SPOTLIGHT

What determines organ size during development andregeneration?Laura Boulan* and Pierre Leopold*

ABSTRACTThe sizes of living organisms span over 20 orders of magnitude or so.This daunting observation could intimidate researchers aiming tounderstand the general mechanisms controlling growth. However,recent progress suggests the existence of principles common toorganisms as diverse as fruit flies, mice and humans. As we reviewhere, these studies have provided insights into both autonomous andnon-autonomous mechanisms controlling organ growth as well assome of the principles underlying growth coordination betweenorgans and across bilaterally symmetrical organisms. This researchtackles several aspects of developmental biology and integratesinputs from physics, mathematical modelling and evolutionarybiology. Although many open questions remain, this work alsohelps to shed light on medically related conditions such as tissue andlimb regeneration, as well as metabolic homeostasis and cancer.

KEY WORDS: Growth, Regeneration, Hormones, Nutrition

IntroductionThe Vitruvian Man, da Vinci’s illustrious drawing, represents withunsurpassed precision the symmetry and proportions found in thehuman body. For renaissance artists, body proportions evokedbeauty and the place of Man at the centre of the universe. Fordevelopmental biologists, these properties relate to a complex seriesof phenomenon bridging genetics, signalling, mechanical feedback,robustness and precision. Mechanistically, the control of organ sizeresults from the complex integration of autonomous programmes,whereby intrinsic signals specify organ identity, patterning andgrowth properties, and systemic controls adjust organ growth todevelopmental and environmental cues. But what are the respectiveroles of these two types of control? What are the contributions ofdeterministic genetic programmes versus self-organizingmechanisms? And what are the growth drivers and the size rulers?These are among the many outstanding questions in this field. In thisshort Spotlight article, we discuss recent advances and futurechallenges for this fascinating discipline of biology, which sits at thecross-road of morphogenesis, metabolism, mechanobiology, tissueregeneration, organoid biology, mathematical modelling andevolutionary biology.

Autonomous processes controlling organ growthThe ability of organs to develop independently of their normalenvironment was first suggested by Ross Harrison in 1924(Harrison, 1924). In a series of elegant grafting experiments,Harrison was able to swap anterior limb buds between two

salamander species of different size and the spectacular result wasthat limbs grafted into a different species kept their size of origin(Fig. 1). This indicates the capacity of some organs to follow agenetically encoded growth programme while in a differentenvironment. Heterotypic ‘grafting’ has since been achievedsuccessfully using induced pluripotent stem cells (iPSCs) to growa mouse pancreas in a rat host unable to make a pancreas(Yamaguchi et al., 2017). This opens up the ethically challengingpossibility of using animal hosts to grow human organs (Cyranoski,2019). Interestingly, in the context of pancreatic replacement,mouse iPSCs injected into a rat blastocyst generate a mousepancreas that has the size of a rat pancreas. This illustrates thevarious tissue-specific balances existing between autonomous andsystemic size control mechanisms, and sets the stage for futureexploration of these regulatory mechanisms.

The autonomous growth control properties of organs can also beexplored through their ability to recover after perturbation. Manyorgans can repair from amputation or injury while they develop, butonly a few species can regenerate organs following amputation ofthat fully developed organ. Studying regeneration has uncoveredfundamental properties of organ patterning, leading to the conceptof intercalary growth. When two distal segments of a cockroach legare joined together after removing an intermediate part, the legregenerates the missing intercalary segment (French et al., 1976).The same happens with salamander limbs (Iten and Bryant, 1976).This fits with the notion of ‘positional information’, which is usedduring development to dictate the shape and size of an organ(Wolpert, 1969, 1989). A discontinuity in positional information iswhat generates growth in order to smooth out these disparities. Thisconcept has been largely supported by the discovery of morphogensand their diffusion along different axes of vertebrate andinvertebrate tissues. Indeed, the field of morphogen signalling hascontributed immensely to our understanding of pattern formationand growth (Briscoe and Small, 2015). However, despite intense

Advocating developmental biologyThis article is part of Development’s Advocacy collection – a series ofreview articles that make compelling arguments for the field’simportance. The series is split into two: one set of articles addressesthe question ‘What has developmental biology ever done for us?’ Wewant to illustrate how discoveries in developmental biology have had awider scientific and societal impact, and thus both celebrate our field’shistory and argue for its continuing place as a core biological discipline. Ina complementary set of articles, we asked authors to explore ‘What arethe big open questions in the field?’ Together, the articles will provide acollection of case studies that look back on the field’s achievements andforwards to its potential, a resource for students, educators, advocatesand researchers alike. To see the full collection as it grows, go to: https://dev.biologists.org/content/advocating-developmental-biology.

