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321 (2014) 71 –76
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Review Article
The control of cell growth and body size in Caenorhabditis elegans Simon Tuckn Umeå Center for Molecular Medicine, Umeå University, SE-901 87 Umeå, Sweden
article information
abstract
Article Chronology:
One of the most important ways in which animal species vary is in their size. Individuals of the
Received 1 August 2013
largest animal ever thought to have lived, the blue whale (Balaenoptera musculus), can reach a weight
Received in revised form
of 190 t and a length of over 30 m. At the other extreme, among the smallest multicellular animals
8 November 2013
are males of the parasitic wasp, Dicopomorpha echmepterygis, which even as adults are just 140 μm in
Accepted 11 November 2013
length. In terms of volume, these species differ by more than 14 orders of magnitude. Since size has
Available online 19 November 2013
such profound effects on an organism's ecology, anatomy and physiology, an important task for
Keywords:
evolutionary biology and ecology is to account for why organisms grow to their characteristic sizes.
Caenorhabditis elegans
Equally, a full description of an organism's development must include an explanation of how its
Body size
growth and body size are regulated. Here I review research on how these processes are controlled in
TGFbeta
the nematode, Caenorhabditis elegans. Analyses of small and long mutants have revealed that in the
dbl-1
worm, DBL-1, a ligand in the TGFβ superfamily family, promotes growth in a dose-dependent
BMP
manner. DBL-1 signaling affects body size by stimulating the growth of syncytial hypodermal cells rather than controlling cell division. Signals from chemosensory neurons and from the gonad also modulate body size, in part, independently of DBL-1-mediated signaling. Organismal size and morphology is heavily influenced by the cuticle, which acts as the exoskeleton. Finally, I summarize research on several genes that appear to regulate body size by cell autonomously regulating cell growth throughout the worm. & 2013 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A substantial amount of growth in C. elegans is the result of increases in cell size. A TGFβ pathway regulates body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins modulating the DBL-1 signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . The BMP pathway regulates endoreduplication of hypodermal nuclei . . . . . . . . . . . Gonad and nutrient signals modulate body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of cuticle structure on body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
n
Fax: þ46 90 785 44 00. E-mail addresses: [email protected], [email protected]
0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.11.007
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The actin cytoskeleton in hypodermal cells has profound effects on organismal shape TFG-1 and CED-4 have antagonistic effects on cell growth and apoptosis . . . . . . . . . . . Other genes required for growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Organismal size is determined to a significant extent genetically. This fact is almost implicit in the observation that characteristic sizes are associated with different species; and it chimes well with our everyday experience: taller (or larger) individuals of a given species tend to have taller (or larger) offspring. At the same time, environment can have an appreciable effect on body size: Historical studies of people living in Britain, for example, have shown that over the past hundred years, a period of time so short that the population cannot have changed appreciably genetically, the average height has risen by almost 10 cm, (6%). Thus, models for how body size is regulated must be able to account both for how this process is controlled genetically, and for how it can be influenced environmentally. Since its genetic make-up has such profound effects on an organism's size, the ability to perform genetic analyses greatly facilitates experimental studies into how body size is regulated. Caenorhabditis elegans is well suited to these types of studies because a wealth of tools has been developed to aid its genetic manipulation.
