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doi: 10.1242/10.1242/dev.00331

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Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs

Department of Biomedical Engineering and Orthopaedic Research Center, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA

View larger version (143K): [in a new window]   Fig. 1. Lack of nuclei pulposi in Sox5–/–/Sox6–/– fetuses. (A) Histological analysis of mid-sagittal sections of the thoracic vertebral column of E15.5 control and Sox5–/–/Sox6–/– fetuses. In the control (top), intervertebral discs (arrows) feature a nucleus pulposus (NP), a cartilaginous inner annulus (IA) and a mesenchymal outer annulus (OA). Vertebral bodies (VB) exhibit a core of hypertrophic chondrocytes, surrounded with chondroblasts juxtaposed to inner annulus chondroblasts. In the mutant (bottom), the segmentation of the vertebral column is only visible in outer annuli. Nuclei pulposi are lacking. The cartilage matrix of inner annuli and vertebral bodies is deficient (pale Alcian Blue staining), and chondroblasts are all small and round. Groups of condensed cells (arrowheads), remnants of the notochord, are seen in vertebral body regions, but not in intervertebral regions. (B) Histology of cross sections of E15.5 control and Sox5–/–/Sox6–/– fetuses in the upper lumbar vertebral column. In control intervertebral regions (top), the nucleus pulposus is surrounded with inner and outer annulus. Notochord cells are no longer seen in vertebral bodies or between intervertebral and vertebral regions (arrowheads). The mutant intervertebral and vertebral regions (bottom) show no nucleus pulposus or notochord cells. Residual notochord cells form trains between the center of cartilages and the outer mesenchyme between vertebral and intervertebral regions (arrow).

View larger version (133K): [in a new window]   Fig. 4. Removal of Sox5–/–/Sox6–/– notochord cells from intervertebral spaces. (A) Histological analysis of mid-sagittal sections of E14.5 and E15.5 control and Sox5–/–/Sox6–/– embryos. At E14.5, in the distal tail, the control notochord is continuous, but the mutant is interrupted in intervertebral regions (arrows in first row). In the intervertebral regions of the proximal tail, the control notochord is bulging (arrow in second row), whereas mutant notochord cells are missing. In both segments of the tail, control vertebral bodies are starting to accumulate cartilage matrix, but mutant vertebral bodies hardly are. In the E14.5 thoracic region and E15.5 lumbar region (third and fourth rows), control notochord cells have been removed from vertebral bodies, but the notochord sheath is still visible (arrow in left panel in third row). Vertebral body and inner annulus cartilage is developing, and nuclei pulposi expanding. In mutants, vertebral bodies are accumulating a deficient cartilage matrix, and notochord cells are forming trains (arrows in right panels in third and fourth rows) between vertebral and intervertebral regions. (B) In situ hybridization of Car3 RNA in an E13.5 Sox5–/–/Sox6–/– embryo. The RNA signal identifies notochord stretches in vertebral bodies, but not in intervertebral regions (higher cell density, arrows). Arrowheads indicate notochord cells found at ectopic positions, i.e. dorsal and ventral (mid-sagittal section) or lateral (para-sagittal section) to the normal axis of the notochord, aside intervertebral regions. (C) Histological analysis of cross-sections of E12.5 control and Sox5–/–/Sox6–/– presumptive intervertebral discs. The control notochord is intact in all regions. The mutant notochord is still visible in the cervical and caudal regions, but not in the lumbar region. (D) In situ hybridization of Shh RNA in cross-sections of E12.5 Sox5+/–/Sox6+/– and Sox5–/–/Sox6–/– embryos through intervertebral regions. Shh RNA is detected in the floor plate of the neural tube in both embryos (arrowheads). It is also detected in notochord cells in both lumbar and cervical control regions and in the Sox5–/–/Sox6–/– cervical region (arrows). By contrast, no Shh RNA signal is seen in the Sox5–/–/Sox6–/– lumbar region (arrow).

