Temperature regulates limb length in homeothermsby directly modulating cartilage growthMaria A. Serrata,1, Donna Kingb, and C. Owen Lovejoya
aDepartment of Anthropology and School of Biomedical Sciences, Kent State University, Kent, OH 44242; and bDepartment of Microbiology, Immunology,and Biochemistry, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272
Edited by Nina Jablonski, Pennsylvania State University, State College, PA, and accepted by the Editorial Board October 13, 2008 (received for reviewApril 4, 2008)
Allen’s Rule documents a century-old biological observation thatstrong positive correlations exist among latitude, ambient tem-perature, and limb length in mammals. Although genetic selectionfor thermoregulatory adaptation is frequently presumed to be theprimary basis of this phenomenon, important but frequently over-looked research has shown that appendage outgrowth is alsomarkedly influenced by environmental temperature. Alteration oflimb blood flow via vasoconstriction/vasodilation is the currentdefault hypothesis for this growth plasticity, but here we showthat tissue perfusion does not fully account for differences inextremity elongation in mice. We show that peripheral tissuetemperature closely reflects housing temperature in vivo, and wedemonstrate that chondrocyte proliferation and extracellular ma-trix volume strongly correlate with tissue temperature in metatar-sals cultured without vasculature in vitro. Taken together, thesedata suggest that vasomotor changes likely modulate extremitygrowth indirectly, via their effects on appendage temperature,rather than vascular nutrient delivery. When combined with classicevolutionary theory, especially genetic assimilation, these resultsprovide a potentially comprehensive explanation of Allen’s Rule,and may substantially impact our understanding of phenotypicvariation in living and extinct mammals, including humans.
Allen’s Rule � bone growth � bone tissue culture � cartilage biology �thermoregulation
Ecogeographical rules relating climate to extremity length andbody mass have long been central tenets of biology, and are
among the best supported observations in natural-dwellingspecies (1). Allen’s Rule codifies the observation that append-ages (ears, limbs, and tails) of animals living in cold geographicalregions are consistently shorter than those of closely relatedcounterparts occupying warmer climes (2). Shortened extremi-ties minimize heat loss by reducing surface area relative tovolume and have long been viewed as genetically determinedthermoregulatory adaptations (1). However, the heritability ofextremity length is largely unknown, because similar phenotypescan be reproduced in laboratory mammals by modifying theirambient rearing temperature (3–6) (Fig. 1). The mechanism bywhich environmental temperature modulates extremity growthhas remained elusive (7, 8). Understanding growth plasticity iscritical to basic evolutionary analyses, because many charactersthat have long been hypothesized to be adaptations may insteadbe partial or even entirely effects of ambient temperature (9, 10).Moreover, knowledge of this phenotypic plasticity will be a keyfactor in ecological conservation strategies for anticipatedchanges in global climate that may have direct impacts on humaneconomics and sustainability (11).
The traditional explanation for temperature-growth effects inskeletal extremities is an altered supply of essential nutrients andgrowth factors via increased or decreased blood flow that resultsfrom changes in vasomotor tone (i.e., temperature-inducedvasoconstriction/vasodilation) (5, 12). However, no study to datehas tested this central hypothesis directly. Impairment of bonegrowth by disruption of blood flow is well-known (13), as are
correlations of enhanced blood flow with bone elongation (14)and ear enlargement (15). It is equally well established thatcold-induced vasoconstriction shunts blood away from theperiphery as a heat-conserving mechanism (1, 9), just as warmtemperature enhances heat dissipation via vasodilation (1). Priorwork has demonstrated that abrupt (acute) heat or cold exposureindeed affects bone perfusion (16), but despite frequent citationin explanatory models of climate-phenotype interactions (5, 12),no study has yet addressed the effect of long-term temperatureexposure on skeletal blood supply and its potential relationshipto bone elongation. Here we address the vascularity hypothesisthat bone perfusion and elongation are directly related toambient temperature.