A substantial amount of growth in C. elegans is the result of increases in cell size C. elegans and other nematodes belong to the ecdysozoa, the clade of animals that molt. Within this clade two types of growth are recognized, continuous and “saltational” (stepwise). Arthropods, which have non-elastic exoskeletons made up largely of chitin, display saltational growth in which increases in body size occur exclusively at the molts. Nematodes, on the other hand, having elastic collagenous cuticles display continuous growth during postembryonic stages. An adult C. elegans hermaphrodite worm has a length of approximately 1.3 mm. Quantitative studies of growth in C. elegans larvae have shown that growth in terms of volume is linear during each of the four larval stages [1]. The rate of growth, however, increases from one larval stage to the next with the result that, seen over the entire course of postembryonic development, organismal volume is best approximated by an exponential growth curve [1]. [The C. elegans egg shell is made of chitin, and is non-elastic. Substantial cell division occurs during embryogenesis but there is no increase in volume.] The increase in growth rate that occurs at each molt is possibly caused by an increase in size of the buccal cavity, a structure at the tip of the head through which food is ingested. Unlike most of the worm, this structure does no grow continuously but stepwise at each molt [1]. C. elegans and many other nematodes are eutelic, that is, as adults, they have fixed numbers of somatic cells. However,
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regarded in terms of an increase in organismal volume, growth in C. elegans is not bound by the strict limit on the number of somatic cells. On the contrary, a substantial proportion of the growth that occurs during development occurs after cell division has ceased [1]. In fact, the major mechanism by which C. elegans worms grow postembryonically is by an increase in the size rather than the number of cells. Between hatching and the 1st day of adulthood there is an approximately 3.5-fold increase in the total number of nuclei (including germ-line nuclei) but a 32-fold increase in organismal volume [1]. Thus the growth in terms of volume in C. elegans is largely a result of an increase in the sizes of some or all cells. While there is little variation in the sizes of C. elegans worms grown under standard laboratory conditions, size is severely reduced when nutrients become limiting [2]. This reduction is observed even when individuals are starved after the time at which cells cease to divide implying that (not surprisingly) a scarcity of nutrients can affect cell growth as well as cell division. Almost all cells are thought to need exogenously supplied nutrients in order to grow. We can, therefore, expect that the reduced organismal size of worms grown in the presence of limiting nutrients is in part the result of cell autonomous effects on cell growth. In addition, however, the genetic analysis of body size in C. elegans, has revealed another layer of control exerted systemically [3]. Specifically, the characterization of body size mutants has revealed the existence of a signaling pathway that regulates growth and body size non-autonomously in response to nutrient status [2,3]. This pathway is separate from other systemically acting pathways that regulate development or metabolism.
A TGFβ pathway regulates body size Mutants with reduced body size were first isolated in the genetic screens for morphological mutants carried out by Sydney Brenner in the 1960s and 1970s [3]. Subsequent directed screens for small (Sma) and long (Lon) mutants, and the molecular characterization of the genes affected revealed that body size is regulated by a signaling pathway activated by a ligand in the TGFβ superfamily, DBL-1. Loss-of-function mutations in dbl-1 or in other genes in the pathway cause a marked (40%) reduction in both length and width throughout postembryonic development [4,5]. Increased DBL-1 signaling activity, on the other hand, causes worms to be more than 25% longer than wild type [5,6]. DBL-1 is highly similar to Decapentaplegic (Dpp) from Drosophila melanogaster, and to two vertebrate members of the bone morphogenetic protein family, BMP2 and BMP4. DBL-1 is expressed predominantly within specific parts of the nervous system notably in motor neurons in the ventral nerve cord (Fig. 1) [4,5]. Studies of the pathway that DBL-1 activates have led to significant advances in
EX P ER I ME NTAL C E LL RE S E ARCH
gonad
ciliated sensory neurons
ciliated sensory neuron ventral cord motor neurons
Fig. 1 – The positions of cells and tissues that modulate growth and body size systemically. DBL-1, a ligand in the TGFβ superfamily that regulates growth and body size in a dosedependent manner, is expressed predominantly in ventral cord motor neurons, coloured grey. Note that only a subset of such neurons is indicated and that although the paths depicted for their processes are representative, they are not strictly anatomically accurate. The focus of the pathway DBL-1 activates to control body size is the hypodermis, a part of which is shown in Fig. 2. The gonad (different shades of blue) inhibits the growth of somatic tissues. Ciliated sensory neurons in the head and tail promote growth. A subset of the 61 ciliated sensory neurons present is indicated (coloured brown). The pharynx, the feeding organ, is shaded green.
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for lon-2 null mutations are more than 25% longer than wild type, a phenotype that is entirely dependent upon BMP pathway activity [13]. lon-2 encodes plasma-membrane anchored proteoglycan in the glypican family that can bind BMPs directly [6]. Recent intriguing results with LON-2 have shown that the protein contains two separate domains that both, independently, are able to inhibit DBL-1 activity [13]. drag-1 encodes the sole member of the repulsive guidance molecule (RGM) family of proteins in C. elegans [14]. Like part of LON-2, RGM family proteins are GPI-linked membraneassociated proteins. DRAG-1 is expressed in the hypodermis, which is also its focus of activity [14]. kin-29 encodes a serine/threonine kinase that may have more than one function in regulating body size [15,16]. Genetically, kin-29 acts downstream of dbl-1. However expression of kin-29 either under the control of dbl-1 promoter, which is principally active in neurons, or under the control of a promoter active in the cells in which DAF-4 and SMA-6 function can rescue the kin-29 body size defect [15], suggesting that kin-29 acts in both signaling and receiving cells. In yeast cells and in vitro, KIN-29 can bind TAX-6, the catalytic subunit of calcineurin [16].
