Mechanisms Limiting Body Growth in Mammals

R E V I E W

Mechanisms Limiting Body Growth in Mammals Julian C. Lui and Jeffrey Baron Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Recent studies have begun to provide insight into a long-standing mystery in biology—why body growth in animals is rapid in early life but then progressively slows, thus imposing a limit on adult body size. This growth deceleration in mammals is caused by potent suppression of cell proliferation in multiple tissues and is driven primarily by local, rather than systemic, mechanisms. Recent evidence suggests that this progressive decline in proliferation results from a genetic program that occurs in multiple organs and involves the down-regulation of a large set of growth-promoting genes. This program does not appear to be driven simply by time, but rather depends on growth itself, suggesting that the limit on adult body size is imposed by a negative feedback loop. Different organs appear to use different types of information to precisely target their adult size. For example, skeletal and cardiac muscle growth are negatively regulated by myostatin, the concentration of which depends on muscle mass itself. Liver growth appears to be modulated by bile acid flux, a parameter that reflects organ function. In pancreas, organ size appears to be limited by the initial number of progenitor cells, suggesting a mechanism based on cell-cycle counting. Further elucidation of the fundamental mechanisms suppressing juvenile growth is likely to yield important insights into the pathophysiology of childhood growth disorders and of the unrestrained growth of cancer. In addition, improved understanding of these growth-suppressing mechanisms may someday allow their therapeutic suspension in adult tissues to facilitate tissue regeneration. (Endocrine Reviews 32: 422– 440, 2011)

I. Introduction

I. Introduction

II. Cellular Events Responsible for Declining Proliferation A. Decreasing growth fraction vs. increasing cell-cycle time B. Changes in stem cells, transit-amplifying cells, and terminally differentiated cells III. Systemic and Local Mechanisms for Body Growth Deceleration A. Coordination of body growth: temporal, conditional, and evolutionary B. Modulation of growth by systemic mechanisms C. Progressive suppression of growth by local mechanisms D. A multiorgan postnatal genetic program IV. How Do Organs “Know” When to Stop Growing? A. Mechanisms that assess time: biological clocks B. Catch-up growth: evidence for mechanisms that assess growth C. Mechanisms that assess organ mass: growth-inhibiting chalones D. Mechanisms that assess organ function E. Mechanisms that count cell divisions V. Clinical Implications VI. Conclusion ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/er.2011-0001 Received January 3, 2011. Accepted March 2, 2011. First Published Online March 25, 2011

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person’s right and left legs almost always end up the same length, and the hearts of mice and elephants each fit the proper rib cage. How genes set limits on cell size and number continues to mystify.” (1) One of the remaining fundamental mysteries in biology is how organs and organisms know when to stop growing. In mammals, by the time of adulthood, body growth has generally ceased or slowed dramatically. Importantly, this cessation or near cessation of growth does not occur abruptly, but rather is the end result of a progressive decline in growth rate that begins in early life. In humans, for example, the linear growth rate (change in body length per unit time) declines dramatically with age (Fig. 1). During fetal life, the linear growth rate is enormous, exceeding 100 cm/yr. By birth, the rate has fallen to approximately 50 cm/yr, and by mid-childhood, 5 cm/yr (2). This decline in growth rate is briefly interrupted by the pubertal growth spurt, raising the growth

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Abbreviations: AgRP, Agouti-related peptide; Cdk, cyclin-dependent kinase; IGFBP-3, IGF binding protein-3; mTOR, mammalian target of rapamycin; NPY, neuropeptide Y; Trp⫺, tryptophan-deficient.

Endocrine Reviews, June 2011, 32(3):422– 440


Fig. 1. The decline in the human linear growth rate is not due to declining circulating IGF-I levels. First row, In humans, height increases rapidly in early childhood but eventually plateaus in adolescence (216). Second row, The linear growth velocity (first derivative of the height curve) decreases dramatically during infancy, more gradually during childhood, briefly rises during the pubertal growth spurt, and then resumes its decline, approaching zero (216). Third to fifth rows, As growth is slowing, there is a general increase in total IGF-I and IGFBP-3, both of which are stimulated by GH, as well as an increase in free IGF-I (derived from reference 52).


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rate to approximately 10 cm/yr, but then the decline in growth rate resumes, causing linear growth to cease by mid to late adolescence (Fig. 1). Similar declines in linear growth rate occur in other mammals. However, the time course varies markedly. In rodents, this decline occurs over weeks, resulting in a small body size, whereas in humans and other large mammals, the decline occurs gradually, over years, resulting in a larger body size (2, 3). Another difference is that some species, such as the rodent, do not undergo epiphyseal fusion at the time of sexual maturation and have persistent linear growth into adulthood, but nonetheless, this persistent growth continues to slow, reaching very low rates. This growth deceleration occurs not only in linear growth, which reflects growth of chondrocytes in the growth plate, but also in growth of other tissues and organs, and consequently in growth of overall body mass. For example, in rodents, body mass increases exponentially during embryonic life, but then this initial exponential growth is restrained to a linear and then a sublinear trajectory (4). Similarly, in humans, body mass increases exponentially for the first 3 wk after conception, and then the body mass curve deviates below the exponential trajectory (5). Postnatally, human body mass doubles in the first 5 months after birth, doubles again in the next 30 months, and then again in the next 6 yr. Eventually, the body mass growth rate approaches zero as the individual approaches adult size (except for the continuing gain in fat mass that occurs in permissive environments). Growth deceleration results from a decline in both the rate of cell proliferation (hyperplasia) and the rate of cell enlargement (hypertrophy). In dividing cells, cell growth and cell division are often coordinated to maintain a constant average cell size while cell number increases (6). In rodents, body growth in early juvenile life is primarily due to proliferation, leading to an increase in cell number (7). With increasing age, this hyperplasia slows, at which time hypertrophy occurs in many tissues but also plateaus subsequently (7). Thus, the decline in body growth velocity is first due to a decrease in the rate of cell proliferation, followed by a decrease in the rate of cell enlargement. In the rat, the net result is that between birth and 1 month of age, the whole body DNA content (which reflects the number of nuclei) increases 7-fold, and the amount of protein per nucleus (which reflects cell size) increases 3-fold. The enormous differences in body size among mammals result primarily from differences in cell numbers rather than cell size. For example, the sizes of most cell types in a mouse do not differ much from that of a dog or a cow. The volume of a hepatocyte in all these animals is approximately 2000 fl (8, 9). In contrast, a 70-kg human contains about 1013

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cells, compared with a 25-g mouse that contains about 3 ⫻ 109 cells (10, 11). Thus, an approximately 3000-fold difference in body mass is accounted for by an approximately 3000-fold difference in cell number (10). In this review, we focus our attention on the mechanisms responsible for the decline in cell proliferation.