Institut Curie, PSL University, CNRS UMR3215, INSERM U934, Genetics andDevelopmental Biology unit, 75005 Paris, France.

*Authors for correspondence (laura.boulan@curie.fr; pierre.leopold@curie.fr)

L.B., 0000-0003-2903-6835; P.L., 0000-0002-2365-2103

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studies, the links between morphogens and organ/tissue growth areonly partially clarified.Organ growth appears to be controlled at a global level. Indeed,

neither cell proliferation nor cell size suffices to drive growth, as adefect in proliferation can easily be compensated for by an increasein cell size in order to maintain normal organ size (Fankhauser,1945; Neufeld et al., 1998). Morphogens could offer such globalcontrol, because they cover relatively large fields of cells bydiffusing from their sites of production. However, the gradeddistributions of morphogens contrast with the almost homogeneousgrowth and mitotic activities observed across developing tissues,and this paradox has been difficult to integrate into ourunderstanding of growth control. Over the last two decades,intense research has led to several models exploring the growth-promoting activity of morphogens, mainly focusing on the BMP-like morphogen Decapentaplegic (Dpp) in the Drosophila imaginalwing disc (a larval epithelial structure that undergoes extensivegrowth and morphogenesis to form an adult wing aftermetamorphosis). In the ‘gradient scaling’ model (Day andLawrence, 2000), the graded activity of Dpp adjusts to the size ofthe disc, but Dpp production at the source remains constant. Thismodel proposes that a cell in the gradient activates growth andproliferation when it reads a difference in Dpp activity in itsneighbours. As the disc expands, the slope of the gradient isreduced. This explains both the homogenous pattern of celldivisions and the progressive arrest of cell proliferation, whichoccurs when the difference in Dpp signalling between two adjacentcells drops below a threshold (Rogulja and Irvine, 2005; Wartlicket al., 2011). An alternative ‘temporal dynamics’ model was laterproposed, based on the observation that Dpp concentrationsincrease with time at the source (Wartlick et al., 2011). Here, eachcell is exposed to a relative increase in Dpp signalling over time,which promotes cell division. This relative increase declinesprogressively with time until proliferation ceases. However,whether the Dpp gradient scales with tissue size is stillcontroversial (Hufnagel et al., 2007) and recent studies indicatethat the graded nature of Dpp activity, although crucial forpatterning, is dispensable for tissue growth (Barrio and Milán,2017; Bosch et al., 2017).

But how do morphogens promote growth? It is known that cellsgrow by increasing their protein, lipid and nucleic acid contents.One possibility could be, therefore, that morphogens simply allowan underlying basal growth machinery to operate. Indeed, Dpp actson growth by repressing the transcription factor Brinker (Brk),which represses the growth activators Myc and Bantam in lateralcells. Co-expression of brk, bantam andMyc in the wing pouch onlypartially rescues the growth inhibition mediated by brk alone,suggesting that Brk could act on other targets for growth inhibition(Doumpas et al., 2013). Similarly, the morphogen Wingless (Wg)promotes growth by counteracting the repressor form of thetranscription factor TCF (also known as Pangolin in Drosophila).When a morphogen, either Dpp or Wg, and its negative effector aresimultaneously inactivated, wing imaginal discs grow almostnormally (Barrio and Milán, 2017, 2020; Schwank et al., 2008).This suggests that Dpp and Wg act by preventing growth inhibitionby Brk and TCF. This forms the basis of the ‘growth equalizationmodel’ whereby Dpp serves to balance the non-uniform growthpotential of wing disc cells by restricting the growth repressor Brk tothe lateral domain (Schwank et al., 2008). How Dpp and Wgfunction independently of one another to drive growth remainsunclear. Their respective overexpression induces distinctanisotropic growth patterns, suggesting that they carry non-interchangeable growth functions (Barrio and Milán, 2020).

The paradigms emerging from studies of patterning and growth inDrosophila have also been interrogated in vertebrates, which exhibitan increased range of organ size. These studies have revealed that, instructures such as the limbs or the spinal cord, early cell-typespecification is dictated by gradients of morphogens (e.g. BMPs,Wnts, sonic hedgehog). This early specification phase precedes agrowth phase (Kicheva and Briscoe, 2015). This allows largerstructures to formwithout being limited by the short-range effects ofmorphogen diffusion. In this context, however, maintaining organproportions during the growth phase requires regulative feedbackstrategies to correct for variability in the number of precursor cells(Lander et al., 2009).

In summary, despite intense research, our understanding of therole of morphogens in tissue growth is far from complete. This isdue to the inherent difficulty of observing morphogen actions invivo, which moving forward will necessitate the use of more-powerful, less-disruptive technologies. These include refined gene-editing techniques to manipulate endogenous gene function, non-disruptive live imaging to evaluate the kinetics of endogenousmorphogens, and fast light-mediated gene activation/repression toavoid long-term adaptation effects due to genetic manipulation.

Non-autonomous processes adapting organ growth to bodyenvironmentAs highlighted above, organ patterning and growth rely onautonomous signals. However, the picture is much more complexthan this. Using inter-species eye-grafting experiments insalamanders, similar to the limb-grafting work of Harrison, VictorTwitty realized over 90 years ago that grafted eyes did not grow onstarved hosts, and that the ability of the graft to grow decreased withage (Twitty, 1930). This led Twitty to conclude that ‘the potentialsize of (an organ) is largely determined by intrinsic factors, but thatthe expression or realization of this potentiality during the growth ofthe animal depends upon its interactions with gradually changingenvironment’. Many would still agree with this conclusion today.Indeed, it is now known that growth strongly relies onenvironmental factors. These include light, humidity, temperature,crowding, oxygen and nutrients; the importance of the last of these

Ambystoma tigrinumAmbystoma punctatum

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Fig. 1. Autonomous determination of limb size.Reciprocal grafts of anteriorlimb buds (marked by asterisks) between two salamander species of differentsize illustrate that limb size is determined autonomously, i.e. the grafted limbsgrow to the size expected for that of the donor species (see Harrison, 1924).

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factors – nutrition – was tragically illustrated by studies linkingnutrient restriction to a decrease in the size of newborns and childrenduring World War II (Angell-Andersen et al., 2004; Stein et al.,2004).The control of organ and body growth by nutrition involves

sensor and effector pathways. Since the discovery of the Target ofRapamycin (TOR) network in yeast and its conservation inmetazoans, we have gained a better knowledge of the cellularmachinery that couples nutrients with cell and tissue growth (Liuand Sabatini, 2020). At the heart of this machinery lies the TORC1complex, which senses energy and nutrient inputs, and controls cellgrowth autonomously through the coordinated activation of protein,lipid and nucleotide synthesis. In Drosophila, hypomorphic Tormutations produce viable small animals, illustrating the essentialrole of this pathway in the general control of cell, organ and bodysize (Zhang et al., 2006). TORC1 also receives inputs from theinsulin/insulin-like growth factor (IGF) signalling pathway, whichcoordinates nutrient availability and growth through circulatinghormones. Indeed, a coordinated reduction in all (or most) bodyparts is observed under nutritional restriction, highlighting thatsystemic hormonal relays play a key role in adjusting organ andbody growth in response to nutrient availability. Outstandingquestions in the field therefore have focussed on the identity ofnutrient sensor tissues and the nature of the nutrients sensed, theidentity of the signals ensuring organ crosstalk for systemic growthregulation, and the definition of the effector pathways.The power of genetic analyses in Drosophila facilitated the study

of nutritional physiology. Specifically, the possibility ofmanipulating different genetic pathways in separate organsallowed the identification of a large number of physiologicalcrosstalk events involved in nutritional response (Droujinine andPerrimon, 2016). The fat body, which is functionally related to thevertebrate liver and fat, is key for nutrient sensing inDrosophila. Fatbody cells use TORC1 and possibly other branches of the nutrient-responding pathways to: (1) store/release nutrients; and (2) produceand send signals triggering a systemic response in peripheral tissues.Fat body-derived signals activate specific centres in the brain, whereDrosophila insulin-like peptides (Dilps) are secreted to promotesystemic growth when conditions are favourable (Agrawal et al.,2016; Delanoue et al., 2016; Koyama and Mirth, 2016; Meschiet al., 2019; Rajan and Perrimon, 2012; Sano et al., 2015). Muscletissue (Katewa et al., 2012) and the gut (Alfa et al., 2015; Redhaiet al., 2020; Rodenfels et al., 2014; Song et al., 2017) are alsoemerging as major nutrient sensors. Interestingly, many factorsidentified in cross-organ communication in Drosophila are alsofound in vertebrates (e.g. Dilps/insulin/IGF, Impl2/IGF-BP, AKH/glucagon, Upd2/Leptin, Egr/TNFα, Myo/GDF11, Daw/TGFβ, Hh/Shh, GBP/EGF), suggesting functional conservation of theirphysiological roles. However, our understanding of how thesefactors come together to achieve systemic growth regulation remainsincomplete.Moving forward in this direction will first require a clear