Temperature and Extremity Growth. We housed outbred micecontinuously at cold (7 °C), control-intermediate (21 °C), orwarm (27 °C) ambient temperatures from weaning throughmaturity (3.5–12 weeks age). Two intermediate endpoints were
Author contributions: M.A.S. and C.O.L. designed research; M.A.S. performed research;M.A.S. and D.K. contributed new reagents/analytic tools; M.A.S. and C.O.L. analyzed data;and M.A.S., and C.O.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. N.J. is a guest editor invited by the Editorial Board.
1To whom correspondence should be addressed at: Cornell University, Department of Bio-medical Sciences, T5–002 VRT, Ithaca, NY 14853-6401. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/0803319105/DCSupplemental.
Fig. 1. Temperature effects on femur length. Representative femora frommice housed at cold (7 °C) and warm (27 °C) temperatures from weaning ageto adulthood showing the effect of ambient temperature on extremity size.The underlying cause of such effects is not immediately obvious becausehomeotherms maintain tightly regulated internal body temperatures inde-pendent of their external environment. For discussion see text.
evaluated (4.5 and 6.5 weeks) in separate trials to assess tem-perature effects during ontogeny (see supporting information(SI) Table S1). Consistent with prior research (3, 4, 6–8), weconfirmed that the ears, limbs, and tails of warm-reared micewere significantly longer than those of siblings raised in the cold(Figs. 1 and 2 A, C, and D and Table S2), but with no change intotal body mass (Fig. 2B). Differences in core organ sizeappeared to account for the latter—hearts and kidneys wereenlarged in cold-reared mice (Table S2)—but differences in limblength were not explicable by diet and/or activity level, becausecold-reared mice consumed substantially more food and weremore active than their warm and control counterparts (Figs. S1and S2), two factors reasonably presumed to be associable withincreased rather than decreased limb length (17). There were,nevertheless, significant positive correlations among ambientand appendage temperatures, and associated growth of thelimbs, tails (Fig. 2C) and ears (Fig. 2D). Although not subject toformal regression analysis, these data suggest that in vivoextremity temperature is a good predictor of extremity growth.
Blood Flow Analysis. To determine whether a cold-induced reduc-tion in skeletal blood supply contributed to extremity foreshorten-ing, we measured relative bone blood flow (BBF) to the hindlimbbones and tail base of juvenile mice by using fluorescent micro-spheres, a reliable standard for quantifying regional organ and boneperfusion (18). We predicted that BBF would positively covary withrearing temperature and bone elongation as previously hypothe-sized. When compared with the two warmer-temperature groups,mice raised at the coldest temperature (7 °C) indeed had reducedfemur, tibia, and hindpaw BBF at 4.5 weeks of age (after 1 week inthe cold) (Fig. 3 A–C), and reduced tibia, paw, and tail BBF at 6.5weeks (Fig. 3 B–D) (all confirmed by ANOVA and Tukey posthoctest at P � 0.05 as detailed in Fig. 3). Because BBF precipitouslydeclines with age (19, 20), we neither expected nor found statisti-cally significant temperature effects on bone perfusion in 12-weekanimals. We did observe an interesting decrease in paw blood flowin cold-reared adults consistent with the younger age points (Fig.
3C), but this was not statistically significant and so should beinterpreted cautiously.
Our results do not fully support the vascularity hypothesisbecause the highest growth rates were observed in the warmestreared mice (i.e., Figs. 1 and 2 A), but these animals did not alsoexhibit the correspondingly highest predicted BBF (Fig. 3 A–D,detailed in legend). The coldest-reared mice, however, did showthe lowest growth rate and markedly reduced BBF. This, to-gether with the fact that the two warmer-reared groups weremore similar to one another in terms of housing temperature,growth rate, and BBF, raises the possibility of a thresholdresponse to cold.
