Hedgehog signaling is required for formation of thenotochord sheath and patterning of nuclei pulposiwithin the intervertebral discsKyung-Suk Choi and Brian D. Harfe1

Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL 32610

Edited by Clifford J. Tabin, Harvard Medical School, Boston, MA, and approved April 27, 2011 (received for review June 7, 2010)

The vertebrae notochord is a transient rod-like structure thatproduces secreted factors that are responsible for patterningsurrounding tissues. During later mouse embryogenesis, the noto-chord gives rise to the middle part of the intervertebral disc, calledthe nucleus pulposus. Currently, very little is known about themolecular mechanisms responsible for forming the intervertebraldiscs. Here we demonstrate that hedgehog signaling is required forformation of the intervertebral discs. Removal of hedgehog signal-ing in the notochord and nearbyfloorplate resulted in the formationof an aberrant notochord sheath that normally surrounds thisstructure. In the absence of the notochord sheath, small nucleipulposi were formed, with most notochord cells dispersed through-out the vertebral bodies during embryogenesis. Our data suggestthat the formation of the notochord sheath requires hedgehogsignaling and that the sheath is essential formaintaining the rod-likestructure of the notochord during early embryonic development. Asnotochord cells form nuclei pulposi, we propose that the notochordsheath functions as a “wrapper” around the notochord to constrainthese cells along the vertebral column.

Sonic Hedgehog | Smoothened

Low back pain will affect most people over the age of 65 inindustrialized countries. In the United States, treatment of low

back pain is estimated to cost 50 to 100 billion dollars per year(reviewed in ref. 1). For the majority of people, bed-rest will re-lieve most symptoms of back pain, but in a small population ofpatients the condition will become chronic. Most back pain isthought to originate from degeneration of the intervertebral discsor through physical damage to the disc. This damage leads eitherto herniation of the middle part of the disc, called the nucleuspulposus, or tears, bulging, and rupture of the annulus fibrosus,which surrounds the nucleus pulposus (reviewed in ref. 2).The intervertebral discs connect two adjacent vertebrae and

provide structural stability and flexibility to the spinal column.The nuclei pulposi, which originate from the embryonic notochord(3), are composed primarily of proteoglycan, water, and collagentype II and are located in the middle of each intervertebral disc(reviewed in ref. 4). As the discs age or are damaged, the nucleuspulposus is dramatically altered. Proteoglycan and water contentdecreases in the nucleus pulposus and collagen type II is re-placed by type I collagen so that nuclei pulposi become fibrousand contain less water.During midembryogenesis in both mice and humans, the no-

tochord—a transient rod-like structure that is located along themidline of embryos—becomes segmented and forms the in-tervertebral discs (3, 5). In embryonic day (E) 12.5 to E15.5mouseembryos, notochord cells are removed from regions of the embryocontaining the vertebral bodies and are relocated to the inter-vertebral mesenchyme through an unknown mechanism. Inter-estingly, mouse mutants formed aberrant cartilage around thevertebral column but a normal notochord and notochord sheathfailed to remove notochord cells from the vertebral bodies,resulting in malformed intervertebral discs (6–9). These datasupport the hypothesis that a mechanical force driven by the

forming vertebral bodies is responsible for pushing notochordcells into the intervertebral discs (6, 10, 11, reviewed in ref. 12).In numerous tissues, hedgehog signaling has been implicated in

regulating pattern formation and cell proliferation and cell sur-vival (13–15). Previous studies have shown that Sonic Hedgehog(Shh) is expressed in nuclei pulposi in both prenatal and postnatalintervertebral discs (16, 17). In addition, Indian Hedgehog (Ihh) isexpressed in condensing chondrocytes of the embryonic vertebralbodies and in the vertebral endplate during later development(17). Both Shh and Ihh produce secreted proteins that bind thetransmembrane protein PATCHED1 (PTCH1), resulting in ac-tivation of the hedgehog signaling pathway (reviewed in ref. 18).In mice containing a null allele of Shh, the notochord formed butwas quickly lost (19). Because the notochord was quickly lost inShh-null animals, the role of hedgehog signaling may play intransforming the notochord into nuclei pulposi remains unknown.Ihh is not expressed in nuclei pulposi (17); however, conditionalremoval of Ihh in chondrocytes of postnatal mice has been shownto result in loss of the annulus fibrosus and enlargement of thenucleus pulposus. These data suggest that Ihh may be requiredwithin growth plates for intervertebral disc homeostasis (20).To directly examine the role hedgehog signaling plays in the