description of nutrient sensors and their respective nutrient stimuli.In invertebrate genetic models, this could reasonably be achievedthrough exhaustive screening of organs and pathways for systemicgrowth defects (for recent examples, see Delanoue et al., 2016;Agrawal et al., 2016; Redhai et al., 2020). Moreover, currentdescriptions of nutrient sensing have thus far been limited to broadcategories, i.e. lipids, carbohydrates and amino acids. However,cells need to distinguish between nutrients with high precision. Aclear illustration of this is the exquisite regionalization of specificamino acid transporter expression in spatially structured organs such

as the gut and the liver (Kandasamy et al., 2018), suggesting thatlocalized nutrient sensing is adjusted to the needs of specific celltypes. Our current view of global nutrient sensor functions shouldthus be revisited to integrate the notion of cell- or region-specificsensors, as recently exemplified in the case of copper-sensing cellsin the fly gut (Redhai et al., 2020).

Another future challenge is to understand the central integrationof nutritional signals. In both mouse and Drosophilamodels, recentfindings indicate that a major part of this integration takes place inthe brain. Nutrients enter the brain and directly activate neuralsensors controlling energy balance and feeding (Augustine et al.,2018). Alternatively, the brain receives relay signals produced bynutrient-sensing organs in the form of hormones (e.g. leptin, insulin,adiponectin and several gut-derived peptides), which participate inthe central control of energy balance (Kim et al., 2018). This has ledto an integrated model in which the vertebrate hypothalamus,together with the central nervous system, forms a complex neuralnetwork controlling energy homeostasis. Our ability to fullydecipher this circuitry controlling nutritional homeostasis is, ofcourse, challenged by the complexity of the mammalian brain.Important insights could therefore come from parallel studies onsimpler brains, such as those of insects for which the elucidation offull neural architecture is nowwithin reach (https://www.janelia.org/project-team/flyem). In addition, recent improvements inoptogenetic and chemogenetic techniques could allow precisemanipulations of nutrient-sensing centres.

An additional and important aspect of nutritional physiology thathas emerged recently is the observation that not all body parts areequally sensitive to nutrient restriction during growth. Indeed, ahierarchy of tissues is established in response to nutrient shortage, asillustrated by early studies on intrauterine growth restriction. Ininfants suffering nutrient deprivation, the redistribution of bloodflow maximizes oxygen and nutrient supply to the brain, allowingthis specific organ to be spared (Cohen et al., 2015). Remarkably,this feature is also observed during development in mice (Gonzalezet al., 2016) and flies (Cheng et al., 2011). In Drosophila, theobservation of phenotypic plasticity in response to nutritionidentified another hierarchy among imaginal discs. Whereasgenital discs appear rather insensitive to nutritional deprivation,others are strongly affected, leading to various degrees of sizedecrease and altered body proportions. Molecularly, this hierarchyrelies on the ability of different organs to ‘read’ nutritionalinformation through insulin signalling (Tang et al., 2011). Thisfinding therefore highlights that the proportions between differentorgans can be modified through a plastic response to changingenvironments.

Coordinating growth between body partsWhat happens when the growth of one body organ is impairedprovides interesting insights into a more global level of regulationtaking place between body parts. In the early 1980s, it was observedthat abnormal growth of Drosophila imaginal discs (triggered byinjury, undergrowth or tumoural growth) delays developmentaltransitions (Simpson et al., 1980). These inspiring findingsindicated that ill-growing tissues can modify body physiologythrough systemic signals. A relaxin-like hormone called Dilp8has since been found to be produced by growth-impaired discs.Dilp8 delays pupal development by inhibiting production ofthe steroid hormone ecdysone through a central brain relay(Colombani et al., 2012, 2015; Garelli et al., 2012, 2015; Vallejoet al., 2015). In addition, for body proportions to be maintainedwhen the growth of one organ is impaired, the growth rates of other