The BMP pathway regulates endoreduplication of hypodermal nuclei the understanding of TGFβ–mediated signaling. For example, Smad proteins were first shown to be the downstream effectors of BMP receptors from studies of DBL-1 and Dpp signaling in C. elegans and Drosophila [3]. In target tissues, DBL-1 activates type I and type II coreceptors, SMA-6 and DAF-4, which are serine kinases [3]. Transduction of the signal to the nucleus requires SMA-2, SMA-3 and SMA-4, which are thought to function together as a complex to regulate the transcription of target genes [3]. SMA-2 and SMA-3 are homologous to receptorregulated Smad (R-Smad) proteins whereas SMA-4 is homologous to common-mediator Smads (Co-Smads). At least for SMA-3, receptor activity promotes accumulation of the protein in the nucleus [7]. Besides, sma-2, sma-3 and sma-4, sma-9 also functions genetically downstream of the BMP (DBL-1) pathway to regulate body size [8]. At least eight different transcripts are generated from the sma-9 locus most of which encode proteins homologous to Schnurri, a zinc-finger transcription factor that acts in the Dpp signaling pathway in the fly [8–10]. The SMAD complex may act together with a RUNX-family transcription factor, RNT-1. rnt-1 mutants are small and RNT-1 protein can bind SMA-4 in vitro [11].
Proteins modulating the DBL-1 signaling pathway Analyses of sma-10, lon-2, drag-1 and kin-29 have revealed that the proteins they encode act by modulating the BMP pathway regulating body size. sma-10 null mutations cause a Sma phenotype very similar to that caused by null mutations in dbl-1 [12]. SMA-10 encodes a widely-expressed protein with leucine-rich repeats and immunoglobulin-like domains (LRIG) that is found in puncta both at the plasma membrane and within cells [12]. In a heterologous system, SMA-10 binds directly to the receptors, SMA-6 and DAF-4, and stimulates their activity [12]. The Drosophila orthologue, lambik, rescues sma-10 phenotypes showing that sma-10 function has been conserved in evolution [12]. Worms homozygous
Consistent with the observation that a large proportion of the increase in volume that occurs during wild-type C. elegans development is the result of increases in cell size rather than cell number, mutations that cause a decrease in the activity of the BMP pathway appear to reduce body size by reducing the sizes of some or all cells rather than their number [4,5]. Although the pathway does have a function in the control of development in the male tail [17], only a small number of cells are affected and the mutants have almost wild-type numbers of cells. Likewise, lon-2 mutants or those with elevated dbl-1 gene copy number are longer than wild type but do not have more cells [6]. Genetic mosaic analysis indicates that a major focus of activity of the receptor with respect to body size (at least in adult worms) is in syncytial hypodermal cells that surround the internal tissues (and which secrete the cuticle) (Fig. 2) [3]. The pathway acts within these cells in part by affecting the process of endoreduplication [18]. In wild type, many of the nuclei in the hypodermal syncytia undergo two or three rounds of endoreduplication so that in 3-day old adults these nuclei have an average ploidy of 12. In mutants with reduced BMP signaling, however, the hypodermal nuclei undergo fewer rounds of endoreduplication with the result that their average ploidy is only 2/3 that in wild type [18]. Body size and hypodermal ploidy is also reduced in worms mutant for CRM-1, a C. elegans homologue of vertebrate Crim1, a transmembrane protein with multiple cysteine rich domains [19]. Blocking endoreduplication in adult wild-type worms by treatment with hydroxyurea (which blocks S phase) leads to a reduction in body size [20]. However, dbl-1 mutants treated with hydroxyurea are not further reduced in size strongly suggesting that the reduced body size of BMP mutants is the result of reduced endoreduplication rather than vice versa [20]. Thus, while in many species growth is controlled by regulation of cell division, which entails replication of chromosomes, nuclear division and then finally cytokinesis, growth in adult C. elegans worms is associated with rounds of chromosomal duplication within certain cells that occurs in the absence of nuclear division or cytokinesis.
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cuticle
annuli
hypodermis
seam cells
ventral nerve cord
furrows
Fig. 2 – Diagram showing part of the hypodermis and cuticle. The hypodermis, shaded yellow in cross-section, is made up of a 12 syncytial hypodermal cells, which together contain 157 nuclei in the adult. These hypodermal cells secrete the cuticle (shaded grey). Circumferential rings called annuli run almost the entire length of the cuticle. Between adjacent annuli are furrows, which form immediately above circumferential bundles of actin (not shown) present in hypodermal cells during embryogenesis and molting. The annuli are discontinuous and contain gaps above two rows of cells termed seam cells (coloured orange) that run along the left and right sides. The position of ventral nerve cord (coloured green) is indicated.
