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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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Lineage study in control and mutant IVDs.A,B. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1αf/f (a,e,i), HIF-1αf/f;mTmG (b,f,j), Foxa2iCre;HIF-1αf/+;mTmG (c,g,k) and Foxa2iCre;HIF-1αf/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.
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pone-0110768-g008: Lineage study in control and mutant IVDs.A,B. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1αf/f (a,e,i), HIF-1αf/f;mTmG (b,f,j), Foxa2iCre;HIF-1αf/+;mTmG (c,g,k) and Foxa2iCre;HIF-1αf/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.

Mentions: In order to unequivocally prove that mutant NP cells truly disappear postnatally, we performed a lineage study by using the mT/mG reporter mouse [18]. These mice have loxP sites on either side of a membrane-targeted tandem dimer Tomato (mT) cassette, and express red fluorescence in all tissues. When crossed with Cre positive mice, the mT cassette is deleted, allowing expression of the membrane-targeted EGFP (mG) cassette located downstream, in the tissues where Cre has been recombining the reporter gene sequence, whereas red fluorescent protein is no longer synthesized. At E15.5, no auto-fluorescence was detected using either red or green filters in HIF-1αf/f NP (Figure 8A a, e, i). Consistent with the lack of Cre activity, E15.5 HIF-1αf/f;mTmG NPs were positive only for the red fluorescent signal (Figure 8A b, f, j). Conversely, NPs from E15.5 Foxa2iCre;HIF-1αf/+;mTmG displayed both red and green signals, which indicates that a mixed population of recombined and non-recombined cells was indeed present in the normal developing NP (Figure 8A c, g, k). Likewise, also the NPs of E15.5 Foxa2iCre;HIF-1αf/f;mTmG mutant mice appeared to be composed of a mixed population of recombined and non-recombined cells (Figure 8A d, h, l).


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)

Lineage study in control and mutant IVDs.A,B. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1αf/f (a,e,i), HIF-1αf/f;mTmG (b,f,j), Foxa2iCre;HIF-1αf/+;mTmG (c,g,k) and Foxa2iCre;HIF-1αf/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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206488&req=5

pone-0110768-g008: Lineage study in control and mutant IVDs.A,B. Detection of fluorescence in frozen sections of NP isolated from E15.5 (A) and 1 month (B) HIF-1αf/f (a,e,i), HIF-1αf/f;mTmG (b,f,j), Foxa2iCre;HIF-1αf/+;mTmG (c,g,k) and Foxa2iCre;HIF-1αf/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.
Mentions: In order to unequivocally prove that mutant NP cells truly disappear postnatally, we performed a lineage study by using the mT/mG reporter mouse [18]. These mice have loxP sites on either side of a membrane-targeted tandem dimer Tomato (mT) cassette, and express red fluorescence in all tissues. When crossed with Cre positive mice, the mT cassette is deleted, allowing expression of the membrane-targeted EGFP (mG) cassette located downstream, in the tissues where Cre has been recombining the reporter gene sequence, whereas red fluorescent protein is no longer synthesized. At E15.5, no auto-fluorescence was detected using either red or green filters in HIF-1αf/f NP (Figure 8A a, e, i). Consistent with the lack of Cre activity, E15.5 HIF-1αf/f;mTmG NPs were positive only for the red fluorescent signal (Figure 8A b, f, j). Conversely, NPs from E15.5 Foxa2iCre;HIF-1αf/+;mTmG displayed both red and green signals, which indicates that a mixed population of recombined and non-recombined cells was indeed present in the normal developing NP (Figure 8A c, g, k). Likewise, also the NPs of E15.5 Foxa2iCre;HIF-1αf/f;mTmG mutant mice appeared to be composed of a mixed population of recombined and non-recombined cells (Figure 8A d, h, l).

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