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body parts must adjust. Such coordination is observed betweenimaginal discs (Parker and Shingleton, 2011), or between regions ofa single disc (Gokhale et al., 2016; Mesquita et al., 2010). In bothcases, this coordination relies on the action of Dilp8 on ecdysonelevels (Boulan et al., 2019; Sanchez et al., 2019). Therefore, thesystemic regulation of both developmental time and growth raterelies on a single molecular mechanism, allowing tight coordinationbetween developmental parameters (Fig. 2A). Despite importantprogress, many aspects of this coordination remain. In particular, itis not known how systemic relays act differentially on the injuredparts (which compensate and grow faster) and the healthy ones(which slow down and ‘wait’).In vertebrates, long bones are the model of choice for studying

limb growth regulation, growth termination and inter-organcommunication (Roselló-Díez and Joyner, 2015). Here again, newtools have extended the power of genetic manipulations and havepaved the way for novel findings. In the mouse model, a complexgenetic set up has been used to allow for unilateral inhibition of cellproliferation in the growth plate of the left limb (Roselló-Díez et al.,2018). This approach revealed that contralateral growthcoordination takes place during early pup development (Fig. 2B),although the molecular mechanisms mediating this crosstalk areunknown.Contralateral limb communication also appears to play a role in

the context of regeneration. Early experiments on salamander andaxolotl have shown that, after amputating two limbs on the sameanimal, regeneration progresses slower when the second amputated

limb is contralateral to the first one, possibly because oftransneuronal connexions (Tweedle, 1971). In Xenopus, unilateralleg amputation triggers a mirrored bioelectric signal on thecontralateral limb, suggesting that depolarization could act as along-range signalling process (Busse et al., 2018). Moving forward,merging the fields of growth control and limb regeneration could beinstrumental, allowing us to revisit the early findings derived fromgrafting and regeneration experiments and provide further insightinto inter-organ communication.

Termination of growthGrowth stops when organs or tissues reach their target size.However, behind this simple statement lies some very complexbiology. As presented earlier, appendage grafting andtransplantation experiments in salamanders and Drosophilasuggest a ‘ruler’ mechanism that is intrinsic to each organ. In fact,different organs use different ways to assess their target size. Thedetermination of liver size, for example, depends on functionalfeedback that follows a liver:body size ratio. Accordingly, iftransplanted into a host with a different size from the donor, the liveradjusts to the size of the host (Kam et al., 1987). The same applies tothe thyroid gland, which increases its size when thyroid hormoneproduction is deficient due to a negative-feedback loop involvingthyroid-stimulating hormone (Ortiga-Carvalho et al., 2016). Theseare examples of ‘regulative’ control, whereby final organ size relieson extrinsic cues, mostly linked to function. By contrast, the gut, thepancreas or the thymus do not adjust to recipient size after

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Fig. 2. Feedback mechanisms coordinating growth across different organs maintain body proportions. (A) Coordination between imaginal discs inDrosophila. When growth is perturbed in the wing disc, Dilp8 triggers non-autonomous growth inhibition in other imaginal discs (here the eye disc) through aneuronal relay controlling ecdysone levels. (B) Coordination between bilateral mouse limbs. When growth is genetically reduced on only one side, contralaterallimb growth inhibition is observed, thereby equalizing limb sizes.

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transplantation, nor do they regrow to normal size after ablationearly in development (Metcalf, 1964, 1963; Stanger et al., 2007). Inthe case of the pancreas, the number of divisions for each progenitorcell appear to be limiting, therefore preventing full regenerationupon ablation (Stanger et al., 2007).Organ mass can also be used as a ruler for final organ size. In this