formation of nuclei pulposi, we conditionally removed Smooth-ened (Smo), which is required for all hedgehog signaling, inall Shh-expressing cells. In the vertebral column, Smowas removedfrom the mouse notochord and floorplate. Removal of Smo fromthe vertebral column, coupled with detailed fate-mapping andmolecular analysis in this mutant background, allowed us to de-termine the role the hedgehog signaling pathway plays in trans-forming the notochord into nuclei pulposi.

ResultsRemoval of Hedgehog Signaling from the Mouse Notochord. Mouseembryos in which hedgehog signaling was removed from all cellsdie before formation of the intervertebral discs (21). Previously,we had shown that the notochord forms the entire nucleus pul-posus of each disc in the mouse vertebral column (3). To de-termine the role of hedgehog signaling during formation of nucleipulposi, hedgehog signaling was removed from all Shh-expressingcells, including notochord and floorplate in the vertebral column,using a floxed mouse allele of Smo and the Shhgfpcre allele (Fig. 1A and B and Figs. S1 and S2) (22, 23).

Hedgehog Signaling Is Required for Formation of Intervertebral Discsand Normal Cell Proliferation in the Notochord. Intervertebral discsare located between each vertebra in wild-type mice. To de-termine the role hedgehog signaling played in formation of the

Author contributions: K.-S.C. and B.D.H. designed research; K.-S.C. performed research;K.-S.C. and B.D.H. analyzed data; and K.-S.C. and B.D.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: BHarfe@mgm.ufl.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1007566108/-/DCSupplemental.

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intervertebral discs, newborn control and mutant vertebral col-umns were analyzed. Sections through mutant vertebral columnsrevealed that nuclei pulposi were smaller compared with controllittermates (Fig. 1 I–L). Mutant intervertebral discs contained anannulus fibrosus but this tissue appeared to have lost concentriclamellae within the annulus fibrosus, possibly as a result of theimproper formation of nuclei pulposi in the center of the discs(Fig. 1 M and N).In a number of organs, hedgehog signaling is required for cell

proliferation and cell survival (13–15). In these tissues, removal ofhedgehog signaling often resulted in a decrease in cell proliferationand an increase in cell death. To determine whether removal ofhedgehog signaling from the notochord resulted in a defect in cellproliferation, BrdU was administrated to pregnant dams 3 h beforeharvest. BrdU assays were performed on the rostral regions ofE11.5 wild-type and mutant embryos. Using this assay, notochordcells in E11.5 embryos were found to proliferate at a lower ratethan controls (Fig. 2). In addition, an increase in proliferation inthe surrounding perichordal mesenchyme was observed in mutantembryos. These data suggest that the observed smaller nucleipulposi located in the rostral vertebral column in mutant mice maypartially result from a decrease in the rate of cell proliferation ofnotochord cells upon the removal of hedgehog signaling.

Removal of Hedgehog Signaling Resulted in Aberrant Migration ofNotochord Cells During Intervertebral Disc Formation. The mousenucleus pulposus and annulus fibrosus are formed in highlycondensed regions of intervertebral mesenchyme along theventral midline of the embryo beginning at E12.5 (10). Over thenext 3 days, the notochord forms the nuclei pulposi of the in-tervertebral discs. Notochord cells are normally excluded fromregions of the vertebral column where vertebrae form. To de-termine if the aberrant nuclei pulposi found in mutant animalswere the result of defects in the transition of notochord cells intonuclei pulposi, the notochord in mutant animals was fate-mappedusing the ROSA26 reporter allele (24). In these animals, all cellsarising from the notochord were marked, allowing for a detailedanalysis of the fate of notochord cells throughout development.At E12.5 a slightly thinner notochord was observed in mutant

