INTRODUCTION: Congenital vertebral synostosis (CVS) and Klippel-Feil syndrome (KFS) cause the fusion of vertebrae and are a rare developmental deformity that widely goes unnoticed. The vertebral column develops from the embryonic notochord, which gives rise to the intervertebral disc's nucleus pulposus (NP) cells. The sclerotome gives rise to the annulus fibrosus (AF) of the disc and the vertebra. Resegmentation of the sclerotome is critical for proper vertebral patterning, regulated by signals from the notochord and neural tube. CVS can result from the complete fusion of two vertebrae or when a disc fails to form, resulting in a rudimentary disc. Very little is known about the cause of CVS or KFS. The occurrence of KFS is reported to be sporadic (reasons not known) or inherited in an autosomal dominant manner. Some cases of KFS are associated with variation in GDF6, GDF3, MEOX1, and RIPPLY2 genes. However, the causes of CVS and many instances of KFS in patients are not associated with either of these reported variants, and their reasons remain unknown.
GLI1 is a downstream target of hedgehog (Hh) signaling. Expression of sonic hedgehog (SHH), a key member of the Hh family of proteins, by the notochord, has both cell-autonomous and non-autonomous functions in embryonic patterning. Here, while studying the spine of the Gli1-loss of function mutant mouse model, we found that complete loss of GLI1 causes CVS at several levels in the mouse spine.
METHODS: The mice were maintained, and experiments were conducted using the approved IACUC protocol. Gli1Lzknock-in allele was used where endogenous GLI1 function is abolished. Notochord cells were lineage-traced using ShhCre; R26TOM alleles in the Gli1Lz/WT (Gli1-het) and Gli1Lz/Lz (Gli1-null) littermates. Spines were assessed soon after birth to 18 months of age, with n=2-6 mice/genotype at each age. Immunofluorescence analyzed the expression of NP markers, cell proliferation, cell death, and extracellular matrix in the IVD. Morphometric and histopathological analysis quantified changes to the IVD. GraphPad Prism was used to perform statistical analyses of the data.
RESULTS: We found vertebral synostosis at several spine levels of the Gli1-null mice at different ages. No fused vertebra or discs were observed in the age-matched Gli1-het littermates. The fused vertebrae had rudimentary IVDs that lacked notochord/ NP cells, which were validated by lineage tracing and immunofluorescence for NP markers. Immunofluorescence revealed that fused IVD lacked essential structural proteins in the NP, and AF—morphometric and histopathological analysis quantified changes in each region of IVD.
DISCUSSION: Our studies have identified GLI1 as a novel gene associated with CVS in mice. Our findings show the importance of GLI1 in notochord and sclerotome patterning and formation of the NP and greater IVD region. Interestingly, the IVDs with notochord/NP cells maintained proper structure in the postnatal stage, indicating that GLI1 is essential for vertebral and IVD patterning but may not be critical for maintaining postnatal NP cells.