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Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

Merceron C, Mangiavini L, Robling A, Wilson TL, Giaccia AJ, Shapiro IM, Schipani E, Risbud MV - PLoS ONE (2014)

Bottom Line: 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.Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical School, University of Michigan, Ann Arbor, Michigan, United States of America; Inserm, UMRS 791-LIOAD, Centre for Osteoarticular and Dental Tissue Engineering, Group STEP 'Skeletal Tissue Engineering and Physiopathology', Nantes, France; LUNAM, Nantes University, Faculty of Dental Surgery, Nantes, France.

ABSTRACT
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.

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Specific and efficient deletion of HIF-1α in the nucleus pulposus of mutant mice.A. Breeding scheme used to generate Foxa2iCre;HIF-1αf/f (mutant), Foxa2iCre;HIF-1αf/+ and HIF-1αf/f (control) mice. B. HIF-1α immunohistochemistry in the nucleus pulposus of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Black and white arrows indicate the presence and the absence of HIF-1α at the nuclear level, respectively. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm. C. HIF-1α immunohistochemistry in the vertebral body of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm.
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pone-0110768-g001: Specific and efficient deletion of HIF-1α in the nucleus pulposus of mutant mice.A. Breeding scheme used to generate Foxa2iCre;HIF-1αf/f (mutant), Foxa2iCre;HIF-1αf/+ and HIF-1αf/f (control) mice. B. HIF-1α immunohistochemistry in the nucleus pulposus of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Black and white arrows indicate the presence and the absence of HIF-1α at the nuclear level, respectively. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm. C. HIF-1α immunohistochemistry in the vertebral body of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm.

Mentions: We conditionally inactivated HIF-1α in the notochord by breeding Foxa2iCre male mice with female mice homozygote for a floxed HIF-1α allele (HIF-1αf/f). Newly generated Foxa2iCre;HIF-1αf/+ male mice were bred with HIF-1αf/f females in order to obtain Foxa2iCre;HIF-1αf/f mutant mice, Foxa2iCre;HIF-1αf/+ and HIF-1αf/f control littermates (Figure 1A). Of note, the Foxa2iCre knock-in mouse expresses Cre recombinase at high levels in notochord starting from E9.5 [19].


Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

Merceron C, Mangiavini L, Robling A, Wilson TL, Giaccia AJ, Shapiro IM, Schipani E, Risbud MV - PLoS ONE (2014)

Specific and efficient deletion of HIF-1α in the nucleus pulposus of mutant mice.A. Breeding scheme used to generate Foxa2iCre;HIF-1αf/f (mutant), Foxa2iCre;HIF-1αf/+ and HIF-1αf/f (control) mice. B. HIF-1α immunohistochemistry in the nucleus pulposus of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Black and white arrows indicate the presence and the absence of HIF-1α at the nuclear level, respectively. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm. C. HIF-1α immunohistochemistry in the vertebral body of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4206488&req=5

pone-0110768-g001: Specific and efficient deletion of HIF-1α in the nucleus pulposus of mutant mice.A. Breeding scheme used to generate Foxa2iCre;HIF-1αf/f (mutant), Foxa2iCre;HIF-1αf/+ and HIF-1αf/f (control) mice. B. HIF-1α immunohistochemistry in the nucleus pulposus of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Black and white arrows indicate the presence and the absence of HIF-1α at the nuclear level, respectively. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm. C. HIF-1α immunohistochemistry in the vertebral body of control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) mice at E15.5. Respective negative controls for which the primary antibody has been omitted are shown (c and d). Bar = 50 µm.
Mentions: We conditionally inactivated HIF-1α in the notochord by breeding Foxa2iCre male mice with female mice homozygote for a floxed HIF-1α allele (HIF-1αf/f). Newly generated Foxa2iCre;HIF-1αf/+ male mice were bred with HIF-1αf/f females in order to obtain Foxa2iCre;HIF-1αf/f mutant mice, Foxa2iCre;HIF-1αf/+ and HIF-1αf/f control littermates (Figure 1A). Of note, the Foxa2iCre knock-in mouse expresses Cre recombinase at high levels in notochord starting from E9.5 [19].

Bottom Line: 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.Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical School, University of Michigan, Ann Arbor, Michigan, United States of America; Inserm, UMRS 791-LIOAD, Centre for Osteoarticular and Dental Tissue Engineering, Group STEP 'Skeletal Tissue Engineering and Physiopathology', Nantes, France; LUNAM, Nantes University, Faculty of Dental Surgery, Nantes, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus