Chitra Lekha Dahia

Shh Signaling from the Nucleus Pulposus Is Required for the Postnatal Growth and Differentiation of the Mouse Intervertebral Disc

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.

Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging.

Study Design. Intervertebral discs at different postnatal ages were assessed for active intercellular signaling pathways. Objective. To generate a spatial and temporal map of the signaling pathways active in the postnatal intervertebral disc (IVD). Summary of Background Data. The postnatal IVD is a complex structure, consisting of 3 histologically distinct components, the nucleus pulposus, fibrous anulus fibrosus, and endplate. These differentiate and grow during the first 9 weeks of age in the mouse. Identification of the major signaling pathways active during and after the growth and differentiation period will allow functional analysis using mouse genetics and identify targets for therapy for individual components of the disc. Methods. Antibodies specific for individual cell signaling pathways were used on cryostat sections of IVD at different postnatal ages to identify which components of the IVD were responding to major classes of intercellular signal, including sonic hedgehog, Wnt, TGFβ, FGF, and BMPs. Results. We present a spatial/temporal map of these signaling pathways during growth, differentiation, and aging of the disc. Conclusion. During growth and differentiation of the disc, its different components respond at different times to different intercellular signaling ligands. Most of these are dramatically downregulated at the end of disc growth.

Postnatal Growth, Differentiation, and Aging of the Mouse Intervertebral Disc

Study Design. This study follows postnatal intervertebral disc (IVD) growth and differentiation in the mouse. Objective. To initiate use of the mouse as a model system for postnatal IVD differentiation and growth, and to serve as a basis for assaying changes caused by disease or genetic or experimental perturbation. Summary of Background Data. Lower back pain caused by disc degeneration is one of the most common clinical conditions worldwide. There is currently no completely effective treatment, largely because of a lack of basic knowledge of the molecular and cellular controls of disc growth, differentiation, and maintenance after birth. Methods. Conventional histology of decalcified IVDs, differential interference contrast, polarizing optics, immunocytochemistry, laser capture microscopy followed by molecular analysis of the dissected cells by reverse transcriptase polymerase chain reaction. Results. There is a single postnatal growth spurt in the mouse IVD, between birth and 9 weeks of age. Cell proliferation was found in the nucleus pulposus (NP) and anulus fibrosus (AF) only until 3 weeks of age. Most of the postnatal growth of the IVD is due to accumulating extracellular matrix. NP cell numbers decline steadily after 2 weeks of age, because of apoptosis. Laser capture microscopy was used to dissect NP cells from the disc, and showed that these cells express markers of the embryonic notochord. The postnatal AF appears initially as a continuous structure surrounding the NP. This structure differentiates, during the first 2 postnatal weeks, to form the mineralized, but nonossified endplate over the surfaces of the vertebral growth plates, and the mature fibrous AF (fAF) passing between adjacent vertebrae. The fact that the mature fAF and the endplate form from an originally continuous layer of cells explains the anatomic relationship between these 2 structures, in which the fAF inserts into the vertebral endplate. Conclusion. Growth of the IVD takes place during the first 9 postnatal weeks, although cell proliferation ceases after 3 weeks. After birth, the early postnatal IVD differentiates into 3 tissue types, the NP, the fAF between the vertebrae, and the mineralized endplates over the surfaces of the vertebrae.

Regulation of FSH receptor, PKIβ, IL-6 and calcium mobilization: Possible mediators of differential action of FSH

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.

Wnt Signaling Activates Shh Signaling in Early Postnatal Intervertebral Discs, and Re-Activates Shh Signaling in Old Discs in the Mouse

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

Demonstration of Follicle-Stimulating Hormone Receptor in Cauda Epididymis of Rat

Abstract FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind 125IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.