animals compared with controls (Fig. 3B), consistent with theobservation that there is a decrease in cell proliferation in thistissue (Fig. 2). In control E13.5 embryos, the notochord formedbulges between each vertebra where the future discs would form.In mutant embryos, this did not occur. Notochord cells contin-ued to reside as a rod along the midline of the embryo. A smallnumber of notochord cells were also found within the vertebralbodies (Fig. 3D). By E14.5 nuclei pulposi had formed from thenotochord in control embryos, with very few notochord cells stillresiding in vertebral bodies. However, in mutants few notochordcells were found to reside in the forming disc with most cellsscattered throughout the vertebral column (Fig. 3F). In postnatalcontrol animals, the nucleus pulposus was located inside the

Fig. 1. Removal of SMO in Shh-expressing cells results in abnormal de-velopment of the intervertebral discs and vertebrae. (A and B) Analysis ofPtch1:lacZ expression revealed that hedgehog signaling was absent in mu-tant notochords. Section of Ptch1:lacZ expression of E9.5 control (Smof/f;Ptch1:lacZ) and mutant (Smof/f;Shhgfpcre;Ptch1:lacZ) embryos. Note thatPtch1:lacZ expression was absent in the mutant caudal notochord (arrow). Inthe neural tube a decrease in Ptch1:lacZ was observed compared with con-

trols. Lower expression may be because of the inability of floorplate cells torespond to SHH secreted from the notochord because the Shhgfpcre alleleremoved SMO from the floorplate in addition to the notochord. (C and D)Bright-field images of E12.5 wild-type and mutant embryos in which Smo hasbeen removed from Shh-expressing cells. An abnormally truncated andthinner tail (asterisk in D) was observed in the E12.5 mutant. (E–H) Lyso-tracker assay of control and mutant E11.5 embryos. (Magnification: E and F,×10 and G and H, ×32.) G and H are higher magnification of the boxedregions shown in E and F. An increase in cell death occurred caudal to thelumbar vertebra (arrowhead in F). (I–N) Histological analysis of a saggitalsection of the intervertebral vertebral column using Picro-sirius red andAlcian blue staining. Close up view of nucleus pulposus (K and L) and annulusfibrosus (M and N). Mutant tissue contained a smaller nucleus pulposus thancontrols and concentric lamellae were absent in the annulus fibrosus. af,annulus fibrosus; np, nucleus pulposus; nt, neural tube; and vb, vertebralbody. (Scale bars, 20 μm in A and B; 100 μm in I and J; and 20 μm in K–N.)

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annulus fibrosus throughout the vertebral column. In contrast,mutant animals contained small nuclei pulposi with the majorityof notochord cells dispersed throughout the vertebral column(Fig. 3 H, J, and L, and Fig. S3).

Hedgehog Signaling Is Required for Notochord Sheath Formation.The notochord sheath is composed of extracellular matrix pro-teins that surround the notochord in E10.0 embryos (25). Todetermine if hedgehog signaling was required for notochordsheath formation, sections of E11.5 vertebral columns werestained with Alcian blue to visualize the extracellular matrixcomposition surrounding the notochord. In control embryos thenotochord sheath formed around the notochord in a rostral tocaudal progression (Fig. 4 A and C). Sheath formation occurredbefore cartilage condensation within the vertebral bodies. Inmutant embryos a thin notochord sheath was observed in therostral region of E11.5 embryos (Fig. 4B); however, no Alcianblue staining was observed in the caudal region of mutant em-bryos (Fig. 4D). In addition, the caudal notochord of mutantswas abnormally flattened (Fig. 4 D, H, and L).To determine if the ultrastructure of the notochord sheath

was affected by the removal of hedgehog signaling, transmissionelectron micrography was performed on the transverse section of