Studies on other species of nematode have shown that a number are not eutelic. In particular, different individuals of the same species can have different numbers of hypodermal nuclei [21]. However, between different species, no strong correlation exists between the average number of hypodermal nuclei and body size. It is possible, therefore, that within these species, body size is determined in part by the ploidy of hypodermal nuclei rather than their number. However, other mechanisms very likely also influence growth. Within C. elegans, while effects on ploidy of hypodermal cells undoubtedly account for part of the reduced size of the BMP mutants, they do not account for all of it: the mutants are reduced in size even during larval stages before most hypodermal endoreduplication has occurred.
Gonad and nutrient signals modulate body size Fundamental studies within evolutionary biology have shown that trade-offs exists between the germ line and the soma that can affect different aspects of the soma including its size. In almost all organisms tested to date (including humans), reducing the size of the germ line by surgical or genetic manipulation, leads to increased lifespan and fat storage, and to significantly increased body size. In keeping with the studies on other organisms, ablation of the gonad (depicted in Fig. 1) in C. elegans and some other nematodes also leads to a dramatic increase in body size [22] and lifespan. In C. elegans, the increase in size is not caused by changes in insulin signaling (which account for the increase in longevity) but by a different (as-yetunknown) mechanism. Gonadectomy of dbl-1 mutant worms also results in an increase in body size implying that the gonad must signal, at least in part, independently of BMP pathway.
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A worm subjected to dietary restriction will fail to grow to its proper size even if it is starved after the time at which somatic cell division has ceased. The reduced body size of worms subjected to dietary restriction is associated with reduced BMP signaling: dietary restriction reduces endoreduplication of hypodermal nuclei in wildtype worms but has much less effect on body size and endoreduplication in dbl-1 mutants [2]. A number of studies indicate, however, that in addition to DBL-1, other signals likely regulate body size in response to nutrient sensation. First, che-2, che-3, egl-4, osm-6, tax-2 and tax-4 gene activities have all been shown to be required in a group of sensory neurons in the head and tail (in which no dbl-1 expression has been observed) for worms to grow to the proper size (Fig. 1) [23]. che-3 and osm-6 are required for the formation of the sensory cilia in 61 ciliated sensory neurons through which worms sense chemicals in (and temperature of) their environment; che-2, egl-4, tax-2 and tax-4 encode proteins that directly mediate chemo- or thermosensation. Other proteins required for correct cilia formation such as certain members of the Bardet-Biedl syndrome (BBS) family of proteins are also required for worms to grow to their proper size [24]. ASNA-1, an ATPase is required in certain sensory neurons to promote cell growth [25]. Although ASNA-1 stimulates the secretion of insulin/insulin-like proteins, ASNA-1’s effect on body size does not appear to be mediated via insulin signaling [25]. Mutations in cnb-1 and tax-6, which encode the regulatory and catalytic subunits of calcineurin respectively can also affect sensory neuron function and body size [16,26]. Together, these studies strongly suggest that, in response to chemosensory cues, ciliated sensory neurons secrete a factor (or factors) that promote growth. Although, such a factor has yet to be identified, EGL-4, a cyclic GMP-dependent protein kinase, might negatively regulate its secretion. Whereas mutations affecting cilia structure, asna-1 or the chemosensory receptor gene, che-2, reduce body size, loss-of-function mutations in egl-4 increase body size [23,27].
The effect of cuticle structure on body size One way in which the BMP pathway affects growth in terms of length and morphology is by directly or indirectly regulating components of the cuticle (part of which is depicted in Fig. 2). Microarray analysis has revealed that the expression of several cuticle collagen genes is altered in mutants with elevated or reduced pathway activity [9,28,29]. Collagens make up over 50% of the cuticle by mass and the C. elegans genome contains 158 cuticle collagen genes [30]. In keeping with its vital function in acting as a barrier to the exterior, and with this surprisingly large number of cuticle collagen genes, the cuticle has a very complex, multi-layered structure; different collagens are found in the different layers. A small subset of the collagens appears to be particularly important for proper organismal length and morphology. Null or reduction-of-function mutations in dpy-2, dpy-3, dpy-5, dpy-7, dpy-8, dpy-10, dpy-13, and sqt-3 all cause a Dpy phenotype (in which length is reduced but width is essentially normal) similar to some hypomorphic BMP pathway mutations [30]. On the other hand, null mutations in just one cuticle collagen, lon-3, cause a Lon phenotype strikingly similar to that caused by elevated levels of dbl-1 [31,32]. The expression of a LON-3::GFP fusion protein is reduced in worms with increased dbl-1 gene copy number [32]. Furthermore, increasing lon-3 gene copy number reduces body length [31].
EX P ER I ME NTAL C E LL RE S E ARCH
Besides LON-3, two other lon proteins, LON-1 and LON-8, likely affect body size by directly or indirectly affecting the cuticle. LON-1 is a cysteine-rich secretory protein that is expressed in hypodermal cells [3]. lon-1 gene expression is inhibited by the BMP pathway [15]. lon-8 encodes a novel secreted protein that is highly conserved throughout nematode evolution [33]. LON-8 is expressed in hypodermal cells, and lon-8 mutations interact strongly genetically with those in dpy-11 and dpy-18, which encode collagen-modifying enzymes. LON-8 is thought to affect body length independently of the BMP pathway [33]. Mutations in adt-2 cause a Sma phenotype but are also thought to affect body size by affecting the cuticle. ADT-2 is a member of the disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted metalloproteases [34]. adt-2 mutations also affect dbl-1 gene transcription suggesting that the cuticle can signal back to the BMP pathway in some way [34].
The actin cytoskeleton in hypodermal cells has profound effects on organismal shape sma-1 encodes the βΗ-spectrin orthologue and is found on the apical surfaces of polarized epithelial cells including hypodermal cells. SMA-1 protein is required to maintain the association between the actin cytoskeleton and apical membranes [35]. During embryogenesis in wild-type larvae, following enclosure, cortical arrays of actin within epidermal cells, dramatically reorganize to form parallel circumferential bundles close to the apical surfaces. The transition from the stocky, so-called comma-stage embryo resulting from enclosure to the lithe first larval (L1) hatchling is driven by dramatic changes in shape of epithelial cells. The cells elongate along the anteriorposterior axis lengthways while at the same time the circumferential bundles of actin within them constrict; the vermiform shape is thus the result of the cells in the middle of the embryo being squeezed out at either end by the epithelial cells tightening around them. In sma-1 mutants, the actin cytoskeleton dissociates from the apical membrane, and, as a result, the extension of the epithelial cells is incomplete. The resulting larva is then considerably shorter than normal [35]. Interestingly, the function of the circumferential rings of actin in maintaining organismal morphology appears not to be restricted to embryonic development but may also be important during larval development. During postembryonic stages, circumferential ridges in the cuticle termed annuli are present along the entire length of the worm (Fig. 2). The troughs (furrows) between these ridges coincide precisely with the circumferential actin bundles that reform periodically during molting [30]. In animals homozygous for mutations in dpy-2, dpy-3, dpy-7, dpy-8 or dpy-10 the annuli are absent suggesting perhaps that the cuticle collagens they encode are required for linking parts of the cuticle to the apical membranes of hypodermal cells adjacent to the positions of the circumferential actin bundles. DPY-7 and DPY-10 are known to be located in the furrows of the annuli [30].
TFG-1 and CED-4 have antagonistic effects on cell growth and apoptosis Mutations in tgf-1 have been described that profoundly affect body size by reducing cell growth through a BMP-independent mechanism. tfg-1 encodes the C. elegans orthologue of the human
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proto-oncogene, TRK-fused gene. Reducing the function of tfg-1 in worms by RNAi results in a 70% reduction in body size caused by a reduction in cell size rather than cell number [36]. Complete loss of tfg-1 activity leads to ectopic activation of the programmed cell death pathway. Remarkably, inactivation of a gene in this pathway, ced-4 efficiently suppresses the tfg-1 size defects. Thus, tfg-1 and ced-4 link pathways regulating cell growth and cell death.
Other genes required for growth sma-5 encodes an ERK5/MAPK7/BMK1 MAP kinase homologue that is required for many cells in the worm to grow to their proper sizes [37]. Within the hypodermis, sma-5 functions cell autonomously but the gene can also affect body size non-autonomously [37]. Mutants lacking the activity of RICT-1, a component of the TORC2 complex, are also smaller [38]. ncl-1, which encodes a homologue of Brain tumour, a Drosophila tumour suppressor, acts cell autonomously to control cell and nucleolar size by negatively regulating the transcription of ribosomal and 5S RNAs [39]. Finally, mutants lacking bec-1 or unc-51, which are required for autophagy, are smaller than wild type [40] implying that autophagy is required for normal growth.
Future studies Considerable progress has been made over the past 15 years in understanding how cell growth and body size are regulated in C. elegans. Many interesting questions remain, however. How is the synthesis, secretion or activity of DBL-1 regulated in response to nutritional cues? How does the BMP pathway regulate endoreduplication of hypodermal nuclei? Does the hypodermis promote the growth of other tissues and, if so, how? The mechanism by which the germ line can affect body size is presently not well understood. Since the trade-offs that exist between the germ line and the soma is known to be of fundamental importance for evolutionary fitness, it is important to understand how the germ line influences the size of the soma. Among other outstanding questions are how ciliated sensory neurons modulate body size, how the actin cytoskeleton within hypodermal cells is regulated and how, in turn, it affects the elasticity of the cuticle. Lastly, it should not be forgotten that an organism's size is of central importance to its ecology. With the exception of the air, nematodes are ubiquitous in most ecological niches. In terms of numbers of individuals, they are by far the most abundant type of animal on the planet. Nematode species (of which around 24,000 have been described to date) vary in length from fractions of a millimeter to greater than 8 m. Research with C. elegans has provided important mechanistic insights into how growth is regulated, and likely will continue to do so in the future. In addition, the results obtained with C. elegans provide a platform for studies aimed at understanding how growth and size in other members of this important and varied group of animals is controlled.
Conflict of interest None.
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Acknowledgments Work in my laboratory is supported by grants from Cancerfonden and Vetenskapsrådet. I apologize to authors whose work on growth and body size, for lack of space, I was unable to cite directly.
references [1] C.G. Knight, et al., A novel mode of ecdysozoan growth in Caenorhabditis elegans, Evol. Dev. 4 (1) (2002) 16–27. [2] L.S. Tain, et al., Dietary regulation of hypodermal polyploidization in C. elegans, BMC Dev. Biol. 8 (2008) 28. [3] T.L. Gumienny, R.W. Padgett, A small issue addressed, Bioessays 25 (4) (2003) 305–308. [4] K. Morita, K.L. Chow, N. Ueno, Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family, Development 126 (6) (1999) 1337–1347. [5] Y. Suzuki, et al., A BMP homolog acts as a dose-dependent regulator of body size and male tail patterning in Caenorhabditis elegans, Development 126 (2) (1999) 241–250. [6] T.L. Gumienny, et al., Glypican LON-2 is a conserved negative regulator of BMP-like signaling in Caenorhabditis elegans, Curr. Biol. 17 (2) (2007) 159–164. [7] J. Wang, W.A. Mohler, C. Savage-Dunn, C-terminal mutants of C. elegans Smads reveal tissue-specific requirements for protein activation by TGF-beta signaling, Development 132 (15) (2005) 3505–3513. [8] J. Liang, et al., The Caenorhabditis elegans schnurri homolog sma9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling, Development 130 (26) (2003) 6453–6464. [9] J. Yin, L. Yu, C. Savage-Dunn, Alternative trans-splicing of Caenorhabditis elegans sma-9/schnurri generates a short transcript that provides tissue-specific function in BMP signaling, BMC Mol. Biol. 11 (2010) 46. [10] M.L. Foehr, et al., An antagonistic role for the C. elegans Schnurri homolog SMA-9 in modulating TGFbeta signaling during mesodermal patterning, Development 133 (15) (2006) 2887–2896. [11] Y.J. Ji, et al., RNT-1, the C. elegans homologue of mammalian RUNX transcription factors, regulates body size and male tail development, Dev. Biol. 274 (2) (2004) 402–412. [12] T.L. Gumienny, et al., Caenorhabditis elegans SMA-10/LRIG is a conserved transmembrane protein that enhances bone morphogenetic protein signaling, PLoS Genet. 6 (5) (2010) e1000963. [13] S. Taneja-Bageshwar, T.L. Gumienny, Two functional domains in C. elegans glypican LON-2 can independently inhibit BMP-like signaling, Dev. Biol. 371 (1) (2012) 66–76. [14] C. Tian, et al., The RGM protein DRAG-1 positively regulates a BMP-like signaling pathway in Caenorhabditis elegans, Development 137 (14) (2010) 2375–2384. [15] L.L. Maduzia, et al., C. elegans serine-threonine kinase KIN-29 modulates TGFbeta signaling and regulates body size formation, BMC Dev. Biol. 5 (2005) 8. [16] G. Singaravelu, et al., Calcineurin interacts with KIN-29, a Ser/Thr kinase, in Caenorhabditis elegans, Biochem. Biophys. Res. Commun. 352 (1) (2007) 29–35. [17] C. Savage, et al., Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components, Proc. Nat. Acad. Sci. U.S.A. 93 (2) (1996) 790–794. [18] A.J. Flemming, et al., Somatic polyploidization and cellular proliferation drive body size evolution in nematodes, Proc. Nat. Acad. Sci. U.S.A. 97 (10) (2000) 5285–5290.
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[19] W.Y. Fung, et al., crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans, Dev. Biol. 311 (1) (2007) 95–105. [20] E. Lozano, et al., Regulation of growth by ploidy in Caenorhabditis elegans, Curr. Biol. 16 (5) (2006) 493–498. [21] R.B. Azevedo, et al., Spontaneous mutational variation for body size in Caenorhabditis elegans, Genetics 162 (2) (2002) 755–765. [22] M.N. Patel, et al., Evolution of germ-line signals that regulate growth and aging in nematodes, Proc. Nat. Acad. Sci. U.S.A. 99 (2) (2002) 769–774. [23] M. Fujiwara, P. Sengupta, S.L. McIntire, Regulation of body size and behavioral state of C. elegans by sensory perception and the EGL-4 cGMP-dependent protein kinase, Neuron 36 (6) (2002) 1091–1102. [24] C.A. Mok, et al., Mutations in a guanylate cyclase GCY-35/GCY-36 modify Bardet-Biedl syndrome-associated phenotypes in Caenorhabditis elegans, PLoS Genet. 7 (10) (2011) e1002335. [25] G. Kao, et al., ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells, Cell 128 (3) (2007) 577–587. [26] J. Bandyopadhyay, et al., Calcineurin, a calcium/calmodulindependent protein phosphatase, is involved in movement, fertility, egg laying, and growth in Caenorhabditis elegans, Mol. Biol. Cell 13 (9) (2002) 3281–3293. [27] T. Hirose, et al., Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans, Development 130 (6) (2003) 1089–1099. [28] A.F. Roberts, et al., Regulation of genes affecting body size and innate immunity by the DBL-1/BMP-like pathway in Caenorhabditis elegans, BMC Dev. Biol. 10 (2010) 61. [29] M. Mochii, et al., Identification of transforming growth factorbeta- regulated genes in Caenorhabditis elegans by differential hybridization of arrayed cDNAs, Proc. Nat. Acad. Sci. U.S.A. 96 (26) (1999) 15020–15025. [30] A.P. Page, I.L. Johnstone, The cuticle, in WormBook, T.C.e.R. Community (Ed.) 2007, WormBook. [31] J. Nystrom, et al., Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen, cause reciprocal changes in body length, Genetics 161 (1) (2002) 83–97. [32] Y. Suzuki, et al., A cuticle collagen encoded by the lon-3 gene may be a target of TGF-beta signaling in determining Caenorhabditis elegans body shape, Genetics 162 (4) (2002) 1631–1639. [33] G. Soete, M.C. Betist, H.C. Korswagen, Regulation of Caenorhabditis elegans body size and male tail development by the novel gene lon-8, BMC Dev. Biol. 7 (2007) 20. [34] T. Fernando, et al., C. elegans ADAMTS ADT-2 regulates body size by modulating TGFbeta signaling and cuticle collagen organization, Dev. Biol. 352 (1) (2011) 92–103. [35] V. Praitis, E. Ciccone, J. Austin, SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans, Dev. Biol. 283 (1) (2005) 157–170. [36] L. Chen, et al., ced-4 and proto-oncogene tfg-1 antagonistically regulate cell size and apoptosis in C. elegans, Curr. Biol. 18 (14) (2008) 1025–1033. [37] N. Watanabe, et al., Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans, Development 132 (14) (2005) 3175–3184. [38] A.A. Soukas, et al., Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans, Genes Dev. 23 (4) (2009) 496–511. [39] D.J. Frank, M.B. Roth, ncl-1 is required for the regulation of cell size and ribosomal RNA synthesis in Caenorhabditis elegans, J. Cell Biol. 140 (6) (1998) 1321–1329. [40] I. Aladzsity, et al., Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans, Genetics 177 (1) (2007) 655–660.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Review Article
The control of cell growth and body size in Caenorhabditis elegans Simon Tuckn Umeå Center for Molecular Medicine, Umeå University, SE-901 87 Umeå, Sweden
article information
abstract
Article Chronology:
One of the most important ways in which animal species vary is in their size. Individuals of the
Received 1 August 2013
largest animal ever thought to have lived, the blue whale (Balaenoptera musculus), can reach a weight
Received in revised form
of 190 t and a length of over 30 m. At the other extreme, among the smallest multicellular animals
8 November 2013
are males of the parasitic wasp, Dicopomorpha echmepterygis, which even as adults are just 140 μm in
Accepted 11 November 2013
length. In terms of volume, these species differ by more than 14 orders of magnitude. Since size has
Available online 19 November 2013
such profound effects on an organism's ecology, anatomy and physiology, an important task for
Keywords:
evolutionary biology and ecology is to account for why organisms grow to their characteristic sizes.
