Mechanisms limiting body growth in mammals The human fetus grows at an enormous rate, increasing in mass more than 100 fold between the end of embryogenesis and birth. Somatic growth then slows progressively in postnatal life and ceases by the end of the second decade. The deceleration in body growth is due primarily to a progressive decline in cell proliferation, but the underlying mechanisms are largely unknown. One clue regarding the mechanism is that growth deceleration occurs coordinately in multiple organs in order to maintain body proportions and yet this coordination does not appear to be orchestrated by a systemic mechanism. We therefore hypothesized that postnatal growth deceleration results from a genetic program that is occurring simultaneously in multiple tissues. Consistent with this hypothesis, we identified an extensive program of gene expression that is occurring between 1 and 8 wk of age simultaneously in lung, kidney, and heart of mice. Multiple lines of evidence suggest that at least part of this program contributes to body growth deceleration. For the downregulated genes in the program, gene ontology analyses indicated strong overrepresentation of genes implicated in cell growth / proliferation. Furthermore, of the age-downregulated genes that have a reported knockout phenotype, more than one third showed a decrease in body size without any detected underlying disease, implying that these age-downregulated genes in the program promote somatic growth. This concept was further supported by in vitro studies using siRNA-mediated knockdown of gene expression, which demonstrated that a subset of age-downregulated genes in the program are required for rapid proliferation of cultured fetal hepatocytes. Taken together, the findings imply that the multi-organ postnatal genetic program involves the downregulation of many genes, some of which are required for rapid proliferation in early life, supporting the hypothesis that part of this program contributes to growth deceleration. The finding that this program occurs concurrently in kidney, lung, heart, and liver provides a potential explanation for how growth slows coordinately in these organs, thus maintaining body proportions. Next, we investigated the physiological mechanisms that drive this multi-organ postnatal genetic program. We showed that delaying growth by inducing tryptophan deficiency in juvenile rats caused a striking delay in the genetic program, suggesting that the multi-organ postnatal genetic program is not simply driven by a biological timing mechanism, but instead depends on growth. Thus, the program could, for example, be driven by increasing cumulative number of cell divisions undergone (a cell division counter) or by increasing tissue mass. The dependence of the program on growth is also supported by preliminary observations in another model of growth inhibition, hypothyroidism. We also investigated the molecular mechanisms driving the growth-limiting genetic program and found that histone 3, lysine 4 trimethylation (H3K4me3) declined with age in multiple age-downregulated genes and multiple organs. Because H3K4me3 is a signature of permissive chromatin, the observed declining H3K4me3 may reflect the conversion of chromatin into a non-permissive state with body growth, which may thus orchestrate the observed downregulation of multiple genes. Taken together, our findings support the following model to explain limitation of organ and body size in mammals. Somatic growth deceleration results from a multi-organ postnatal genetic program, primarily involving downregulation of a large set of growth-promoting genes. Because this growth-limiting genetic program is occurring simultaneously in multiple tissues, the decline in growth rate of various organs occurs in a concerted fashion, which serves to maintain body proportions. This growth-limiting program depends not simply on age but on somatic growth itself and may be orchestrated by epigenetic mechanisms including declining H3K4me3. Therefore, growth leads to progression of this program with downregulation of many growth-promoting genes, which in turn causes growth of these organs to slow and eventually cease, thus setting a fundamental limit on adult organ size. Spatial and Temporal Regulation of Gene Expression in the Mammalian Growth Plate Longitudinal bone growth in mammals occurs at the growth plates. These cartilaginous structures are organized into three distinct layers -- the resting zone, the proliferative zone, and the hypertrophic zone. Growth plate chondrocytes undergo sequential differentiation from the resting to the proliferative to the hypertrophic state as their spatial position shifts. To explore the mechanisms responsible for spatial regulation in the growth plate in an unbiased manner, we microdissected postnatal rat growth plates into their constituent zones and then used microarray analysis to characterize the changes in gene expression that occur as chondrocytes undergo spatially-associated differentiation. Findings were confirmed using real-time PCR and in situ hybridization. We then used bioinformatic approaches to identify functional pathways that may regulate these processes. In the transition from the resting to the proliferative zone, this analysis implicated several functional pathways: VDR/RXR activation, PDGF signaling, BMP signaling, and notch signaling. In the transition from the proliferative to the hypertrophic zone, the microarray analysis implicated other functional pathways: p53 signaling, cell cycle: G2/M regulation, cell cycle: G1/S regulation, ephrin receptor signaling, oncostatin M signaling, and BMP signaling. We also used the microarray findings to identify potential molecular markers for each zone of the growth plate. Using an empirical formula to rank genes based on their spatial expression pattern, we identified markers that show greater than 10-fold specificity for each of the growth plate zones. These markers of chondrocyte differentiation are likely to prove useful in future studies. In addition to spatial regulation, the growth plate also undergoes important temporal regulation. Over time, proliferation slows in the growth plate, causing the rate of longitudinal bone growth to decrease and approach zero as the organism approaches its adult size. This decline in proliferation is accompanied by gradual structural involution of the growth plate. To explore the underlying mechanisms, we used microdissected growth plates from 3-, 6-, 9-, and 12-wk rats and analyzed gene expression using microarray. This analysis implicated several functional pathways in the developmental program of growth plate senescence: eisosanoid signaling, VDR/RXR activation, p38 MAPK signaling, and Wnt/-catenin signaling. We also identified molecular markers for growth plate senescence, which can now be used as indicators of the maturational state of the growth plate chondrocytes.
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