E11.5 embryos. In wild-type embryos, the notochord sheathconsisted of a basal lamina layer and loosely organized collagenfibrils (Fig. 4 I and K). In the rostral region of mutants, the no-tochord sheath consisted of basal lamina and a thin layer of col-

Fig. 2. Removal of hedgehog signaling resulted in a decrease in cell pro-liferation in rostral mutant notochords. Representative transverse sections ofthe rostral vertebral column of E11.5 embryos are shown. (A and B) BrdUstaining of control (A) and mutant (B) sections. (Scale bars, 200 μm.) (C and D)A merged picture of BrdU, DAPI, and laminin (green). Laminin stainingmarked the inner layer of the notochord sheath and outlined the location ofthe notochord. At E11.5, the number of anti-BrdU+ cells in mutant noto-chords was decreased (D). (E) Quantification of the number of anti-BrdU+

cells/total cells in the notochord demonstrated that the number of pro-liferating cells in mutant notochords was significantly decreased. Quantifi-cation of the number of anti-BrdU+ cells in surrounding perchordalmesenchyme (dotted circle in C and D) indicated that the proliferation ratein surrounding mesenchyme was slightly increased in mutant embryos. Dataare represented as means and the error bars represent the SD. *P < 0.05.

Fig. 3. Aberrant migration of notochord cells throughout the vertebralcolumn upon removal of hedgehog signaling in the notochord. (A–L) Cellswere fate-mapped using the Cre-inducible R26R allele. (A and B) Notochord(blue cells) formed a rod-like structure in both wild-type and mutant animalsuntil E12.5. In wild-type E13.5 embryos (C) the notochord started to forma bulge between the vertebrae in regions where the intervertebral discs wereforming. In mutants (D) the notochord remained as a rod-like structure anda few cells were found to reside outside the notochord (arrows). By E14.5,notochord cells had formed nuclei pulposi in wild-type animals (E) butretained a rod-like structure in the mutant (F). A number of mutant noto-chord cells (arrows) resided outside the notochord. In P0 wild-type animals (Gand I), notochord cells had formed the nucleus pulposus of each intervertebraldisc. In contrast, mutant notochord cells (H and J) were randomly foundthroughout the intervertebral mesenchyme and vertebrae. (K and L) Histo-logical analysis of thoracic vertebrae from newborn mice demonstrated se-vere defects in nucleus pulposus structure and an increase in notochord cellsresiding in surrounding tissues. im, intervertebral mesenchyme; and vb,vertebral body. (Scale bars, 50 μm in A–F and 100 μm in K and L.)

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lagen fibrils (Fig. 4J). In the caudal region of mutant embryos noelectron-dense material was observed (Fig. 4L). Laminin, an in-ner component of the notochord sheath, was found to surroundthe caudal notochord in both wild-type and mutant embryos, in-dicating that at least some components of the sheath are stillpresent in the absence of hedgehog signaling within the caudalnotochord (Fig. 4 E–H).

Removal of Hedgehog Signaling After Formation of the NotochordSheath Does Not Affect Nuclei Pulposi Patterning or Growth. Re-moval of hedgehog signaling before sheath formation resulted ina deformed sheath and aberrant formation of nuclei pulposi.These data suggested that proper formation of a notochord sheathwas essential for normal patterning of nuclei pulposi along thevertebral column. However, it was possible that nuclei pulposiwere not patterned correctly because of loss of hedgehog signalingand not because of the absence of a notochord sheath. To test thishypothesis, hedgehog signaling was removed after sheath forma-tion using the tamoxifen-inducible Cre allele ShhcreERT2 and thefloxed Shh allele (22, 26). Tamoxifen was administered at E8.5(before sheath formation), E9.5 (during sheath formation), E10.5or E11.5 (after the sheath formation; in a normal embryo, thesheath is first observed at E10.0 surrounding the notochord (25).To determine if hedgehog signaling was efficiently removed inShh-expressing cells after tamoxifen exposure, we analyzed ex-pression of Shh and Ptch1 in E9.5 embryos that had been exposedto tamoxifen at E8.5 (27). Both Shh and Ptch1 expression wereabsent in the mutant notochord of treated embryos (Fig. 5 A–D).Examination of the vertebral column of E11.5 control and Shhf/ShhcreERT2 embryos, in which hedgehog signaling had been re-moved after sheath formation occurred, demonstrated that per-durance of the notochord sheath did not require hedgehogsignaling (Fig. S4). To determine if removal of hedgehog signalingfrom the notochord after sheath formation affected nuclei pulposipatterning, control and Shhf/ShhcreERT2 notochords exposed totamoxifen at E11.5 were fate-mapped using the R26R reporterallele (24). In control and tamoxifen-treated E11.5 embryos har-vested at E18.5, no difference in nuclei pulposi formation wasobserved in rostral to sacral vertebrae (Fig. 5 E and I and Fig. S5),indicating that hedgehog signaling is not required for formation ofnuclei pulposi after the sheath has formed.