II. Cellular Events Responsible for Declining Proliferation A. Decreasing growth fraction vs. increasing cell-cycle time

The decline in cell proliferation in multiple tissues during juvenile life could be due to an increase in the cell-cycle time, a decrease in the growth fraction, or a combination of both. An increase in the cell-cycle time indicates that a longer time is required to pass through the entire cell cycle. The growth fraction represents the number of cells that remain in the cell cycle divided by the total number of cells in an organ. Therefore, a decrease in growth fraction indicates that fewer cells remain actively dividing, whereas more cells have dropped out of the cell cycle or remain in the G0 phase. In juvenile mice, as proliferation declines in kidney and liver, the cell-cycle time only increases modestly in renal epithelial cells and does not increase appreciably in hepatocytes (12). In contrast, the growth fraction in these organs decreases markedly (12). Similarly, a decrease in growth fraction with age has been reported in the rat liver postnatally (13, 14). These results therefore suggest that declining cell proliferation in juvenile life primarily results not from an increase in cell-cycle time but from a decrease in growth fraction. B. Changes in stem cells, transit-amplifying cells, and terminally differentiated cells

It has been hypothesized that stem cells exist in adult organs for tissue maintenance purposes (15). The role of these residential stem cells in juvenile growth and growth deceleration is not known. Several possible roles might be imagined. First, growth deceleration might result from a decrease in stem cell proliferation; and second, from a decline in the number of stem cells. Third, the proliferation rate of transit-amplifying progeny derived from these stem cells might decline with age. A fourth possibility is that, in the embryo, proliferation occurs in most cells in each organ, but that with age many cells exit the cell cycle, such that proliferation is eventually restricted to a small set of cells, the residential stem cells. The mammalian growth plate provides a useful system to analyze these issues because, in this cartilaginous structure, stem-like progenitor cells, transit-amplifying cells, and terminally differentiated cells are spatially segregated


into the resting, proliferative, and hypertrophic zones, respectively (16). Declining growth in the juvenile rabbit growth plate is associated with depletion of the stem-like resting zone chondrocytes, both quantitatively (number of cells) and qualitatively (proliferation rate) (17). Interestingly, proliferation also slows with age in the transit-amplifying proliferative chondrocytes (17, 18), which could reflect the declining proliferative capacity of the stem-like cells from which they derive. The role of stem cells and transit-amplifying cells in growth deceleration has been less well studied in other tissues. In muscle, satellite cells, which function as residential myogenic stem cells, show declining proliferation rates by 2–3 months after birth in rats (19) and turkeys (20). In liver, a hepatic progenitor population, the oval cells (21), contributes to regeneration when injury occurs simultaneously with impaired hepatocyte proliferation (usually due to viruses, toxins, or drugs) (22–24). However, regeneration after partial hepatectomy and normal tissue turnover are driven by proliferation of fully differentiated hepatocytes (25). In organs such as kidney and pancreas, where evidence of adult stem cells is conflicting, it is unclear whether the decrease in cell proliferation during postnatal life is due to the depletion of progenitor cells. The observations that existing fully differentiated cells in kidney (26) and pancreatic ␤-cells (27) can undergo cell division for tissue repair argue against the existence of adult stem cells and thus suggest that the decrease in proliferation rate with age may not be attributable to a single progenitor cell population.

III. Systemic and Local Mechanisms for Body Growth Deceleration A. Coordination of body growth: temporal, conditional, and evolutionary

One clue regarding the mechanisms responsible for limiting growth is that growth of different organs and structures appears to be coordinated to maintain proportionality of these body parts. Growth of these various organs appears to be coordinated temporally, conditionally, and evolutionarily (28). Growth is coordinated temporally in that, as growth slows with age, the deceleration occurs in multiple different organs. The time course of this deceleration is similar in different organs but not identical (29). For example, the central nervous system tends to show more rapid declines in growth than most other tissues (30, 31). Conditional coordination of growth is observed during conditions such as GH deficiency, hypothyroidism, and malnutrition. Such conditions generally cause widespread inhibition of growth in multiple organs such that body


proportions tend to be maintained. Similarly, when growth-inhibiting conditions resolve, catch-up growth is observed in multiple organs, again tending to maintain body proportions (32, 33). Furthermore, targeted ablation of a wide variety of growth-regulating genes in mice either inhibits or stimulates growth of multiple organs. This tendency to maintain body proportions is not absolute in that the growth rates of different organs are typically affected in the same direction but not to an identical extent. For example, in mice lacking the GH receptor, the weights of major organs are reduced proportionately, except for the kidney and spleen, which show a greater reduction in size, and the brain, which shows less reduction in size (34). Similarly, IGF-I deficiency and thyroid hormone deficiency in mice inhibit both brain growth and overall body growth but, in both disorders, brain growth is inhibited less, which results in an increase in brain to body weight (35, 36). In humans, uteroplacental insufficiency similarly causes less inhibition of brain growth compared with overall body growth (37). Growth also appears to be coordinated during evolution. In large mammals, such as the elephant, the size of multiple organs is proportionally increased compared with small mammals, such as mice. Less dramatic examples can also be found within a species; organs in large dogs are bigger than organs in small dogs (38), and similarly, in comparing organ sizes between chickens of light and heavy breeds (39). B. Modulation of growth by systemic mechanisms

One possible explanation for coordinated growth of different organs is that body growth is orchestrated by a hormonal or other systemic mechanism. In fact, a hormonal mechanism plays a significant role in growth deceleration of insects. When the larvae reach a “critical size,” juvenile hormone levels fall and ecdysone levels rise, which signals juvenile growth to stop and metamorphosis to commence (40). Might GH play an analogous role in mammalian growth deceleration? Several observations appear to support this hypothesis. Certainly, GH is a potent regulator of postnatal growth and can affect final body size. Mice lacking functional GH receptors reach an adult body mass approximately 60% that of wild-type mice, and mice lacking IGF-I reach an adult body mass approximately 30% of normal (34). Conversely, overexpression of GH and IGF-I in mice leads to an increase in body size (41, 42). Analogous effects on body size are seen in humans with decreased GH, increased GH, or decreased IGF-I. GH deficiency affects primarily postnatal growth, whereas IGF-I deficiency affects both pre- and postnatal somatic growth (43). Interestingly, size differences among different breeds of domestic dogs are associated with different IGF-I alleles (44) and with serum


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IGF-I levels (45). Furthermore, the normal rapid proliferation rate in early life is temporally associated with a high level of GH. Circulating GH levels in the human, ovine, and rodent fetus are substantially higher than in the adult organism (46). These levels then decline sharply during the neonatal period (46 – 48). Similarly, the human pubertal growth spurt is driven in large part by an increase in serum GH levels. However, multiple lines of evidence suggest that growth deceleration is not primarily driven by declining GH levels. First, the elevated fetal levels of GH are not required for the normal rapid pace of fetal growth, which is maintained even in the absence of functional GH receptors in both mice and humans (34, 49). Furthermore, in these mice and humans lacking functional GH receptors, overall postnatal growth is diminished, but the normal pattern of deceleration is maintained (34, 49). Second, in GH-deficient humans, administration of a fixed dose of GH per kilogram of body weight produces a growth curve similar to that of normal individuals, including the normal decline and cessation in growth. Third, overexpression of GH or IGF-I in transgenic mice leads to a substantial increase in body and organ size, but postnatal growth still slows progressively (42, 50, 51). Fourth, the decline in GH levels during early infancy is not accompanied by declines in circulating IGF-I or of IGF binding protein-3 (IGFBP-3) (47, 52), both of which reflect GH action on the liver. Indeed, IGF-I, which mediates most of the growth-promoting effect of GH (34), generally increases in concentration from infancy through early adolescence, as growth is slowing (53) (Fig. 1). This increase in IGF-I is coupled to an increase in IGFBP-3 (47, 52), which stabilizes circulating IGF-I but may also decrease IGF-I bioavailability. However, free IGF-I rises with age (52) (Fig. 1), suggesting that bioavailable IGF-I also likely rises and thus could not account for a declining growth rate. Fifth, both circulating GH and IGF-I concentrations are quite elevated during mid to late adolescence, as growth slows and the individual approaches adult size (47, 52, 54, 55). Serum GH and IGF-I levels do reach a peak during midpuberty and then decline (55, 56), but even when body growth is ceasing, levels are still considerably higher than levels during the rapid growth of earlier childhood. These findings argue that declining GH and IGF-I, acting in an endocrine manner, do not play major roles in growth deceleration. However, none of these arguments exclude the possibility that IGF-I, acting in a paracrine fashion, plays a role in growth deceleration. Indeed, IGF-I is expressed by many tissues under complex regulation (including, but not limited to, GH) and in mice, IGF-I promotes growth through both endocrine

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actions (57) and paracrine actions (58). Thus, the role of paracrine IGF-I in growth deceleration remains to be determined. Nutritional intake is another systemic factor that strongly modulates growth but does not appear to be the predominant mechanism driving normal growth deceleration. Although undernutrition inhibits juvenile growth (29, 59 – 62), overnutrition does not prevent growth deceleration. In rats (63, 64), pigs (65), and chickens (66, 67), forced overfeeding accelerates weight gain. However, with increasing age, more of the weight gain is attributed to fat deposition rather than protein content (63, 64). Similarly, in transgenic animal models that exhibit hyperphagia and obesity, such as leptin-deficient mice (68) and melanocortin-4 receptor (Mc4r)-deficient mice (69), the majority of weight difference is due to increased fat mass, rather than changes in lean mass or visceral organ mass (69, 70). A systemic factor, estrogen, does plays a role in growth deceleration, specifically in the growth plate. In humans, the 20-fold decline in linear growth rate that occurs between fetal life and preadolescence appears to be largely independent of sex steroids. However, with puberty, in both males and females, estrogen levels increase, causing linear growth acceleration (due in part to increased GH secretion). Soon afterward, the decline in growth resumes, so that the growth rate approaches zero at approximately 15 yr of age in girls and 18 yr of age in boys. Once linear growth has essentially ceased, the inactive growth plates are converted into bone (71), a process called epiphyseal fusion. In the absence of estrogen effect, this latter phase of linear growth deceleration occurs more gradually. In a man with mutations in estrogen receptor-␣, and thus estrogen resistance, persistent growth plate function was observed well into adulthood (72). Importantly, however, in this individual, linear growth did progressively slow and eventually cease. At 30 yr of age, the growth rate had fallen to approximately 0.5 cm/yr (73), and at 35 yr of age, ossification of the growth plates finally occurred (74). Similar slowly progressive declines in linear growth rate have been observed in men with estrogen deficiency due to aromatase mutations (75) and, indeed, in normal rodents whose growth plates appear not to respond to estrogen in a manner similar to humans (76). Studies in rabbits indicate that the normal developmental program of growth plate senescence, which includes a decline in chondrocyte proliferation rate, is accelerated by estrogen, causing earlier exhaustion of the proliferative capacity and consequently earlier fusion (77). Thus, estrogen promotes growth deceleration in the growth plate but is not required for this process.


C. Progressive suppression of growth by local mechanisms

Might some unknown systemic factor other than GH, IGF, or nutritional status drive postnatal growth deceleration? Transplantation experiments between animals of different ages argue against this conjecture. When juvenile organs, including intestine (78, 79), kidney (80), heart (81), thymus (82, 83), spleen (84), and the growth plate (85, 86) are transplanted into adult recipients, these organs continue to grow rapidly as if they were still situated in a juvenile host, and then later show growth deceleration when approaching their adult sizes (78, 80, 82, 84), suggesting that growth deceleration is an intrinsic property of the organ rather than the systemic environment. In contrast, parabiosis between young (2–3 months old) and old (19 –26 months old) mice restores the proliferation and regenerative capacity of aged muscle satellite cells and hepatocytes in the older mice (87). However, it is unclear how these studies on regenerative capacity of adult cell proliferation apply to juvenile growth. In summary, existing evidence does not exclude the possibility that systemic mechanisms contribute to physiological growth deceleration but does indicate that local mechanisms play a central role in this process. Thus, growth deceleration appears to be primarily an intrinsic property of growing organs. It remains possible that such local mechanisms may depend on a systemic signal. For example, although circulating GH and IGF-I concentrations do not decline substantially during human growth deceleration, the local downstream effectors of GH or IGF-I at the organ level, such as receptors and the associated signaling pathways, could be involved. Similarly, declining nutritional intake does not appear to be a principal mechanism driving growth deceleration, but it is possible that local, downstream mediators of this nutritional effect might be involved. For instance, nutritional availability regulates cell growth in part through the mammalian target of rapamycin (mTOR) signaling pathway at the molecular level (88 –90). Activation of mTOR kinase under high nutrient/energy conditions leads to the phosphorylation of two major targets, S6K and 4E-BP (91). Phosphorylated S6K in turn acts on a variety of effectors (92) to promote cell growth, cell-cycle progression, and protein synthesis (93–96). Phosphorylated 4E-BP is released from the translation initiation factor eIF4E, allowing its binding with 5⬘-capped mRNAs to initiate translation (90, 97, 98). Conversely, nutrient deprivation leads to suppression of mTOR kinase activity, which in turn causes inhibition of protein synthesis and cell growth (90, 97, 98). It is possible that the mTOR kinase activity is gradually suppressed in growing organs, thus contributing to growth deceleration. This hypothesis has been tested recently by injecting rapa-



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mycin into fetal (embryonic d 19) and postnatal (10-d-old) mice and examining the effects on proliferation (99). Surprising, cellular proliferation in tissues from both ages was resistant to rapamycin treatment and thus occurs independently of mTOR/S6K signaling (99). These findings therefore argue against a role of mTOR signaling in regulating normal organ growth with age. Although the mechanisms responsible for limiting organ growth appear to be local, rather than systemic, they may not be cell autonomous but rather may involve local interactions among cells, such as paracrine signals. In muscle, experiments involving coculture of myofiber explants with satellite cells suggest that the proliferative capacity of satellite cells depends on the age of the myofiber rather than the satellite cells themselves, suggesting a local, nichedependent mechanism (100). Similarly, resting zone chondrocytes, which are thought to act as progenitor cells for the growth plate (16), show marked declines in cell proliferation in vivo (17), but when these cells are placed in cell culture, their proliferative capacity is independent of the age of the donor animal (101), suggesting that the decline in proliferative activity is not cell-autonomous but depends on interaction of the cell with neighboring cells, extracellular matrix, or other extracellular factors. D. A multiorgan postnatal genetic program

Growth retardation and overgrowth phenotypes have been reported in knockout mouse models of many different genes, indicating important roles of these genes and associated signaling pathways in modulating body growth. However, it is yet unclear how these findings can be used to explain juvenile growth deceleration. For example, as discussed above, manipulating GH expression in transgenic animals affects growth rate and adult body size, but it does not imply that GH is responsible for the physiological deceleration and cessation of body growth. Similarly, for most signaling pathways that affect body size, like the oncogenic c-Myc pathway (102–104) and the recently discovered Hippo kinase cascade (105–110), it is not known whether modulation of these regulatory systems contributes to growth deceleration. As discussed in Section III.C, body growth deceleration occurs coordinately in many organs throughout the body, and yet this coordination does not appear to be orchestrated by a systemic mechanism. Another possible explanation for this coordination is that multiple organs employ the same or similar local growth-decelerating mechanisms. Consistent with this line of reasoning, recent evidence suggests that there exists a complex growth-limiting genetic program that occurs simultaneously in multiple organs during juvenile life. The presence of this program was first suggested by microarray analysis of global changes in gene expression as juvenile growth slows in

Fig. 2. A complex growth-related genetic program occurs in multiple organs during juvenile life. A, Venn diagrams showing the number of genes down-regulated and up-regulated with age by microarray analysis in mouse and rats. The analysis included genes that showed age regulation (P ⬍ 0.05; ⱖ2.0-fold) in mouse kidney, lung, and heart or in both rat kidney and lung. The substantial overlap indicates that the program was highly conserved during the 20 million yr since the two species diverged. B, Heat maps based on the same set of genes. Each row corresponds to a single gene. Green, Downregulation with age; red, up-regulation. Scale values are log2 (fold difference). C, A knockout phenotype was reported for 139 of the genes in this same gene set. For the down-regulated genes, knockout frequently resulted in decreased body size, suggesting that many down-regulated genes in this program are growth promoting. D, Bioinformatic analyses of these age-regulated genes using Ingenuity Pathway Analysis (IPA) 7.1 and GeneGO also suggest that many of the genes that are down-regulated with age serve to regulate proliferation. For IPA, the five most overrepresented molecular, cellular, or physiological functions are shown (solid bars, P value; striped bars, number of significant genes involved). For GeneGO, all significant (P ⬍ 0.05) map folders are shown. [Reproduced from J. C. Lui et al.: FASEB J 24:3083–3092, 2010 (113).]

mice (111, 112) and rats (113). These studies have uncovered an extensive genetic program that occurs during growth deceleration; is common among kidney, lung, and heart; and is conserved between mice and rats (Fig. 2, A and B). This common program involves the down-regulation of hundreds of genes with age. For many of these

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genes, knockdown of expression in vitro inhibits cell proliferation, and knockout in vivo inhibits body growth, suggesting that they normally promote growth (Fig. 2, C and D) (113). These down-regulated, growth-promoting genes include growth factors such as Igf2, Mdk, and Ptn and transcription factors such as Ezh2, Mycn, Peg3, and Plagl1. More limited data suggest that this program also occurs in liver (113). Taken together, these findings suggest that juvenile growth deceleration is caused by the down-regulation of a large set of growth-promoting genes, some of which may involve local paracrine mechanisms. Because this genetic program is common to multiple organs, it may explain how growth in different organs slows concordantly, therefore maintaining body proportions. This complex program may also help explain why genetic disorders that produce indefinite, unrestrained body growth have not been observed; the existence of multiple “brakes” ensures that growth deceleration occurs despite the mutation of one or a few genes. The molecular mechanisms that orchestrate the downregulation of hundreds of genes in this juvenile program are not known, but recent evidence suggests that epigenetic mechanisms may be involved. In mice between 1 and 4 wk of age, histone H3K4 trimethylation decreases in the promoter region of many down-regulated genes in the multiorgan genetic program (113). Because H3K4 trimethylation is associated with an open chromatin state and increased transcription, its decline with age may contribute to the observed declines in gene expression.

IV. How Do Organs “Know” When to Stop Growing? The identification of this genetic program could help explain how growth in different organs slows concordantly, but it is insufficient to explain how the organs “know” that they have reached the right sizes to stop growing. The ability of the organs and organism to target a specific adult body size suggests the acquisition of information about elapsed time, organ mass, organ function, or number of cell divisions undergone. In principle, mechanisms to assess any of these types of information would allow the organ to “know” when to stop growing. A. Mechanisms that assess time: biological clocks

The existence of a biological clock would allow the body to grow for a defined period of time before slowing, thus achieving a certain size. Theoretically, under this mechanism, if growth is transiently inhibited, the final body size would be reduced. Such timing mechanisms may be employed during embryonic development in certain cell types. Embryonic rat cardiac myocytes cultured under dif-


ferent conditions tend to stop dividing at a set time, despite having undergone different numbers of cell division (114). Similar studies in oligodendrocyte precursor cells in the central nervous system (115–117) suggest that time, rather than the number of cell divisions, signals the start of differentiation, indicating the presence of a cell-intrinsic timing mechanism. In particular, cyclin-dependent kinase (Cdk) inhibitor p27/Kip1 has been proposed to act as the timing mechanism because it accumulates progressively with time in cultured oligodendrocyte precursor cells (117). However, when these cells are isolated from p27-deficient mice, they only undergo one or two more cell divisions than wild-type cells before differentiation (118), suggesting that p27/Kip1 is only part of a more complex regulatory mechanism that consists of multiple components with overlapping functions, such that defects in one component do not totally abolish the timing function (119). B. Catch-up growth: evidence for mechanisms that assess growth

An alternative mechanism for organs and organisms to grow to a reproducible size is by gathering information about growth itself. It is perhaps best demonstrated by the phenomenon of catch-up growth. In humans and other mammals, juvenile growth is inhibited by conditions such as GH deficiency, hypothyroidism, or malnutrition. If such conditions transiently inhibit growth but then resolve, the growth rate generally does not just return to normal but rather exceeds the normal rate for chronological age (120). Even when postnatal growth is delayed for more than 1 yr in rats fed a tryptophan-deficient (Trp⫺) diet (121) or a corn gluten-based diet (122), switching to a replete diet induces marked catch-up growth, although the animals are well into adulthood. As ingeniously suggested in 1914, this finding suggests that the loss of growth capacity results from the execution of growth itself, rather than increasing age (122). The mechanisms responsible for catch-up growth have been best studied in the growth plate. Previously, the prevailing explanation for catch-up growth was that it results from a mechanism within the central nervous system that compares the actual body size to an age-appropriate setpoint and then adjusts the growth rate accordingly. Thus, if a child is too small for age, the mechanism would sense this deficit and turn up the growth rate, causing catch-up growth (123). However, subsequent studies showed that local growth inhibition, in a single growth plate, is followed by local catch-up growth within that growth plate. This anatomic specificity suggests that the mechanism responsible for catch-up resides not in the central nervous system but rather within the growth plates themselves (124). Studies in rats, rabbits, and humans, after various



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slow the progression of the genetic program and thereby conserve future growth potential. This prediction has recently been tested by slowing growth experimentally in newborn rats for 4 wk using a Trp⫺ diet that suppresses food intake and therefore induces malnutrition (113). At 5 wk of age, 1 wk after release from the Trp⫺ diet, these rats showed increased proliferation in kidney, lung, and liver compared with control rats that had received a replete diet, consistent with the concept that growth-inhibiting conditions slow the loss of growth capacity (Fig. 3). In addition, expression microarray analysis demonstrated that the putative growth-limiting genetic program had been delayed; for a large set of growth-promoting genes that are normally down-regulated with age, the expression levels in rats with prior tryptophan deficiency were higher than in control rats of the same age (113) (Fig. 3). These findings are further supported by a previous study suggesting that hypothyroidism-induced growth retardation also delays down-regulaFig. 3. Transient inhibition of growth slows the multiorgan juvenile genetic program and tion of growth-promoting genes, alalso slows the normal loss of growth capacity. Left column represent conceptual diagrams, though far fewer genes were studied whereas middle and right columns represent experimental data. During normal mammalian (111). These findings therefore suggest juvenile growth (black curves), multiple growth-promoting genes show declining expression (top left panel). This decline is associated with declining proliferation rate (middle left panel), that the putative growth-limiting geand therefore plateauing of body mass (bottom left panel). The gray curves in these panels netic program is not simply a function depict the hypothesis that transient growth inhibition (gray box just above x-axis) would delay of age, but rather depends on the extent the normal decline in expression of growth-promoting genes and consequently delay the decline in proliferation rate and thus the increase in body mass. The middle and right columns of organ growth undergone. show data from newborn rats that were placed on a Trp⫺ diet for 4 wk (gray bars) to restrict Thus, current findings suggest that growth (113). In kidney and lung, transient tryptophan deficiency delayed the decline in mammalian body growth is limited, at expression of many genes, including Igf2 (top row), delayed the decline in proliferation (middle row), and therefore the gain in organ mass (bottom row). [Middle and right panels of least in part, by a negative feedback loop top two rows reproduced from J. C. Lui et al.: FASEB J 24:3038 –3092, 2010 (113).] (Fig. 4). Growth causes the down-regulation of a large set of growth-promoting growth-inhibiting conditions, suggest that this local catch-up genes, which in turn causes body growth to progressively growth occurs because growth-inhibiting conditions slow slow and eventually cease, thus setting a fundamental limit the normal loss of proliferative capacity in the growth on adult body size. This negative feedback appears to act in plate (125–128). Thus, the loss of growth capacity in the skeletal growth plate is driven, not simply by time, but multiple organs, providing a possible explanation for how growth slows concordantly throughout the body, thus mainrather by growth itself. As discussed above, current evidence suggests that the taining body proportions. As discussed above, organs appear to stop growing normal process of growth deceleration is driven, at a physbased not simply on information about the amount of time iological level, by growth itself and, at a molecular level, elapsed, but based at least in part on the amount of growth by a multiorgan genetic program. Thus, one attractive hypothesis is that growth drives the juvenile multiorgan ge- undergone. This information about prior growth could netic program, which in turn drives growth deceleration. involve any of several possible variables. One possibility is This hypothesis predicts that experimentally slowing that a mechanism exists to assess organ size, either the growth by imposing growth-inhibiting conditions would tissue mass or the number of cells that have accumulated