case, feedback factors called ‘chalones’ have been proposed to besecreted proportionally to organ mass and inhibit growth in anautocrine manner. Although this concept is attractive, only a fewexperimentally confirmed examples of chalones have beendescribed to date. These include the TGFβ myostatin in skeletaland cardiac muscles (McPherron et al., 1997) and GDF11 in theolfactory neural epithelium (Gamer et al., 2003). In the vertebratelimb bud, the accumulation of tissue mass displaces signallingcentres and, as a consequence, blocks a positive-feedback loopbetween the morphogens Shh and Fgf, inducing proliferation arrest(Scherz et al., 2004; Verheyden and Sun, 2008). As discussedabove, morphogens are essential for patterning and growth duringdevelopment. Two separate models, the gradient scaling model andthe temporal dynamics model, propose that morphogen signallingalso contributes to growth arrest and the determination of final organsize in the fly. Although supported by elegant experiments, thesemodels are challenged by contradicting experimental evidence(Vollmer et al., 2017). In the gradient scaling model, proliferationshould stop when the Dpp signal is uniform. Yet, wing growth isobserved when Dpp activity is constant throughout the disc (Boschet al., 2017; Schwank et al., 2008). In the temporal dynamics model,a relative increase of Dpp signalling is needed to sustainproliferation. However, clones of cells lacking Mad (theintracellular mediator of Dpp) and the growth inhibitor Brk growto a normal size despite being unable to transduce an increase inDpp signal (Schwank et al., 2012).Mechanical forces exerted on cells within tissues form an

additional level of regulation. At the tissue scale, cell proliferationand growth increase local pressure, inducing mechanical feedbackthat inhibits growth in compressed zones and stimulates growth instretched ones. Two distinct mathematical models integrating bothmorphogen signalling and mechanical feedback predict uniformproliferation and growth termination in tissues (Aegerter-Wilmsenet al., 2007, 2012; Hufnagel et al., 2007; Shraiman, 2005). In thesemodels, growth stops when compression eventually overcomes thenon-uniform growth-promoting activity of morphogens.Experimental measurements comparing patterns of proliferation,mechanical stress and cell deformation in the central and lateralregions of the Drosophila wing disc support this model (LeGoffet al., 2013; Mao et al., 2013; Nienhaus et al., 2009).Mechanistically, this feedback involves the regulation of cellproliferation and apoptosis via changes in the acto-myosincytoskeleton and the canonical effectors of the growth-promotingHippo/Yorkie (Hpo/Yki) pathway (Pan et al., 2016; Rauskolb et al.,2014). However, a recent report showing that releasing tension inthe wing disc does not affect Hippo signalling or wing size (Maet al., 2017) challenges the role of mechanical feedback incontrolling final appendage size and leaves the door open forfurther studies. Although the Hippo effectors Yap/Taz were firstshown to transduce mechanical information in vertebrate cells(Dupont et al., 2011), a role for this coupling in controlling organsize in vertebrates has not yet been established.Regardless of its underlying mechanism, growth termination

seems to be controlled organ autonomously. However, final organsize is largely influenced by systemic cues that superimpose a layerof control on autonomous regulatory circuits. In Drosophila,

nutritional status, acting via the PI3K and TOR signalling relays,modulates Yki localization and hence the sensitivity of cells tomechanical feedback and final organ size (Borreguero-Muñoz et al.,2019). Interestingly, when the size of the wing is modified byalterations to insulin signalling, the resulting appendages areperfectly patterned, suggesting that morphogen activityaccommodates for varying tissue size (Leevers et al., 1996).Indeed, when PI3K activity is specifically modified in the posteriorcompartment of the wing disc, the domain of phosphorylation of thetransducer MAD and the domain of expression of the Dpp targetSpalt adapt to compartment size early during wing discdevelopment (Teleman and Cohen, 2000). Nutrient-responsivepathways also modulate the expression of Dally, a proteoglycaninvolved in the spreading and activity of Dpp, providing amechanism for morphogen gradient scaling by nutrition (Ferreiraand Milán, 2015). Therefore, in addition to promoting growth, Dppsignalling can mediate modifications in tissue size driven byenvironmental cues.

Finally, an intriguing aspect of growth arrest comes with theobservation that the final size of organisms could be limited by therate of energy flux towards organs and cells. Pioneering work in thelate 1940s indicated that the metabolic rate of many animal speciesscales with body mass with a power law of ¾ (B∝B0M3/4; Kleiber,1947). Remarkably, this so-called ‘Kleiber’s law’ applies toorganisms as diverse as mammals, birds, fish, insects, molluscsand plants, and for masses ranging across more than 16 orders ofmagnitude (see Box 1). The theoretical modelling of this empiricalfinding has remained elusive. Recently, an attractive model wasproposed to explain the so called ‘¾ rule’ relying in part on thephysical properties of branched, fractal networks that are required toequally allocate metabolic energy to all cells of an organism (Westet al., 1997). An important consequence of Kleiber’s law is that,whereas energy needs (proportional to cell number) scale to thepower of 1 with mass, energy allocation scales to the power of ¾.This imbalance is expected to ultimately limit growth to anasymptotic body mass specific for each species. This led West,Brown and Enquist to propose a universal, dimensionless, growthcurve on which growth parameters collected from animal species,including shrimp, shrew, salmon, hen, caw and others, all alignperfectly (West et al., 2001). Therefore, allometric scaling ofmetabolic rate and the energy cost of producing/maintainingbiomass could explain body growth arrest. Current research in thefield of growth control has only begun to explore the physiologicalbasis of Kleiber’s law (Thommen et al., 2019). Future researchcould help to decipher whether similar principles could also beapplied to autonomously limit the growth of individual organs.