Proper Formation of Vertebrae Is Required for the Transition of theNotochord into Nuclei Pulposi.Removal of Shh from Shh-expressingcells at E8.5 or E9.5 resulted in the continued presence of thenotochord throughout the vertebral column and an absence ofnuclei pulposi throughout embryonic development (Fig. 5 F andG). Unlike when Smo was removed from Shh-expressing cells,notochord cells were not observed scattered throughout thevertebral column, even when the notochord sheath was abnor-mal. In addition, removal of Shh resulted in defects in formationof the vertebrae. An increasing severity in defective nuclei pul-posi and vertebrae formation correlated with earlier removal ofShh. Removal of Shh at E10.5 resulted in formation of vertebraebut they lacked condensations. In this experiment, nuclei pulposibegan to form but notochord cells were still found to reside withthe vertebral bodies (Fig. 5H). These data support the proposalthat vertebrae may be responsible for forcing notochord cellsinto the forming intervertebral bodies (Fig. 6).

DiscussionRole of Hedgehog Signaling Within the Mouse Notochord. Duringnormal mouse development the notochord sheath surrounds theentire notochord, beginning at E10.0 (26). As the notochordbegins to form visible nuclei pulposi at E12.5, the sheath remainsaround notochord cells. Our data directly addresses the rolehedgehog signaling plays in formation of the notochord sheath.In Shh-null embryos, the notochord forms but then quickly dis-appears before sheath formation, suggesting that hedgehog sig-naling is essential for maintaining a functional notochord (19).Because Shh-null embryos are defective in hedgehog signalingthroughout the entire embryo, it was not possible to determine ifloss of the notochord in these mutant animals was an indirectconsequence of loss of hedgehog signaling in other tissues. In ourexperiments, hedgehog signaling was removed from the noto-chord but was still present in tissues surrounding this structure.In these embryos, notochord cells persisted throughout embry-

Fig. 4. Hedgehog signaling is required for notochord sheath formation.Asterisks (*) indicate the location of the notochord sheath in all panels. (A–D)Histological analysis of transverse sections of rostral and caudal notochordsfrom E11.5 embryos. Control (A and C) notochords were surrounded by thenotochord sheath, which was visualized with Alcian blue stain. In mutants, athin layer of the notochord sheathwas observed in the rostral notochord (B). Inthe mutant caudal notochord, the notochord sheath was absent (D). In bothcontrols (E and G) and mutants (F and H) immnohistochemistry revealed thatlaminin surrounded the notochord. (I–L) Transmission electron micrograph ofthe notochord sheath. Insets in I to L are lowermagnification of the notochord.(Magnification: I–L, 30,000×.) The notochord sheath of controls containedbasal lamina and collagen fibrils (I and K). In the mutant, basal lamina anda thinner layer of collagen fibrils formed in the rostral notochord (J) but thecollagen fibrils appeared to be absent in the caudal notochord (L). n, noto-chord; and ns, notochord sheath. (Scale bars, 10 μm in A–H and 0.5 μm in I–L.)