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function, or the number of cell divisions may depend on the organ involved. Even for a particular organ, more than one type of information might be employed to limit growth. C. Mechanisms that assess organ mass: growth-inhibiting chalones

Fig. 4. Model for a mechanism that restricts juvenile body growth. In early life, multiple growth-promoting genes are well expressed, leading to rapid growth. However, growth causes down-regulation of these growth-promoting genes (perhaps through epigenetic mechanisms) which causes growth to slow. Progression of this negative feedback loop would eventually cause the growth rate to approach zero.

in the organ. A second possibility is that growth continues until the organ achieves a certain level of function. A third possibility is that a mechanism exists to count the number of cell divisions undergone. There is an important distinction between counting the number of cells present and counting the number of cell divisions that have occurred. If growth deceleration is determined by counting the number of cells that have accumulated, it would imply that if we experimentally remove a certain fraction of cells from an organ during the period of growth, the organ will continue to grow until the normal size is achieved. In contrast, if growth deceleration is determined by counting the number of cell divisions undergone, the organ will not sense any experimental removal of cells and would thereafter attain a smaller than normal size. Whether growth is limited by tissue mass, number of cells accumulated, organ

Classical studies in salamanders suggest that body growth in these amphibians is dependent on the ultimate tissue mass (129). A tetraploid salamander has cells that are twice as large as those of a diploid salamander, but the tetraploid adult has half as many cells, resulting in a similar body size (129). Similarly, in mice, generation of tetraploids by electrofusion (130) results in embryos with cells of approximately twice the normal size but half the normal cell number, thus achieving an overall size comparable to normal embryos (131). Conversely, in Drosophila, when the cell cycle pace is accelerated or slowed in an imaginal disc by overexpression or ablation of dE2f, the changes in proliferation rate are offset by changes in cell size to achieve a constant overall organ size (132). Recent studies suggest that an additional mechanism called cell competition helps maintain a constant organ mass (133). When growth-promoting dMyc is expressed in some but not all cells in the imaginal wing disc, cell competition occurs such that more slowly proliferating wild-type cells are selectively eliminated by apoptosis, thus, compensating for the increased number of dMycexpressing cells (103, 134) to maintain a constant cell number. How might organ size regulate growth? One old theory postulates the existence of “chalones”—soluble factors that are secreted by cells in an organ and negatively regulate growth of that organ (135) (Table 1). According to this theory, because the concentration of a chalone is directly proportional to the cell number in an organ, when the organ reaches a certain size, the concentration of the chalone is sufficient to stop growth (135). Such growthinhibiting signals could act locally or could circulate and act systemically. Elegant studies demonstrate that myostatin serves as a chalone (136). Myostatin is a TGF-␤ family protein, which is secreted by muscle cells and acts

TABLE 1. Regulation of growth by chalones and chalone-like signals Tissue

Chalone/chalone-like signal

Muscle

Myostatin

Heart Adipose

Activin A Myostatin Leptin

Olfactory epithelium Hair follicle

GDF11 BMP2/BMP4

Effect Inhibits muscle fiber proliferation Inhibits satellite cell renewal Inhibits muscle fiber proliferation Inhibits cardiomyocyte proliferation Suppresses appetite by activating MSH secretion and inhibiting NPY and AgRP secretion at the hypothalamus Inhibits olfactory receptor neuron production Inhibits hair follicle stem cell proliferation


to inhibit muscle growth (137–139) and myogenic satellite cell renewal (140, 141). Homozygous deletion of the myostatin gene (Mstn) in mice results in dramatic increases in skeletal muscle mass, attributed to both muscle cell hyperplasia and hypertrophy (137, 139, 142). This effect is not limited to the embryonic formation of muscle fibers because postnatal loss or blockade of myostatin has the same effect (143–149). Similar studies in dogs (150), pig (151), sheep (152, 153), cattle (154 –156), and human (157) suggest that myostatin is an evolutionarily conserved negative regulator of skeletal muscle growth. Although myostatin was initially reported to be expressed exclusively in skeletal muscle (137), more recent evidence suggests that it is also expressed in cardiomyocytes, with its expression up-regulated postnatally (158), and serves to inhibit cardiomyocyte proliferation (158, 159). These findings therefore suggest that myostatin might also be involved in regulating the size of the heart. Another member of the TGF-␤ family, activin A, has recently been shown to function alongside myostatin to negatively regulate muscle growth (160). Interestingly, the activity of both activin A and myostatin could be inhibited by follistatin (161, 162), which makes follistatin a potential therapeutic agent for improving muscle strength (163, 164). It is yet unclear how muscle mass itself might regulate the expression levels of activin A and follistatin as it regulates myostatin. For many major organs, such as liver, kidney, and lung, it is not known whether chalones are involved in growth regulation. However, recent studies have identified chalone-like growth-inhibiting signals in a limited number of cell types. For example, GDF11 is produced by cells of the neuronal lineage of the olfactory epithelium and inhibit the production of olfactory receptor neurons (165, 166). Similarly, BMP2 and BMP4 serve as local refractory signals to inhibit proliferation of hair follicle stem cells, thus coordinating waves of hair regeneration (167, 168). Leptin is another hormone that functions similarly to a chalone but acts through a systemic negative-feedback loop. Leptin is primarily produced by adipocytes, and the level of leptin in the circulation is directly proportional to whole-body adipose tissue mass (169, 170). At the hypothalamus, leptin then acts on proopiomelanocortin neurons to activate MSH secretion (171–173) and inhibits neuropeptide Y (NPY) and agouti-related peptide (AgRP) secretion by the AgRP/NPY neurons (174, 175). Collectively, these changes suppress appetite, stimulate energy expenditure, and therefore inhibit accumulation of adipose tissue (169, 170). In the recently identified multiorgan juvenile genetic program (111), some of the growth-promoting genes that are down-regulated with increasing body growth are


