-value <0.05 were considered as statistically significant.</p

Histomorphometric analysis of control and mutant NP, VB and AF at E15.5 and at birth.

<p>Surface measurements of VB, AF and NP of control (white bars) and mutant (black bars) mice at E15.5 and birth. Statistical analysis was performed using the Student's t test. Differences with a p-value <0.05 were considered as statistically significant.</p

Progressive disappearance of the mutant NP postnatally.

<p><b>A</b>. H&E staining of NB (a,b), 1 month (c,d) and 4 months (e,f) NP in control (Foxa2^iCre;HIF-1α^f/+) (a,c,e) and mutant (Foxa2^iCre;HIF-1α^f/f) (b,d,f) mice. Bar = 50 µm. <b>B</b>. Safranin O staining of 1 month (a,b) NP in control (HIF-1α^f/f) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) mice. Bar = 100 µm.</p

Morphological abnormalities of the mutant NP at E15.5.

<p><b>A</b>. H&E staining of E15.5 NP in control (Foxa2^iCre;HIF-1α^f/+) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) mice. Bar = 50 µm. <b>B</b>. Safranin O staining of E15.5 NP in control (HIF-1α^f/f) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) specimens. Bar = 50 µm. <b>C</b>. In <i>situ</i> hybridization for aggrecan (a,b), collagen II (c,d), collagen X (e,f), and brachyury (g,h) mRNAs in control (HIF-1α^f/f) (a,c,e,g) and mutant (Foxa2^iCre;HIF-1α^f/f) (b,d,f,h) NP at E15.5. Brightfield (a-f) and darkfield (g,h) pictures are shown. Bar = 50 µm.</p

Normal differentiation of the notochord in absence of HIF-1α.

<p><b>A</b>. H&E staining of E13.5 spine in control (Foxa2^iCre;HIF-1α^f/+) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) mice. High magnification of E13.5 NP in control (Foxa2^iCre;HIF-1α^f/+) (c) and mutant (Foxa2^iCre;HIF-1α^f/f) (d) mice. Bar = 50 µm. <b>B</b>. <i>In situ</i> hybridization for brachyury mRNA in control (HIF-1α^f/f) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) spine at E13.5. Darkfield pictures are shown. Bar = 50 µm.</p

Lineage study in control and mutant IVDs.

<p><b>A,B</b>. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1α^f/f (a,e,i), HIF-1α^f/f;mTmG (b,f,j), Foxa2^iCre;HIF-1α^f/+;mTmG (c,g,k) and Foxa2^iCre;HIF-1α^f/f;mTmG (d,h,l) mice, respectively. Red fluorescence (a-d), green fluorescence (e-h) and merged filters (i-l) are shown. Bar = 100 µm.</p

No impaired proliferation of mutant NP cells.

<p><b>A</b>. BrdU staining of E15.5 NP in control (Foxa2^iCre;HIF-1α^f/+) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) mice, respectively. Bar = 50 µm. <b>B</b>. Quantification of BrdU staining in VB, AF, NP of control (white bars) and mutant (black bars) specimens.</p

Altered biomechanical properties of the mutant IVD lacking the NP.

<p><b>A</b>. Load damping capacity in control (HIF-1α^f/f) (a) and mutant (Foxa2^iCre;HIF-1α^f/f) (b) IVD at 4 months. <b>B</b>. Phase shift angle (a) and energy dissipation (b) based on the load damping test in control (HIF-1α^f/f) and mutant (Foxa2^iCre;HIF-1α^f/f) IVD at 4 months. Statistical analysis performed between control and mutant groups: rmANOVA p<0.001 (a) and p<0.05 (b).</p