Bilateral symmetry and developmental stabilityThe precision of organ size determination is particularly visible forbilateral organs, which in many cases differ in size by less than 1%.These observations raise outstanding questions with regard to themechanisms of size adjustment. Bilateral organs follow identicalgenetic programmes and develop within a shared physiologicalenvironment. They are therefore ideal biological models for studyingprecision and stochastic variations in growth during development.Experimentally, these variations can be quantified by measuring thefluctuating asymmetry (FA) index of bilateral traits within individuals(Juarez-Carreño et al., 2018). FA can be readily assessed for manytraits in many species, including humans (Graham and Özener,2016). However, very little is known about the molecularmechanisms controlling FA, developmental stability and precision.In particular, it is still debated whether developmental precision relies

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on intrinsic properties of gene regulatory networks controllingdevelopmental processes, or on dedicated molecular mechanismsmeasuring and correcting errors (Félix and Barkoulas, 2015).The recent identification of genes involved in developmental

precision in genetically tractable models paves the way toaddressing these questions (Debat and Peronnet, 2013; Félix andBarkoulas, 2015). InDrosophila, the chaperone Hsp90 (encoded byHsp83) was first shown to correspond to the definition of a‘robustness factor’, impairment of which induces low-penetrancedevelopmental abnormalities (Rutherford and Lindquist, 1998).Interestingly, Hsp90 was also proposed to serve as an ‘evolutionarycapacitor’, allowing the accumulation of cryptic genetic variations.Overexpression of the transcriptional regulator Cyclin G in wing

imaginal discs is also sufficient to induce FA autonomously in adultwings (Dardalhon-Cuménal et al., 2018; Debat et al., 2011). CyclinG acts through the chromatin modifier complexes PRC1 and PR-DUB to coordinate expression of a large number of genes,supporting the idea that specific gene regulatory networks ensuredevelopmental stability. By contrast, the Dilp8 hormone and itsreceptor Lgr3 control developmental stability at a systemic levelthrough a central relay, and animals mutant for Dilp8 or Lgr3accumulate developmental errors, quantified by high wing FA(Boone et al., 2016; Colombani et al., 2015; Garelli et al., 2012,2015; Vallejo et al., 2015). As mentioned above, the Dilp8-Lgr3axis also mediates inter-organ communication in response to growthperturbation, suggesting that organ crosstalk could participate indevelopmental stability (Juarez-Carreño et al., 2018). Specificmetabolic adaptations can also buffer the effect of dietary stress ondevelopment. For example, trehalose metabolism in Drosophilabuffers variations in glycemia and ensures homeostasis upon dietarystress (Matsushita and Nishimura, 2020), potentially linkingdevelopmental noise with metabolic processes. Interestingly, fliesmutant for the proapoptotic gene hid also show increased wing FA(Neto-Silva et al., 2009), suggesting that cell elimination could beused to fine-tune organ size. In future work, genetic and tissue-specific analyses could help to establish how these different inputsact together to control robustness in a biological system.

To gain insights into the temporal and developmental basis ofbody symmetry, one would ultimately need to follow bilateral organgrowth over time with high precision. This type of analysis is nowpossible with the help of sophisticated imaging techniques. Suchwork has shown that, in the zebrafish inner ear, left-right sizevariability is initially high and progressively resolves to achievesymmetry (Green et al., 2017). Similar experiments performed inthe zebrafish otic vesicle reveal that mechanical feedback bylumenal hydraulic pressure is required to adjust organ growth rate(Mosaliganti et al., 2019). Vertebrate limbs are initially subjected tointrinsic and extrinsic symmetry breaking signals (Allard and Tabin,2009; Grimes, 2019). In some genetic backgrounds, symmetricorgans become directionally asymmetric, as observed in Holt–Oramsyndrome in humans, where only the left arm exhibits severedevelopmental defects (Newbury-Ecob et al., 1996). This suggests amodel in which symmetry is achieved through mechanismsprotecting against asymmetric cues (Grimes, 2019). Whethercommunication between the two bilateral sides is required forproper size adjustment is not known, but identification of thesignal(s) required for contralateral coordination upon growthperturbation could aid our understanding of how developmentalprecision is achieved at a molecular level in mammals.