Blood not only provides developing limbs with a reservoir ofessential growth factors, nutrients, and oxygen (13), it is also animportant source of heat (1, 9). If this heat source is altered,temperature-sensitive processes will be affected. Cartilage growth,which largely determines bone growth rate via endochondral ossi-fication, is one process that may be sensitive to ambient tempera-ture. Therefore, the amount of blood arriving at an extremity mightmodulate its temperature and thereby its rate of bone elongation viathis route (7). Indeed, many arctic mammals rely on countercurrentvascular heat exchange mechanisms to prevent excessive heat lossin their extremities, and by doing so reduce limb temperatures tonear ambient conditions (1, 9).
Metatarsal Organ Cultures. We examined whether temperature, asa controlled independent variable, could itself enhance or reducebone elongation. We compared growth of neonatal mousemetatarsals (MTs) in culture maintained at cold (32 °C), control(37 °C) or warm (39 °C) temperatures, holding all other factorsconstant. This rodent MT model should be largely free ofmechanical, dietary, vascular and systemic hormonal influences,save those impacted directly or indirectly by tissue temperature.Incubation temperature is already known to affect osteoblastactivity (21), glycosaminoglycan synthesis (22), and collagencalcification (23) in vitro; however, these studies were conductedat the cellular level, whereas our experiments, that rely on a
Fig. 2. Extremity growth under different ambient rearing temperatures. Mouse tail length (A) and body mass (B) measured at weekly intervals beginning atweaning. Means � 1 SEM shown (n � 7/group). Tail elongation, significantly reduced in the cold after only 1 week (P � 0.05, one-way ANOVA), follows apredictable temperature gradient, but body mass remains similar among all 3 groups (P � 0.10). Average peripheral temperature, (C) and (D), measured weeklyby using a noncontact thermometer at the tail base and ear show strong positive correlations with growth (Pearson’s r in figure). These results suggest directtemperature effects on developing cartilaginous tissues.
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heterogeneous tissue complex (entire MTs), provide a modelmore similar to an intact organism.
In vitro temperature had an extraordinary impact on MTelongation (Fig. 4A). MTs from all three groups showed in-creases in length from baseline at two- and four-day timeintervals, but total accrued growth was strikingly temperaturedependent (Fig. 4B). Interestingly, although the control andwarm groups differed only slightly in temperature (2 °C), thissmall increase was still sufficient to produce significantly greatergrowth in warm MTs compared with both other groups (Fig. 4B,pairwise comparisons significant at P � 0.01 by using a Tukeytest at each time point). All increases in MT size (length andwidth) occurred at the cartilaginous ends of the bone; asexpected, the mineralized diaphyses did not differ among groups(Fig. 4A), nor did they elongate under these avascular cultureconditions (data not shown). Histological analyses (Fig. 4 C–E)demonstrated higher rates of mitosis measured by BrdU incor-poration (Fig. 4D) and increased extracellular matrix volume(Fig. 4E) at warmer temperatures, suggesting that chondrocyteproliferation and matrix (synthesis, secretion, and/or stability)respond directly to temperature and likely underlie the observedgrowth differences. In all cases, there were more BrdU-positivecells in the epiphyseal region of the MTs when compared withthe columnar region of the growth plate (Fig. 4 C and D), as isexpected because much early proliferation in vivo occurs in therounded epiphyseal chondrocytes before the establishment of amature growth plate (24, 25). Our experiments did not extendbeyond four days in culture, and so it is possible that a differ-ential temperature effect on the cell populations might occurlater in growth.
A compelling comparison with our whole animal experimentsreveals that these in vitro results closely match data from livingmice in that peripheral ear and tail temperatures strongly
correlated with extremity growth (Fig. 2 C and D). This providesevidence of similar direct and local tissue responses to ambienttemperature in both live mice and MT cultures. However, caremust be taken in interpreting our results with regard to theeffects of temperature on cartilage (as a precursor to bone)versus bone itself. Our data do not negate possible temperatureeffects on vascular perfusion of the periosteum or other regionsof actively growing bone, and such effects might well be syner-gistic to the ones that we have shown. Our experiments did notspecifically address this issue, but previous work does suggestthat it clearly merits further investigation (21, 23). Nevertheless,the marked growth response in non-osseous tissue (i.e., ears)confirms the important role of temperature on the chondrocyte.