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growth factors, such as IGF-II, midkine, and pleiotrophin. These findings suggest that local paracrine factors are involved in deceleration of juvenile growth. However, these soluble factors are functionally distinct from chalones. Although chalones are proposed to be growth-inhibitory factors that increase with growth, these are growth-promoting factors that decrease expression with growth, and therefore, in contrast to chalones, their abundance does not reflect the number of cells. However, it is possible that chalones in different organs drive the common genetic program. D. Mechanisms that assess organ function

Another strategy to target the appropriate adult size of an organ is based on the function of the organ. For example, if the organ is too small to provide adequate function, a mechanism to stimulate growth might be activated. When the organ achieves a size sufficient to provide adequate function, the growth-stimulatory function would cease. Conversely, when the organ achieves adequate size and therefore function, a growth-inhibiting mechanism might be activated. The existence of a mechanism that senses organ function is suggested by the phenomenon of liver regeneration after partial hepatectomy in mammals (176 –178). This regeneration involves a brief burst of cell replication by a large number of differentiated hepatocytes (25). Similarly, the liver also adjusts its size relative to the overall body size after orthotopic transplantation from a small dog to a larger dog (38). Furthermore, in rats connected by parabiosis, partial hepatectomy in one animal stimulates hepatocyte proliferation in the other (179), indicating that liver regeneration involves circulating factors. The concept that liver size may be regulated by function is further supported by its ability to undergo hyperplasia upon increased xenobiotic stress, such as administration of phenobarbital and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (180), without liver injury (181, 182). More recent studies suggest that the liver senses organ functionality, at least in part, through the flux of bile acids (183). Bile acids are synthesized in the liver and secreted into the small intestine, where they facilitate digestion. The bile acids are subsequently reabsorbed and returned to the liver, where they are taken up by hepatocytes, thus completing the enterohepatic circulation. When exposed to a mild increase in bile acid flux, hepatocyte proliferation is induced, causing an increase in liver size (183). Similarly, liver regeneration after partial hepatectomy is potentiated in the presence of bile acids (183, 184). These findings were further supported by a complementary study, showing that disruption of the enterohepatic circulation by draining the common bile duct externally during the 7 d before hepatectomy delays subsequent liver

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of the remaining kidney (187), perhaps involving a temporary increase in serum creatinine that signals the increased functional need (187). However, this increase in kidney size is primarily due to hypertrophy (cell enlargement) rather than hyperplasia (cell proliferation) and thus may involve mechanisms different from those primarily regulating juvenile growth (188). E. Mechanisms that count cell divisions

Insomedevelopmentalsystems,growth appears to be limited by a cell-division counter. During the early development of Xenopus laevis, a counting mechanism is Fig. 5. Organ size is limited by the number of progenitor cells in pancreas but not in liver. in place to ensure that the zygote divides Transgenic mice were generated to allow ablation of Pdx1-expressing (Pdx1⫹) pancreatic exactly 12 times before cell division slows ⫹ progenitor cells or liver-enriched transcriptional activator protein expressing (LAP ) hepatic (189, 190). In mice, reducing the number progenitor cells (191). Tetracycline could be administered to repress progenitor cell ablation at any time point. In the pancreas (upper panels), progenitor cells were ablated through of pancreatic progenitor cells in early deembryonic day (E) 10.5, reducing pancreas mass to 36% of control values at E18.5. velopment is not compensated in later deAfterward, the affected pancreases remained proportionately smaller than those of controls. velopment and thus results in a smaller In the liver (lower panels), progenitor cells were ablated through E13.5, causing liver mass to be reduced to 33% of control values. When ablation was repressed by tetracycline pancreas (Fig. 5) (191). This observation administration from E13.5 to E17.5, almost complete recovery of liver size occurred. [Right argues against a mechanism that senses panels derived from reference 191.] organ size or function but is consistent with a mechanism based on cell-cycle regeneration (185). Thus, collectively compelling evidence counting. As the authors suggested, pancreatic progenitor suggests that liver regeneration and adult liver size can be cell proliferation may be autonomously restricted, and each modulated by organ function. One possibility is that this progenitor cell may be capable of giving rise to only a fixed same mechanism alone could be used to drive rapid juve- amount of tissue. In contrast, ablation of progenitor cells nile growth of the liver and its subsequent growth decel- during liver development appears not to affect the ultimate eration, because the mechanism would cause liver size to organ size, suggesting that hepatic growth is not primarily keep pace with the growth rate of the rest of the body. controlled by cell division counting (Fig. 5). A cell-cycle Alternatively, organ function might be used only to finecounting mechanism may also exist in the growth plate. In tune the adult liver size, and a separate mechanism might this structure, the normal decline in chondrocyte proliferabe used to suppress proliferation in early juvenile life. tion is driven by growth. Because newly formed cartilage For some endocrine organs, such as the thyroid, adrein the growth plate is continuously remodeled into bone nal, and parathyroid glands, size is also regulated in part tissue, the size of the cartilaginous growth plate does not by organ function. If the organ produces insufficient horincrease with age, arguing against a size-sensing mechmone, a negative feedback loop serves to stimulate not only increased secretion but also proliferation and growth anism and suggesting instead a mechanism that senses of the endocrine gland. Although this mechanism may be number of cell divisions undergone (17). A cell division counting mechanism might involve some used to precisely target the optimal adult organ size based characteristic of the cell that changes with each cell divion function, other mechanisms may also contribute to jusion and then directly or indirectly regulates cellular venile growth deceleration and size determination. For example, the size of the thyroid gland is regulated by a growth and/or cell division. In principle, this characteristic negative feedback loop involving thyroid hormone and might involve, for example, DNA, proteins, or other regTSH, and yet, in mice lacking a functional TSH receptor, ulatory molecules. One of the most well-studied cell-cycle the thyroid still grows to approximately 50% of the nor- counting mechanisms is telomere shortening (192). Telomeres are regions of tandem repeats of TTAGGG found at mal size (186). Kidney size also seems to be regulated in part by func- the ends of chromosomes. In all eukaryotes, telomeres tion. Unilateral nephrectomy causes an increase in the size shorten with each round of cell division, owing to the