Indian hedgehog signaling and the role of graft tension in tendon‐to‐bone healing: Evaluation in a rat ACL reconstruction model

The structure and composition of the native enthesis is not recapitulated following tendon‐to‐bone repair. Indian Hedgehog (IHH) signaling has recently been shown to be important in enthesis development in a mouse model but no studies have evaluated IHH signaling in a healing model. Fourteen adult male rats underwent ACL reconstruction using a flexor tendon graft. Rats were assigned to two groups based on whether or not they received 0N or 10N of pre‐tension of the graft. Specimens were evaluated at 3 and 6 weeks post‐operatively using immunohistochemistry for three different protein markers of IHH signaling. Quantitative analysis of staining area and intensity using custom software demonstrated that IHH signaling was active in interface tissue formed at the healing tendon‐bone interface. We also found increased staining area and intensity of IHH signaling proteins at 3 weeks in animals that received a pre‐tensioned tendon graft. No significant differences were seen between the 3‐week and 6‐week time points. Our data suggests that the IHH signaling pathway is active during the tendon‐bone healing process and appears to be mechanosensitive, as pre‐tensioning of the graft at the time of surgery resulted in increased IHH signaling at three weeks.

Intercellular signaling pathways active during and after growth and differentiation of the lumbar vertebral growth plate.

Study Design. Vertebral growth plates at different postnatal ages were assessed for active intercellular signaling pathways. Objective. To generate a spatial and temporal map of the major signaling pathways active in the postnatal mouse lumbar vertebral growth plate. Summary of Background Data. The growth of all long bones is known to occur by cartilaginous growth plates. The growth plate is composed of layers of chondrocyets that actively proliferate, differentiate, die and, are replaced by bone. The role of major cell signaling pathways has been suggested for regulation of the fetal long bones. But not much is known about the molecular or cellular signals that control the postnatal vertebral growth plate and hence postnatal vertebral bone growth. Understanding such molecular mechanisms will help design therapeutic treatments for vertebral growth disorders such as scoliosis. Methods. Antibodies against activated downstream intermediates were used to identify cells in the growth plate responding to BMP, TGF&bgr;, and FGF in cryosections of lumbar vertebrae from different postnatal age mice to identify the zones that were responding to these signals. Reporter mice were used to identify the chondrocytes responding to hedgehog (Ihh), and Wnt signaling. Results. We present a spatial/temporal map of these signaling pathways during growth, and differentiation of the mouse lumbar vertebral growth plate. Conclusion. During growth and differentiation of the vertebral growth plate, its different components respond at different times to different intercellular signaling ligands. Response to most of these signals is dramatically downregulated at the end of vertebral growth.

Effect of deprivation of endogenous follicle stimulating hormone on rat epididymis: a histological evaluation

The growth and function of the epididymis are regulated by testosterone produced by Leydig cells in the testes. In the present study it was observed that neutralization of endogenous follicle stimulating hormone (FSH) in immature rats using a highly specific antiserum to ovine FSH resulted in changes in the histology of the epididymis along with a decrease (50-60%) in its weight compared with the normal serum-treated controls. These changes were observed in both rat and monkey epididymis without any decrease in serum testosterone, on which epididymis is known to be dependent. A detailed study was therefore carried out on the effects of deprivation of FSH or testosterone on the histology of epididymis. The changes in epididymal histology following FSH deprivation included a decrease in the size of the tubule lumen in the rat as well as in the adult male bonnet monkey in which the antiserum against ovine FSH was raised. Intensive vacuolization and uneven surface of the luminal epithelium was also observed. In contrast, the effect of deprivation of testosterone support by way of administration of LH antiserum or fiutamide resulted in a decrease in the size of the lumen and degenerative changes. These results suggest that cauda epididymidis is a target for FSH action.

A comparative analysis of expression of selected genes during induction of differentiation in neonatal rats by deprival of FSH or by hyperthyroidism

The effect of Follicle Stimulating Hormone (FSH) deprivation, or hyperthyroidism, on the expression of selected genes in Sertoli cells of neonatal rats was compared, and significant differences were revealed. While hyperthyroidism resulted in both arrest of proliferation and expression of differentiation markers, FSH deprivation only resulted in arrest of proliferation, as assessed by a decrease in such proliferation markers as connective tissue growth factor (CTGF) and collagen 12A1 and the expression of differentiation markers, such as androgen binding protein, transferrin and clusterin. In Sertoli cells isolated from hyperthyroid rats, a decrease in the expression of FSH receptor was also observed. Microarray analysis of RNA from FSH-deprived or hyperthyroid neonatal rat Sertoli cells suggests that molecular events associated with arrest of proliferation by hyperthyroidism appear to be different from arrest of proliferation by FSH deprivation.