DiscussionThese data suggest that environmental temperature may mod-ulate extremity growth by inducing physiological responses inperipheral tissue temperature, rather than by affecting vascularnutrient delivery, and that such temperature lability may thenimpact extremity size via its direct effect on cell proliferation andmatrix production in cartilage (i.e., epiphyseal plates in longbones and tails, and cartilage mass in ears). Thus, the effects ofvascular modification on bone growth are likely indirect. That is,vasoconstriction and vasodilation may modulate limb growth notby affecting arrival rate of nutrients and hormones, but bymodulating temperature within developing cartilage. From anevolutionary perspective, Allen’s ‘‘extremity size rule’’ may notactually reflect a functional genotypic adaptation in some or evenmany homeotherms (9, 10), but may instead be partially or whollydependent on environmental temperature; that is, a secondarygrowth response to ‘‘facultative extremity heterothermy’’ in mam-mals that maintain constant core body temperatures.
Fig. 3. Relative hindlimb and tail blood flow measured by fluorescent microsphere deposition. Effects of chronic rearing temperature on blood flow to thefemur (A), tibia (B), hindpaw (C), and tail base (D) by using the thoracic spine as an internal reference. Rearing temperature is shown by shaded bars within eachage cluster. Means � 1 SEM plotted (n � 9 minimum per age and group; Table S1). For each age, a single asterisk indicates significant reduction in blood flowin the cold (P � 0.05, Tukey test) compared with either control or warm groups (e.g., 4.5-week femur, tibia, and paw). An asterisk overlying a horizontal bracketindicates that pairwise comparisons among all 3 groups were significant (e.g., 6.5-week paw and tail), and a crossbar (†) denotes significantly increased bloodflow in the warm group compared with both cold and control (6.5-week tibia), all confirmed by Tukey test at � � 0.05. Tail samples were not collected at 12 weeks.
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Our results are particularly intriguing because cold-dwellinghumans, especially Neandertals, possess disproportionately short-ened long bones (12). Moreover the crural index (tibia/femur lengthratio) exhibits a strikingly positive correlation with mean annualtemperature (tibias shorten relative to the femur with decreasingtemperature) (12). This relationship is especially relevant becausethe tibia is directly exposed to ambient temperature by virtue of its
immediately subcutaneous anterior surface and relatively poorblood supply (19). This results in the shank having markedly coolertemperatures than more proximal bones of the limb.
Our experiments, however, may not fully resolve the complexgenetic basis of observed clines in mammalian limb length.Clearly the degree of genotype-environment interaction remainsan unaddressed issue, and future experiments will be required to
Fig. 4. Effects of incubation temperature on metatarsal growth in vitro. Representative comparison of metatarsals from the same individual (left–rightantimeres) grown in culture at cold and control temperatures for 4 days (A). Percentage growth from baseline plotted at 2- and 4-day time points (B) shows thataccrued growth is directly proportionate to temperature (P � 0.01 as noted in text). In histological analyses of left–right antimeres from 2-day cultures kept atcold or control temperatures (C), 150-�m � 100-�m sample regions in the growth plate columnar zone (light gray) and distal epiphysis (dark gray), showedincreased chondrocyte proliferation (C and D) (BrdU positive cells indicated by dark stained nuclei in C) and extracellular matrix area (E) at warmer temperatures,closely matching the growth patterns revealed in (B). Within regions, BrdU staining was significantly increased in the warm group relative to cold in the growthplate (region 1) and in both control and warm groups in the distal epiphysis (region 2) (D). Matrix area (E) was also significantly increased in control and warmgroups relative to cold in the distal epiphysis. Significance verified at � � 0.05 by using ANOVA and Tukey test. Boxes show interquartile range (middle 50%),whiskers denote upper 10th and 90th percentiles, circles indicate outliers, and horizontal line denotes the median. Sample size (n) listed in the horizontal axis ofeach graph.