View larger version (123K): [in a new window]   Fig. 2. Co-expression of Sox5 and Sox6 in the notochord and nucleus pulposus. Mid-sagittal and cross-sections were made in the lumbar vertebral column of E11.5-E15.5 wild-type embryos. Cross-sections were chosen in intervertebral regions. Each row shows pictures of adjacent sections. Left-hand panels are sections stained with Alcian Blue and nuclear Fast Red to identify the neural tube (NT), notochord (arrows), intervertebral mesenchyme (IM), inner annulus (IA), outer annulus (OA), nucleus pulposus (NP) and vertebral bodies (VB). The other panels show Sox5 (middle) and Sox6 (right) RNA in situ hybridization (arrows indicate notochord). The second row displays high-magnification pictures of the boxed areas in the first row. The left-hand pictures for each hybridization were photographed under standard conditions of blue fluorescence (cell nuclei) and dark-field exposure (RNA signals). Fluorescence only was used for the right-hand pictures to visualize the notochord better. At all stages, Sox5 and Sox6 are co-expressed in the notochord, nucleus pulposus, prechondrocytes (IM) and chondroblasts (IA, VB). The signal for Sox5 RNA is slightly weaker in the notochord and nucleus pulposus than in surrounding tissues, whereas the signal for Sox6 RNA is similar is all these tissues.

View larger version (121K): [in a new window]   Fig. 3. Lack of notochord sheath in Sox5–/–/Sox6–/– embryos. (A,B) Histological analysis of mid-sagittal sections of control (left) and Sox5–/–/Sox6–/– (right) embryos at E11.5 (A) and E13.5 (B). Top panels show the notochord surrounded with vertebral bodies (VB) and intervertebral mesenchyme (IM). Bottom panels show the notochord at higher magnification. The control notochord is surrounded with extracellular matrix (arrows) that stains with Alcian Blue. The mutant notochord has fewer cells and lacks the matrix sheath. (C) In situ hybridization of RNAs for extracellular matrix proteins in mid-sagittal sections of control and Sox5–/–/Sox6–/– E13.5 embryos. The left and right panel pairs are low- and high-magnification pictures, respectively, of the same areas. Arrows indicate the notochord. Col2a1 expression is partially downregulated in mutant notochord cells and chondroblasts. Agc1 and Hspg2 expression is severely downregulated in both mutant cell types. Lamc1 expression is not significantly altered or is only slightly downregulated in mutant notochord and intervertebral cells. (D) In situ hybridization of RNAs for notochord-specific non-matrix proteins in control and Sox5–/–/Sox6–/– E13.5 embryos. Shh RNA was tested in cross-sections, and T and Car3 RNA in mid-sagittal sections. The Sox5–/–/Sox6–/– mutation does not affect expression of these RNAs.

View larger version (83K): [in a new window]   Fig. 5. Normal proliferation, but massive apoptosis of Sox5–/–/Sox6–/– notochord cells. (A) BrdU labeling of proliferating cells in E11.5 and E13.5 control and Sox5–/–/Sox6–/– embryos. Three pairs of littermates were tested per age (1-6). For each embryo, notochord cells were counted in five to nine mid-sagittal sections in the thoracolumbar region, totaling 300-1000 cells. The percentage of BrdU-positive cells is presented as the mean±s.d. for all sections. Representative pictures are shown underneath. BrdU-labeled cells are brown. At both stages, mutant notochord cells proliferate at a rate that is similar to or higher than that of control cells. (B) TUNEL assay in the thoracolumbar region of control and Sox5–/–/Sox6–/– embryos. At E11.5, few cells are dying in the control notochord (red, arrowheads), and many more in the mutant notochord. Cells are also dying in intervertebral mesenchyme (IM) in both embryos (arrows). At E13.5, no cells are dying in the control notochord, but massive apoptosis is occurring in the mutant notochord. No cells are dying in control cartilages, but few cells are dying in mutant intervertebral mesenchyme (arrow). VB, vertebral bodies.

View larger version (75K): [in a new window]   Fig. 6. Impaired development of Sox5–/–/Sox6–/– intervertebral mesenchyme. (A) In situ hybridization of Pax1 RNA in mid-sagittal sections of control and Sox5–/–/Sox6–/– embryos in the lower lumbar vertebral column. At E11.5, Pax1 is expressed in pre-intervertebral cell condensations in both embryos (arrowheads). At E12.5 and E13.5, Pax1 is expressed in control embryos in presumptive annuli (arrowheads) and perichondrium (arrows). These expression domains are wider at E12.5 than at E13.5 in the control, but similar in the mutant. At E15.5, Pax1 expression is restricted to outer annuli in control embryos (asterisk). In Sox5–/–/Sox6–/– embryos, Pax1 is still expressed in intervertebral mesenchyme (arrowheads). (B) In situ hybridization of RNAs for extracellular matrix proteins in mid-sagittal sections of the cervical region of E15.5 control and Sox5–/–/Sox6–/– embryos. Wild-type inner annulus cells (arrowheads) are differentiated chondroblasts: they are no longer expressing Col3a1, and are expressing Agc1 and Fmod. Sox5–/–/Sox6–/– intervertebral cells (arrowheads) are still exhibiting an undifferentiated mesenchymal phenotype: they are expressing Col3a1 at high levels, and Agc1 and Fmod at low or undetectable levels.

