Shh Signaling from the Nucleus Pulposus Is Required for the Postnatal Growth and Differentiation of the Mouse Intervertebral Disc Chitra Lekha Dahia1*, Eric Mahoney1, Christopher Wylie2* 1 Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 2 Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America Abstract Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord- derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function. Citation: Dahia CL, Mahoney E, Wylie C (2012) Shh Signaling from the Nucleus Pulposus Is Required for the Postnatal Growth and Differentiation of the Mouse Intervertebral Disc. PLoS ONE 7(4): e35944. doi:10.1371/journal.pone.0035944 Editor: Christoph Englert, Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Germany Received January 12, 2012; Accepted March 23, 2012; Published April 27, 2012 Copyright: 2012 Dahia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Orthopaedic Research and Education Foundation (OREF) Career Development Grant to CD (Grant number 11-228; http://www.oref.org/site/PageServer). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Chitra.Dahia@cchmc.org (CD); Christopher.Wylie@cchmc.org (CW) Introduction Intervertebral discs (IVDs) allow movement and resistance to tension and compression forces between each vertebrae, and maintain a constant intervertebral space that prevents compres- sion of the spinal nerves. Each disc consists of an annulus fibrosus (AF); a series of orthogonally arranged fibrocartilagenous layers passing between adjacent vertebrae, surrounding a more cellular central area, the nucleus pulposus (NP) containing large reticular cells embedded in a dense aqueous matrix of proteoglycans and collagens [1,2]. Between the NP and growth plate of the adjacent vertebral body is a layer of tissue several cell layers thick, which form a mineralized layer of cartilage known as the end plate (EP) during the first few weeks of postnatal life in the mouse [3]. Damaged IVDs are a major cause of lower back pain in humans, and of days lost from work [4–6]. Their surgical repair is complex, expensive, prone to failure, and does not cure the underlying pathogenesis [5]. Despite its clinical importance, the intervertebral disc is a somewhat neglected structure. Billions of dollars are spent each year on its repair, and yet we know surprisingly little about the mechanisms of its growth, differenti- ation, and maintenance, nor how these are affected by aging or acute injury [5]. We have shown previously that many intercellular signaling pathways are active during postnatal IVD growth in the mouse [7]. Elucidation of the functions of these would offer new opportunities for biological therapies for both acute and chronic disorders of the IVD. For example, sonic hedgehog (Shh) is synthesized in abundance by the NP cells [3,7–10], consistent with their derivation from the embryonic notochord [8]. In addition to its structural role in the embryo, the notochord is known to be a major signaling center that controls differentiation of the adjacent spinal cord and somites. We have shown previously that during early postnatal growth and differentiation of the mouse IVD, cells of the disc respond to Hedgehog (Hh) signaling [7]. This signaling becomes inactive a few weeks after birth, suggesting that it is primarily required for IVD growth and/or differentiation. These data raised the interesting hypothesis that the NP in each disc is a local signaling center that controls the growth and/or differentiation of the IVD. We tested this hypothesis in two ways. First, we cultured mouse lumbar IVD’s from postnatal day 4 (P4), a stage at which the IVD is growing and differentiating, and when Shh is being synthesized at high levels by the NP cells [3,7,9]. Discs cultured for up to five days in the absence of serum maintained their differentiated state in culture, suggesting that endogenous signals are sufficient for this. When cultured in the presence of cyclopamine, a specific inhibitor of Hh signaling [11– 14], cell proliferation in the NP stopped, and cell differentiation of NP, AF, and EP was inhibited, as shown by the loss of molecular markers of IVD differentiation. All these effects were rescued by replacing cyclopamine with recombinant Shh (rShh) in the culture PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35944