Regulation of Childhood Growth

Jeffrey Baron, MD, Head, Section on Growth and Development
Kevin Barnes, PhD, Senior Research Assistant
Gabriella Finkielstain, MD, Postdoctoral Fellow
Julian Lui, PhD, Postdoctoral Fellow
Maria Chang, BS, Predoctoral Fellow
Patricia Forcinito, BS, Special Volunteer

We investigate the cellular and molecular mechanisms governing childhood growth and development, focusing particularly on the skeletal system. We are especially interested in the mechanisms that allow rapid proliferation in the young organism and then suppress proliferation later in life. One goal of our work is to improve the medical treatment of childhood growth disorders. In addition, we seek to uncover general principles of developmental biology, given that the cellular processes underlying bone growth, such as cell proliferation, terminal differentiation, angiogenesis, and cell migration, are also essential for development of other tissues.

Spatial organization of the growth plate

Barnes, Baron

Longitudinal bone growth occurs at the growth plate, which consists of three principal layers: the resting zone, the proliferative zone, and the hypertrophic zone. Studies in our laboratory indicate that stem-like cells in the resting zone differentiate into rapidly dividing chondrocytes of the proliferative zone. The proliferative chondrocytes then terminally differentiate into the nondividing chondrocytes of the hypertrophic zone.

We have investigated the molecular mechanisms responsible for spatial organization of the growth plate. Microarray analysis followed by real-time PCR analysis identified genes whose expression changed dramatically during the differentiation program, including several genes functionally related to bone morphogenetic proteins (BMPs). Additional investigation suggests the existence of a BMP signaling gradient across the growth plate, which is established by differential expression of several BMPs and BMP inhibitors in specific zones. Previous functional studies suggest that the BMP signaling gradient may be a key mechanism responsible for spatial regulation of chondrocyte proliferation and differentiation in the growth plate. Using a similar approach that combined expression and functional studies, we explored the role of fibroblast growth factor (FGF) signaling in the postnatal growth plate. The studies revealed a complex pattern of spatial regulation of FGFs and FGR receptors (FGFRs) in the different zones of the growth plate that appears to help regulate proliferation and differentiation. Understanding the FGF system is of particular importance because abnormalities in FGF signaling cause human skeletal dysplasias, including achondroplasia.

More recently, we analyzed the role of Wnt signaling in the spatial organization of the growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only 6 were expressed in postnatal growth plate. Of these, Wnt-2b, Wnt-4, and Wnt-10b signal through the canonical β-catenin pathway, whereas Wnt-5a, Wnt-5b, and Wnt-11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state, and then (except for Wnt-2b) declining expression as the chondrocytes underwent hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. Thus, we identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.

Andrade AC, Nilsson O, Barnes KM, Baron J. Wnt gene expression in the postnatal growth plate: regulation with chondrocyte differentiation. Bone 2007;40:1361-1369.
Lazarus JE, Hegde A, Andrade AC, Nilsson O, Baron J. Fibroblast growth factor expression in the postnatal growth plate. Bone 2007;40:577-586.
Nilsson O, Parker EA, Hegde A, Chau M, Barnes KM, Baron J. Gradients in bone morphogenetic proteinrelated gene expression across the growth plate. J Endocrinol 2007;193:75-84.
Parker EA, Hegde A, Buckley M, Barnes KM, Baron J, Nilsson O. Spatial and temporal regulation of GHIGF-related gene expression in growth plate cartilage. J Endocrinol 2007;194:31-40.

Temporal regulation of growth plate chondrogenesis

Lui, Barnes, Baron

With age, growth plate chondrocyte proliferation slows, causing longitudinal bone growth to slow and eventually stop. This functional change in the growth plate is accompanied by structural changes; with age, the number of resting, proliferative, and hypertrophic chondrocytes declines, as does the size of individual hypertrophic cells. The chondrocyte columns also become more widely spaced. We have termed this developmental program growth plate senescence. It appears to be caused by a mechanism intrinsic to the growth plate.

The developmental program of growth plate senescence could be a function of time per se. Thus, a biological timing mechanism could drive the change in chondrocyte physiology, including the decline in proliferation rate. Alternatively, growth plate senescence could be a function of growth itself. In this case, the system might be driven by a cell-cycle counter, that is, a cellular mechanism that progressively changes with each cell cycle, instead of by a biological timing mechanism.

To distinguish between the two alternative hypotheses, we treated newborn rats with propylthiouracil (PTU) for 8 weeks to induce hypothyroidism and thus inhibit growth at the growth plate. After discontinuation of the PTU, we studied several functional, structural, and molecular markers in both treated animals and untreated controls to determine whether the previous period of hypothyroidism had delayed the programmed senescence of the growth plate.

Control animals showed the normal senescent decline in tibial growth rate, chondrocyte proliferation rate, growth plate height, resting zone height, number of resting zone chondrocytes, number of proliferative zone chondrocytes per column, number of hypertrophic chondrocytes per column, terminal hypertrophic cell height, and column density. In the previously hypothyroid animals, the senescent decline in every one of these variables was delayed. We also studied molecular markers of growth plate senescence—genes whose mRNA expression in growth plate chondrocytes changed markedly during growth plate senescence. These genes included those encoding chondroadherin, osteoprotegerin, secreted frizzled-related protein 4, reelin, and nuclear protein 1. In control animals, mRNA levels of all of these genes increased markedly with age. Previously hypothyroid animals expressed chondroadherin, osteoprotegerin, secreted frizzled-related protein 4, and nuclear protein 1 mRNAs at levels similar to those in younger control animals. Thus, molecular markers of senescence, like the structural and functional markers of senescence, also appeared to be delayed by the previous hypothyroidism.