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onic and postnatal development. These data indicate thathedgehog signaling is not required for maintenance of thisstructure but instead is essential for normal formation of thenotochord sheath that surrounds the embryonic notochord. It isimportant to note that the cre allele used in these experiments,Shhcre, removes hedgehog signaling from the floorplate in ad-dition to the notochord. Although we currently have no evidenceto support a role for hedgehog signaling within the floorplate informing the notochord sheath, it is possible that signaling mol-

ecules within the floorplate that are downstream of the hedgehogsignaling pathway may be important for forming at least someaspects of the notochord sheath.Upon removal of all hedgehog activity, the sheath was dis-

rupted but a ring of basal lamina was still found surrounding thenotochord, indicating that hedgehog signaling is not responsiblefor producing all components of the sheath. Laminin proteinsurrounding the hedgehog-null notochord could be producedfrom nonnotochord cells, as suggested by experiments in zebra-fish (29). A second possibility is that laminin is produced directlyby notochord cells but does not require hedgehog signaling.

Role of the Notochord Sheath During Intervertebral Disc Formation.Although it is clear that a notochord sheath forms around thenotochord in a number of different species, including zebrafish,chicken, mice, and humans, the function this structure playsduring development has remained elusive (25, 29–31). Duringthe transition of the notochord into nuclei pulposi, notochordcells have been proposed to be “squeezed” along the midline ofthe embryo by the condensing vertebra into the forming discs (6,10, 11, reviewed in ref. 12). In embryos in which hedgehog sig-naling was removed from the notochord but contained normalvertebral bodies, notochord cells were observed to be scatteredthroughout the vertebral column. Mutant embryos that hada defective vertebral column, irrespective of whether they hada normal notochord sheath or contained a rod-like notochord,suggesting that vertebrae are needed to form normal discs.We propose that a possible function of the notochord sheath

may be to form a “wrapper” around the notochord (see modelpresented in Fig. 6). Before the notochord-forming nuclei pul-posi, our model suggests that the sheath is required to maintainthe rod-like structure of the notochord. In our experiments, lossof a functional sheath caused the notochord to flatten. Duringlater wild-type development, we propose that the sheath is flex-ible enough so that when the forming vertebrae exert swellingpressure the sheath expands but still constrains notochord cellsto the dorsal midline of the embryo.

Fig. 5. Shh is required for patterning the intervertebraldiscs. (A–D) SectionRNA in situhybridizationof Shh (using aprobeagainst thefloxedexon2)and Ptc1of E9.5control (ShhcreERT2) andmutant (Shhf/ShhcreERT2) embryos. Shh and Ptc1 transcripts in E9.5mutant embryos (B andD) were not detected 24 h after tamoxifen (TM)injection. (E–I) Fate-mapping of cells that have expressed Shh during intervertebral disc formation. Control (ShhcreERT2;R26R) and mutant (Shhf/ShhcreERT2;R26R)embryoswereharvestedatE18.5aftera single TM injectionat eitherE8.5,E9.5, E10.5,or E11.5.All imagesare ventral viewsof the vertebral column. (E′–I′) Ventral viewof control (ShhcreERT2) and mutant (Shhf/ShhcreERT2) vertebral columns. Removal of Shh from E8.5 to E10.5 in Shh-expressing cells resulted in severe defects information of vertebral columns and lack of formation of ossification centers. (I′) Removal of Shh fromE11.5 embryos did not result in any phenotypic abnormalities inthe thoracic vertebral region. (Scale bars, 50 μm.)

Fig. 6. Proposed role for the notochord sheath in forming nuclei pulposi ofthe intervertebral discs. The notochord sheath (red line) begins to formaround the notochord (blue line) at E10.0 (early). By E14.5 (late) most no-tochord cells reside within the intervertebral discs. It has been proposed thatswelling pressure (denoted by arrows) exerted by the vertebral bodies servesto push notochord cells into the space between each vertebrae. Loss of afunctional sheath or “wrapper” around the notochord (denoted by a thinred line) results in notochord cells being scattered throughout the vertebralcolumn and formation of small and misshapen nuclei pulposi. Loss of afunctional sheath in the absence of swelling pressure results in the contin-ued presence of the rod-like notochord throughout embryonic de-velopment. It is important to note that the proposed model does not ruleout the possibility that a currently unknown molecular or chemical pathwayis responsible for moving notochord cells into the forming discs.

