Identification of nucleus pulposus precursor cells and notochordal remnants in the mouse: implications for disk degeneration and chordoma formation

Abstract

A classically identified "notochordal" cell population in the nucleus pulposus is thought to regulate disk homeostasis. However, the embryonic origin of these cells has been under dispute for >60 years. Here we provide the first direct evidence that all cell types in the adult mouse nucleus pulposus are derived from the embryonic notochord. Additionally, rare isolated embryonic notochord cells remained in the vertebral column and resembled "notochordal remnants," which in humans have been proposed to give rise to a rare type of late-onset cancer called chordoma. Previously, this cell type had not been identified in the mouse model system. The development and characterization of a mouse model that can be used to fate map nucleus pulposus precursor cells in any mutant background will be useful for uncovering the cellular and molecular mechanisms of disk degeneration. In addition, the identification of notochordal remnants in mice is the first step towards generating an in vivo model of chordoma.

Fig. 1

Fate mapping Shh-expressing cells in the axial skeleton using the Shhcre allele. The Shhcre allele (Harfe et al., 2004) was used to constitutively activate R26R::EYFP in the notochord. EYFP is observed as green. A–D: Merged fluorescent and bright-field images of the vertebral column. In all samples, a ventral view of the spinal column is shown. Embryos were harvested and all gut tissue was dissected away to visualize the underlying vertebral column. All images are of unfixed tissue. A: At E12.5, the notochord begins to form “bulges” in locations where the nucleus pulposus of the intervertebral disk will form (the arrows denote the notochord). B: By E15.5, clearly demarcated nuclei pulposi have formed from Shhcre-expressing cells. Part of the notochord is still observed between the disks (arrow). C, D: Cells that have expressed Shhcre are restricted to the nucleus pulposus and are mostly excluded from the vertebrae (v) at E16.5 (C) and at P0 (D). See Figure 2a and Figure 3b for exceptions. NP, nucleus pulposus.

Fig. 2

Fate map of Shh-expressing cells using the Shhcre and the tamoxifen-inducible Shh-creERT2 alleles. A: Ten-micrometer transverse frozen section of the intervertebral disk of a Shhcre;R26R newborn mouse. Tissue was stained for the presence of β-galactosidase (blue). Note the entire nucleus pulposus is stained. Rare notochord descendants, denoted with an “*”, are found in the annulus fibrosus (AF). B–D: Distribution of Shh descendent cells in ShhcreERT2;R26R mice pulse-labeled with tamoxifen at E8.0. Progeny were stained for β-galactosidase at E13.5. B: At E13.5, the nuclei pulposi of the intervertebral disks are forming. Ventral whole mount view is shown. All gut tissue has been dissected away so that the vertebral column is visible. The inset in B shows three intervertebral disks (ventral view). Note that some cells of the notochord (arrows) are still present between the forming disks. C, D: Whole-mount images of genital tubercles at E13.5. At this stage of development, male and female external genitalia are indistinguishable. The preputial glands (asterisks) are labeled in embryos constitutively expressing CRE in all Shh-producing cells (C). The absence of β-ga-lactosidase in preputial glands of ShhcreERT2; R26R embryos exposed to tamoxifen at E8.0 (D) indicates that the tamoxifen has been cleared from these embryos prior to E13.5, when Shh expression is initiated in these glands (Perriton et al., 2002). The line of staining down the middle of the external genitalia in C and D is the urethra, which expresses Shh beginning at E9.75 (Perriton et al., 2002).

Fig. 3

The nucleus pulposus and notochordal remnants in adult mice are composed of cells that have expressed Shh. A: In a 19-month-old Shhcre;R26R mouse, the nucleus pulposus is labeled and the annulus fibrosus is negative. A 10-μm transverse paraffin section of an intervertebral disk stained for LacZ is shown. Cells were counterstained with fast red, which clearly marked the nuclei of cells. B: Magnification (40×) of the boxed region in A. In all samples examined (four disks from adult mice), all cells of the nucleus pulposus were stained blue indicating that the nucleus pulposus is composed of cells that have expressed Shh. C: Notochordal remnants were found in adult vertebrae between each intervertebral disk in the Shhgfpcre;R26R mouse. Inset in C shows notochord remnants. Ventral view of the vertebral column is shown. Adult animals were stained in whole-mount for LacZ and then dissected. Whole-mount picture is shown. V, vertebrae between the disks; NP, nucleus pulposus; AF, annulus fibrosus.