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assess the effects of heritability. Studies that incorporategenomic screening in models such as those presented here maybe particularly informative. In fact, environmentally-inducedphysiological changes are not limited to bone or cartilage.Temperature has been shown to have direct effects on skinpigmentation in rabbits (26), cell proliferation in turtle brains(27), tumor metastases (28), slime mold differentiation (29),plant leaf growth (30), mammalian hair growth (7), and inver-tebrate cell size (31). Indeed, temperature effects have beensuggested to impact mutation rates (32), and thus molecularclocks based on them.
In addition to reported temperature effects on the expression ofheat-shock proteins (21) and cell-cycle control genes in culturedbone cells (33), there are several plausible mechanisms by whichtemperature may impact limb elongation via effects on growth platechondrocytes. Temperature variation may induce physiochemicalchanges in extracellular matrix proteins and thereby lead to alteredmatrix-integrin interactions and/or altered diffusion rates of para-crine growth regulators. Recent work has shown that specificmolecular signaling pathways regulate the chondrocyte response toloss of homeostasis, including endoplasmic reticulum stress (34),and these pathways modulate responses such as proliferation,apoptosis, and extracellular matrix synthesis (35). Altered temper-ature could directly or indirectly trigger these stress pathways,resulting in downstream effects on limb elongation. Concordantly,induction of endoplasmic reticulum stress in the growth plate hasrecently been linked to disrupted chondrocyte function and limbgrowth (36). Additional pathways regulating growth plate cell-cycleand metabolism, such as the Ihh–PTHrP feedback loop (37) orexpression of growth hormone, IGF1, and/or its receptor (38) mayalso have temperature-sensitive elements. Elucidation of thesemechanisms may be key to unveiling environment-genotypeinteractions.
Some years ago, Waddington imposed artificial selection inDrosophila by exposing multiple generations to near-fatal hottemperatures (39). This resulted in an eventual fixation of noveltraits first elicited in response to heat but subsequently expressedin its absence, a phenomenon he defined as ‘‘genetic assimila-tion’’ (39). Clinal distributions of limb length may thereforerepresent a complex amalgam of genetic assimilation aftergenerations of selection in combination with direct temperatureresponses in growing cartilage. Future investigations of similartemperature response pathways are likely to provide key insightsinto the evolutionary history of mammals, including humans.
Materials and MethodsIn Vivo Temperature Experiments. All procedures were carried out in accor-dance with IACUC guidelines at the Northeastern Ohio Universities College ofMedicine. Male out-bred CF-1 mice (n � 95 partitioned into age and group,Table S1) were shipped from Charles River Laboratories at weaning. Based ondescribed methods (6), 3.5-week-old mice were randomly assorted into 3sized-matched groups and individually housed at 7 °C, 21 °C, or 27 °C incontrolled environmental chambers (Serrat Heating and Cooling) under oth-erwise identical conditions: Plastic caging with pine bedding, 12-hour light/dark cycle, and ad lib access to food and water. Body mass and tail length weremeasured once weekly. At that time skin temperatures (ear and tail base) werealso recorded by using a noncontact infrared thermometer (Kent Scientific).