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In regions of the embryo where the discs are forming, the no-tochord bulges outward and forms the nucleus pulposus of eachintervertebral disc. We propose that in the absence of a functionalsheath, notochord cells are not constrained and become scatteredthroughout the vertebral column. Consistent with the proposedrole for the notochord sheath in constraining notochord cellswithin the midline of the vertebrate embryo, an increase in theaberrant migration of notochord cells correlated with the ob-served increasing caudal severity of defects in sheath formation.It is possible that abnormal nuclei pulposi formation observed

upon removal of hedgehog signaling results from some other,nonsheath role for hedgehog signaling in this tissue. We cannotrule out the possibility that the hedgehog signaling pathway isresponsible for activation of unknown pathways that are requiredfor proper migration of notochord cells into the forming nucleipulposi, independent of the presence of a notochord sheath.Mechanical removal of the notochord sheath from around thenotochord in normal embryos could directly address this ques-tion; however, this experiment is technically challenging becauseof the inaccessibility of the notochord during vertebrate embry-onic development.Instead, we have taken a genetic approach to address this issue

by removing hedgehog signaling after the notochord sheath hasformed. In these embryos, the sheath was maintained and nor-mal nuclei pulposi formation was observed. These data suggestthat hedgehog signaling is required to specify formation of thenotochord sheath but is not needed to maintain this structureduring later embryogenesis. In wild-type mice, the hedgehogsignaling pathway remains present in nuclei pulposi throughoutearly postnatal life (16). The role this signaling pathway plays inthe postnatal intervertebral discs is unknown.

Materials and MethodsMice. Animals were handled in accordance with the University of FloridaInstitutional Animal Care and Use Committee. Mice containing the condi-

tional floxed allele of Smo (Smof/f), Shhgfpcre, ShhcreERT2, and Shhf/f havebeen described previously (22, 23, 26). For Shhf/ShcreERT2 embryos, tamox-ifen (Sigma) was gavaged at a concentration of 3 mg/40 g body weight ina pregnant female. Detailed information is available in SI Materials andMethods.

RNA in Situ Hybridization, β-Galactosidase Staining, and Skeleton Preparation.Whole-mount RNA in situ hybridization and Xgal staining were performed asdescribed previously (22, 32, 33). Skeleton preparations were performed aspreviously described (34). At least three animals for each genotype wereexamined in all experiments.

Histology and Immunohistochemistry. For histological analysis, embryos werefixed in 4% paraformaldehyde at 4 °C overnight and then E16.5 and olderembryos were decalcified overnight using Cal-Ex (Fisher). Histology andimmunohistochemistry were performed as described in SI Materials andMethods.

Cell Proliferation and Death Assay. To detect cell proliferation in E11.5 no-tochords, pregnant dams were injected with BrdU (50 μg/g bodyweight) for3 h before harvest. Cell proliferation and death assay were performed asdescribed in SI Materials and Methods.

Electron Microscopy. Tissue samples were fixed in 4% paraformaldehyde +2% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.24 and processedfor imaging. A detailed procedure is described in SI Materials andMethods.

ACKNOWLEDGMENTS. We thank members of the B.D.H. and M. Cohnlaboratories for helpful discussions regarding the experiments in this manu-script, and B. Kang and K. Kelley in the University of Florida InterdisciplinaryCenter for Biotechnology Research ElectronMicroscopy and Bioimaging Core.This work was supported by National Institutes of Health/National Insti-tute on Aging Grant AG029353 and National Institutes of Health/NationalInstitute of Arthritis andMusculoskeletal and Skin Diseases Grant AR055568 (toB.D.H.). K.-S.C. was partially supported by the Korea Science and EngineeringFoundation (C00105).

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