View larger version (103K): [in a new window]   Fig. 7. Deficient development of the notochord in Sox5/Sox6 compound mutants. (A) Histological analysis of notochord sheath formation in E12.5 embryos with all combinations of Sox5 and Sox6 wild-type and mutant alleles. The notochord sheath (arrow) becomes thinner as the number of mutant alleles increases. The mutation of Sox6 is more detrimental than that of Sox5. (B) Histological analysis of nucleus pulposus formation in control and Sox5+/–/Sox6–/– littermates. Mid-sagittal sections were photographed in the thoracic region. At E13.5, the notochord is starting to bulge in intervertebral spaces in both embryos (arrows). By E14.5, notochord cells have been completely removed from vertebral bodies (VB) in the control embryo, but incompletely removed in the mutant (arrow). The formation of cartilage matrix is delayed in E13.5 and E14.5 mutant embryos. By E16.5, the notochord has completed its transformation into nuclei pulposi in both embryos. The notochord sheath (arrow) is still visible in the mutant vertebral bodies. The mutant nuclei pulposi are smaller, eccentric and often fragmented. IA, inner annulus; IM, intervertebral mesenchyme; NP, nucleus pulposus. (C) High-magnification pictures of nuclei pulposi in E16.5 embryos. Control cells are highly swollen, as indicated by a large volume of cytoplasm (white) per cell nucleus (red). Sox5–/–/Sox6+/– cells are incompletely swollen and Sox5+/–/Sox6–/– cells hardly swollen. (D) Skeletal preparations of the vertebral column of newborn mice (P0) with various combinations of Sox5 and Sox6 null alleles. Non-mineralized cartilages and nuclei pulposi are stained with Alcian Blue, and mineralized cartilages and bones with Alizarin Red. Pictures show the L1 lumbar vertebra and flanking intervertebral discs. The nuclei pulposi (arrows) of Sox5+/–/Sox6+/– and Sox5–/–/Sox6+/+ and Sox5–/–/Sox6+/– mice are less expanded in width than those of the control (Sox5+/–/Sox6+/+) mouse. The nuclei pulposi of Sox5+/+/Sox6–/– and Sox5+/–/Sox6–/– mice are severely reduced in both width and height, and opaque instead of translucent. (E) Histological analysis of nuclei pulposi in control (Sox5+/–/Sox6+/+) and Sox5+/–/Sox6–/– newborn littermates. Mutant cells are less swollen than control cells. (F) In situ hybridization of Shh RNA (top) and histological analysis (bottom) of sagittal sections of control (Sox5+/–/Sox6+/+) and Sox5+/–/Sox6–/– newborn littermates. Pictures show a lumbar intervertebral disc and segments of flanking vertebrae. Nucleus pulposus cells (arrows) are still expressing Shh in the mutant, but not in the control. Hypertrophic chondrocytes (arrowheads) are expressing Shh in both mice. (G) In situ hybridization of Shh RNA in cross-sections of wild-type embryos. Pictures show a presumptive intervertebral disc region and the ventral part of the neural tube. Shh is expressed in E13.5 and E14.5 notochord cells (arrows), but no longer in E15.5 nucleus pulposus cells (arrow). At all three stages, Shh is expressed in the neural tube floor plate (arrowheads). (H) Picture of 1-week-old (P7) control and Sox6–/– littermates. The Sox6–/– mouse is developmentally delayed and exhibits a kinked tail (arrow). (I) Histological analysis of longitudinal sections through the tail of 4-day-old (P4) control and Sox6–/– mice. Control nuclei pulposi (arrows) occupy the core region of intervertebral spaces. Mutant nuclei pulposi are small and located eccentrically, where the tail forms kinks.

View larger version (26K): [in a new window]   Fig. 8. Sox5 and Sox6 control essential steps in the notochord and chondrocyte differentiation pathways. Chondrocytes and notochord cells progress through four major differentiation steps during development. Major transcription factors known to control one or several of these steps are indicated. Biochemical (Shh and noggin) and physical (mechanical pressure) interactions occur between the two lineages at several steps. See the end of Discussion for details.

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