ConclusionsThe complex and rich phenomenology of growth control hasinspired research spanning molecular and physical studies of thecell, to integrated organ physiology and evolutionary perspectives.As such, the challenge for future work will be to integrate this multi-scale information into a unified conceptual framework. The variousaspects of size control presented in this Spotlight define two distinctvisions on how size is coded in biological systems. Genetic andpatterning aspects suggest a deterministic mode of control, wherebyinitial parameters encoded in the early embryo determine the finalsize of future organs and organisms. This gene-centric view ofgrowth processes is supported in the fly model by the role ofmorphogen signalling networks in growth and patterning. However,accumulating evidence for feedback mechanisms involvingphysical or metabolic inputs supports the existence of a self-

Box 1. Kleiber’s LawIn the early 1930s, Max Kleiber established that the metabolic rate(measured as heat production per day) of animals scales to the ¾ powerwith their mass. This empirical law applies from small crustaceans tolarge turtles in ectotherms, and frommouse to whales in endotherms, i.e.species varying in mass over 16 orders of magnitude. This suggests that,independently of habitat, physiology or lifestyle, all living species aresubjected to inherent, common metabolic constrains. The universalnature of the ¾ power scaling law also explains heart beat rate andlifespan differences. It also helps scientists work out, for example, how toadjust drug doses in rats for use in humans. In 1997, James Brown, BrianEnquist and Geoffrey West proposed a remarkable theoreticalframework for this allometric scaling, based on evolutionary constraintson the network that distributes energy to cells in a living organism (Westet al., 1997). This model presents three key features: (1) a fractal networkdistributing oxygen fills the space in order to reach all cells of anorganism; (2) terminal units of the network (capillaries) are invariant in allsystems, because the final unit is the cell itself; and (3) energy dissipationof the system isminimized. This study triggered a revival of the debate onKleiber’s law and stimulated further research (Banavar et al., 1999). Itwas soon followed by a second study based on geometry rather thanhydrodynamics of networks, allowing the ¾ power law to be applied toone-celled organisms lacking circulating systems (West et al., 1999). In2001, temperature was added to the model as a modifier of metabolicactivity. In this study, the authors demonstrated that, when compensatedfor temperature and scaled to mass, metabolic rates are similar for livingspecies such as microbes, ectotherms, endotherms (including those inhibernation) and plants, for temperatures ranging from 0°C to 40°C(Gillooly et al., 2001). This provides a universal framework forunderstanding energy storage and flux in living organisms, in whichthe two main variables appear to be mass and temperature. Imageadapted from Norris (1998).

10−6 10−3 100 103 106 109

Mass (g; log scale)

10−6

10−3

100

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Ectotherms(cold-blooded

organisms)

Endotherms(warm-blooded

organisms)

Ectotherms(cold-blooded

organisms)

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organisms)

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ic r

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(kca

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determined mode of organ growth/size control. Future research willno doubt help to integrate these two ideas. This research should alsofocus on system energetics and push forward our understanding ofthe physiological implications of Kleiber’s law, in particular at theorgan level. Finally, the study of non-physiological conditions, suchas growth impairment, regeneration or uncontrolled growth, will beneeded to help shed new light on systemic or cross-organ regulatorymechanisms that are, at present, poorly understood.

AcknowledgementsWe thank Yohanns Bellaiche and Paula Santabarbara-Ruiz for useful comments onthe manuscript, and Bertsy Goic for drawing the figures.

Competing interestsThe authors declare no competing or financial interests.

FundingThe work in our laboratory is funded by Institut Curie, Centre National de laRecherche Scientifique, Institut National de la Sante et de la Recherche Medicale,Fondation pour la Recherche Medicale, European Research Council (AdvancedGrant 694677 to P.L.) and the Labex DEEP program (ANR-11-LABX-0044, ANR-10-IDEX-0001-02).

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