The observed delay in several functional, structural, and molecular markers of growth plate senescence in the previously hypothyroid animals strongly supports the hypothesis that hypothyroidism slows the developmental program of growth plate senescence. We previously found evidence, though less extensive, that glucocorticoid excess in the rabbit also slows growth plate senescence. The combined finding, that growth plate senescence is slowed by two growth-inhibiting conditions in two species, supports our model that growth plate senescence is not a function of time per se but rather of growth; therefore, inhibition of growth slows this developmental process.

Emons JA, Marino R, Nilsson O, Barnes KM, Even-Zohar N, Andrade AC, Chatterjee NA, Wit JM, Karperien M, Baron J. The role of p27 kip1 in the regulation of growth plate chondrocyte proliferation in mice. Pediatr Res 2006;60:288-293.
Marino R, Hegde A, Barnes KM, Schrier L, Emons JA, Nilsson O, Baron J. Catch-up growth after hypothyroidism is caused by delayed growth plate senescence. Endocrinology 2008;149:1820-1828.
Schrier L, Ferns SP, Barnes KM, Emons JAM, Newman E, Nilsson O, Baron J. Depletion of resting zone chondrocytes during growth plate senescence. J Endocrinol 2006;189:27-36.

Temporal regulation of growth in non-skeletal tissues

Barnes, Finkielstain, Lui, Chang, Forcinito, Baron

The rate of cell proliferation and consequent somatic growth slows with age and ceases not only in the growth plate but also in several other tissues. To investigate the underlying changes in cell-cycle kinetics, we used [methyl-3H]thymidine and 5΄-bromo-2΄deoxyuridine to double-label proliferating cells in 1-, 2-, and 3-week-old mice for 4 weeks. Proliferation of renal tubular epithelial cells and hepatocytes declined with age. The average cell-cycle time did not increase in liver and increased only 1.7-fold in kidney. The fraction of cells in S-phase that divided again declined approximately 10-fold with age. Concurrently, average cell area increased approximately 2-fold. The findings suggest that somatic growth deceleration primarily results not from a rise in cell-cycle time but from a reduced growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cell volume increases, perhaps because cells are relieved of the size constraint imposed by cell division. In summary, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size.

The rate of cell proliferation that causes somatic growth to decline simultaneously in several organs does not appear to be coordinated by a systemic mechanism. We therefore hypothesized that growth deceleration results from a growth-limiting genetic program that is common to several tissues. To test this hypothesis, we performed microarray analysis to identify changes in gene expression in mice during early postnatal life. We focused on genes that were up- or downregulated in several organs and thus are more likely to contribute to the putative common program of growth deceleration. We noticed that some of the genes showing the greatest changes in expression with age are imprinted; that is, genes show differential expression from the maternal and paternal alleles. Earlier research demonstrated that some imprinted genes positively regulate fetal growth. Our microarray observations raised the possibility that expression of these genes persists into early postnatal life and that their subsequent downregulation contributes to the dramatic decline in proliferation and somatic growth that determines adult body size.

We therefore systematically assessed the age-related changes in gene expression of all known imprinted genes by using microarray analysis for kidney, lung, and heart. We identified a set of 11 imprinted genes that show downregulation of mRNA expression with age in several organs. For these genes—Igf2, H19, Plagl1, Mest, Peg3, Dlk1, Gtl2, Grb10, Ndn, Cdkn1c, and SLC38a4—the declines show a temporal pattern similar to the decline in growth rate. All 11 genes have been implicated in the control of cell proliferation or somatic growth. Thus, our findings suggest that the declining expression of the genes contributes to coordinate growth deceleration in several tissues. We next hypothesized that the coordinate decline in expression of these imprinted genes is caused by altered methylation and consequent silencing of the expressed allele. However, the methylation status of the promoter regions of Mest, Peg3, and Plagl1 did not change with age. In summary, our findings suggest that a set of growth-regulating imprinted genes are expressed at high levels in several tissues in early postnatal life, contributing to rapid somatic growth, but that these genes are subsequently and simultaneously downregulated in several tissues, contributing to coordinate growth deceleration and cessation, thus imposing a fundamental limit on adult body size.

Chang M, Parker EA, Muller T, Haenen C, Mistry M, Finkielstain G, Murphy-Ryan M, Barnes KM, Sundaram R, Baron J. Changes in cell cycle kinetics responsible for limiting somatic growth in mice. Pediatr Res 2008;64:240-245.
Lui JC, Finkielstain GP, Barnes KM, Baron J. An imprinted gene network that controls mammalian somatic growth is down-regulated during postnatal growth deceleration in multiple organs. Am J Physiol Regul Integr Comp Physiol 2008;295:R189-196.

Collaborators

Ola Nilsson, MD, PhD, Karolinska University Hospital, Stockholm, Sweden
Rajeshwari Sundaram, PhD, Biometry and Mathematical Statistics Branch, NICHD, Rockville, MD

For further information, contact jeffrey_baron@nih.gov or visit http://ugd.nichd.nih.gov.