Blood Flow Measurements by Using Fluorescent Microspheres. At 4.5-, 6.5-, and12- week age points, anesthetized mice received an intracardiac injection offluorescent-labeled microspheres (15 �m, Molecular Probes) to measure re-gional blood flow as previously described (40). Femur, tibia, paw, and tailvertebrae (caudal 1–4) were immediately harvested and cleaned of soft tissue.Microspheres were recovered after published digestion/filtration methods(18, 41–43). In brief, individual bones were weighed, demineralized in Cal-Ex(Fisher Scientific), and digested in ethanolic KOH. The homogenate wasfiltered through polyamide mesh (Sefar Nitex 03–7/2), which was soaked inCellosolve acetate (Sigma Aldrich) to dissolve the spheres and release thefluorescent dyes. Aliquots of the solvent containing the dissolved sphereswere transferred in triplicate to a 96-microwell plate (Nunc) and fluorescencewas measured by using a Typhoon 8610 Variable Mode Imager (AmershamBiosciences) and quantified by using ImageQuant software (Molecular Dy-namics). For conventional size-correction (43), the fluorescence of each samplewas standardized by its mass to create a relative density. The thoracic spine(T10–13), similarly sized among groups (P � 0.49, one-way ANOVA) andlocated within the body core, served as a standard reference for evaluatingmicrosphere density in the hindlimb and tail.
Whole-Bone Metatarsal Organ Cultures. The middle 3 metatarsal bones(MT2–4) were harvested from left and right hindpaws of neonatal C57BL/6Jmice (housed at room temperature) on the day of birth after decapitationunder deep halothane-induced anesthesia as detailed elsewhere (24). Eachside was cultured separately at 32 °C, 37 °C, or 39 °C in DMEM growth medium(10% FBS, L-glutamine, fungizone, and gentamycin; Sigma) in humidifiedincubators with 5% CO2. At 2- or 4-day endpoints, culture medium wasreplaced with new containing 1:100 BrdU (Zymed) to identify proliferatingcells. After a 12-hour labeling period, bones were removed from culture toassess their longitudinal growth (expressed as a percentage change frombaseline averaged among MT2–4), fixed and decalcified in Cal-Ex II (Fisher),and processed for routine histology. Serial sections were stained with eitherSafranin-O for morphological evaluation (25) or BrdU by using a commerciallyavailable kit (BD Biosciences). Cells stained positively for BrdU were manuallycounted in 150-�m � 100-�m sample regions from the growth plate columnarzone and distal epiphysis of MT3 by using an Olympus BH-2 microscopeinterfaced to Bioquant Osteo-II image analysis software (BQIAC) (Fig. 4C).Matrix area was quantified in the same regions based on Safranin-O-definedthresholds in Bioquant.
Statistical Analysis. Among-group differences were assessed by using one-wayanalysis of variance (ANOVA). No significant temperature-age interactionswere present in the sample (confirmed by two-way factorial ANOVA), so agegroups were analyzed separately to best discriminate temperature effects.Pairwise differences were revealed by using Tukey posthoc test. Product-moment correlation (Pearson) was applied to test for significant correlationsbetween variables. Analyses were performed by using SPSS 11.0 software and� � 0.05 for all procedures.
ACKNOWLEDGMENTS. We thank the National Science Foundation, Kent StateUniversity Graduate Student Senate, and NEOUCOM Skeletal Biology Re-search Focus Area for generous financial support to M.A.S. Environmentalchambers were designed and built by Marcos Serrat (Serrat Heating andCooling, N. Royalton, OH). W. Horne, J. Jacobi, D. Rurak, R. Glenny, and theFluorescent Microsphere Resource Center (Seattle, WA) provided valuableinput on microsphere injection and recovery protocols, and A. Carey (SefarAmerica, Depew, NY) helped obtain essential filtration material. W. Hortonand P. Reno suggested and facilitated the in vitro experiments. We thank J.Kriz, C. Vinyard, J. Stalvey, R. Meindl, C. Farnum, H. Thewissen, J. Hardwick, L.Yang, D. Dutton, M. Moser, J. Thomas, D. McBurney, B. Lowder, A. Nugent, B.Armfield, H. McEwen, and many others for discussions and technical assistance.We thank P. Reno, M. McCollum, W. Horton, M. Cohn, the Editor, and twoanonymous reviewers for critical reading of the manuscript and helpful commen-tary. We are grateful to Evan Bailey for executing graphic file conversions.
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