incomplete replication of linear chromosomes by conventional DNA polymerases (193). With the exception of a few cell types, including germ cells, stem cells, and certain leukocytes, in which telomerase remains active in adulthood (194), telomeres gradually shorten until a critical length is reached, leading to genome instability and loss of cell viability (192, 195). Telomere shortening plays an important role in cellular senescence, protection against cancer (196), and possibly organismal aging (197, 198) but is unlikely to be centrally involved in regulating juvenile growth. Mice with mutations in Terc, which specifies the RNA component of telomerase, exhibit a variety of phenotypic abnormalities, including decreased life span in late generations, but show normal body growth (199). Similarly, in dyskeratosis congenita, a disorder characterized by abnormally short telomeres due to deficient telomerase activity, short stature does occur but only in a minority of patients (200), particularly those with a severe form of the disease (201). Another cellular characteristic that might change with cell division is epigenetic changes, such as DNA methylation or histone modification. As mentioned earlier, decreases in H3K4 trimethylation in the promoter region of multiple growth-promoting genes have been observed in multiple organs during juvenile growth deceleration (113). This finding is similar to observations in organismal aging where histone acetylation, which favors gene activation, is found to decline with age (202). It remains unclear whether these epigenetic changes are direct consequences of the cumulative number of cell division and thus might provide a cell-cycle counting mechanism that affects subsequent growth.

V. Clinical Implications Understanding the fundamental mechanisms that cause growth deceleration and thus govern adult body size is likely to yield insights into the many human genetic disorders that cause childhood growth failure and overgrowth, many of which remain poorly understood. For example, Beckwith-Wiedemann syndrome, an overgrowth disorder, is associated with abnormalities in two imprinted genes that are growth-regulating and down-regulated with age in juvenile life (112)—IGF2, which is growth promoting, and CDKN1C, which inhibits cyclin E-Cdk2 but also activates cyclin D-Cdk4 for cell cycle G1 phase progression (203–205). Conversely, abnormal imprinting and decreased IGF2 expression have been implicated in the growth retardation of Silver-Russell syndrome (206). Simpson-Golabi-Behmel syndrome, an overgrowth disorder, is caused by loss-of-function mutations in GPC3


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(207), a growth-inhibiting gene that is down-regulated with age in multiple tissues (113). Because body growth limitation involves complex, potent mechanisms to suppress cell proliferation, disruption of these mechanisms may contribute to oncogenesis. Multiple growth-promoting genes that are down-regulated with age in multiple tissues and are therefore part of the putative juvenile growth-limiting genetic program have previously been implicated in cancer pathogenesis, for example, N-MYC, a transcription factor (208); EZH2, a histone methyltransferase (209); and midkine and pleiotrophin, two related cytokines (210). Additionally, elucidating the physiological mechanisms that normally suppress proliferation in juvenile life might suggest new approaches to achieve therapeutic tissue regeneration, either in vivo or ex vivo. It has long been suggested that the regeneration process shares similarities with molecular events of embryonic development. For example, in liver (211, 212) and kidney (213–215), expression of developmental genes reportedly reactivates during injury and regeneration. Interestingly, one gene that is down-regulated postnatally but then reactivated during liver regeneration is Mdk (211). We envision that regeneration may be similarly achieved if quiescent adult cells could be stimulated to proliferate by transiently suspending the mechanisms that suppressed growth during juvenile life.

VI. Conclusion In summary, mammalian body growth is rapid in early life but slows with age, thus setting a limit on adult body size. This growth deceleration reflects declining proliferation in multiple tissues and is driven in large part by local mechanisms within the growing tissues themselves. Recent findings suggest that body growth deceleration is driven by a growth-limiting genetic program that occurs in multiple organs. This growth-limiting program, which involves the down-regulation of a large set of growth-promoting genes, depends not simply on age but also on organ growth itself. Therefore, the adult body size limit appears to be imposed by a negative feedback loop; organ growth leads to progression of a genetic program, which in turn causes growth of the organ to slow and eventually cease. These findings provide insights into long-standing mysteries regarding body size determination, maintenance of body proportionality, and catch-up growth. Different organs seem to use different types of information to precisely target the adult body size. For example, skeletal and cardiac muscle growth are negatively regulated by myostatin, the concentration of which depends on muscle mass itself. Liver growth appears to be modu-

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lated by bile acid flux, a parameter that reflects organ function. In pancreas, organ size seems to be limited by the initial number of progenitor cells, suggesting a mechanism based on cell-cycle counting. Further elucidation of the fundamental mechanisms suppressing juvenile growth is likely to yield important insights into the pathophysiology of childhood growth disorders and of the unrestrained growth of cancer. In addition, improved understanding of these growthsuppressing mechanisms may someday allow their therapeutic suspension in adult tissues to facilitate tissue regeneration.

Acknowledgments Address all correspondence and requests for reprints to: Jeffrey Baron, M.D., National Institutes of Health, CRC, Room 1-3330, 10 Center Drive MSC 1103, Bethesda, Maryland 20892-1103. E-mail: [email protected]. This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. Disclosure Summary: The authors have nothing to disclose.

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