Caenorhabditis elegans
Equally, a full description of an organism's development must include an explanation of how its
Body size
growth and body size are regulated. Here I review research on how these processes are controlled in
TGFbeta
the nematode, Caenorhabditis elegans. Analyses of small and long mutants have revealed that in the
dbl-1
worm, DBL-1, a ligand in the TGFβ superfamily family, promotes growth in a dose-dependent
BMP
manner. DBL-1 signaling affects body size by stimulating the growth of syncytial hypodermal cells rather than controlling cell division. Signals from chemosensory neurons and from the gonad also modulate body size, in part, independently of DBL-1-mediated signaling. Organismal size and morphology is heavily influenced by the cuticle, which acts as the exoskeleton. Finally, I summarize research on several genes that appear to regulate body size by cell autonomously regulating cell growth throughout the worm. & 2013 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A substantial amount of growth in C. elegans is the result of increases in cell size. A TGFβ pathway regulates body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins modulating the DBL-1 signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . The BMP pathway regulates endoreduplication of hypodermal nuclei . . . . . . . . . . . Gonad and nutrient signals modulate body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of cuticle structure on body size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
n
Fax: þ46 90 785 44 00. E-mail addresses: [email protected], [email protected]
0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.11.007
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3 21 (2 014 ) 71 – 76
The actin cytoskeleton in hypodermal cells has profound effects on organismal shape TFG-1 and CED-4 have antagonistic effects on cell growth and apoptosis . . . . . . . . . . . Other genes required for growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Organismal size is determined to a significant extent genetically. This fact is almost implicit in the observation that characteristic sizes are associated with different species; and it chimes well with our everyday experience: taller (or larger) individuals of a given species tend to have taller (or larger) offspring. At the same time, environment can have an appreciable effect on body size: Historical studies of people living in Britain, for example, have shown that over the past hundred years, a period of time so short that the population cannot have changed appreciably genetically, the average height has risen by almost 10 cm, (6%). Thus, models for how body size is regulated must be able to account both for how this process is controlled genetically, and for how it can be influenced environmentally. Since its genetic make-up has such profound effects on an organism's size, the ability to perform genetic analyses greatly facilitates experimental studies into how body size is regulated. Caenorhabditis elegans is well suited to these types of studies because a wealth of tools has been developed to aid its genetic manipulation.
A substantial amount of growth in C. elegans is the result of increases in cell size C. elegans and other nematodes belong to the ecdysozoa, the clade of animals that molt. Within this clade two types of growth are recognized, continuous and “saltational” (stepwise). Arthropods, which have non-elastic exoskeletons made up largely of chitin, display saltational growth in which increases in body size occur exclusively at the molts. Nematodes, on the other hand, having elastic collagenous cuticles display continuous growth during postembryonic stages. An adult C. elegans hermaphrodite worm has a length of approximately 1.3 mm. Quantitative studies of growth in C. elegans larvae have shown that growth in terms of volume is linear during each of the four larval stages [1]. The rate of growth, however, increases from one larval stage to the next with the result that, seen over the entire course of postembryonic development, organismal volume is best approximated by an exponential growth curve [1]. [The C. elegans egg shell is made of chitin, and is non-elastic. Substantial cell division occurs during embryogenesis but there is no increase in volume.] The increase in growth rate that occurs at each molt is possibly caused by an increase in size of the buccal cavity, a structure at the tip of the head through which food is ingested. Unlike most of the worm, this structure does no grow continuously but stepwise at each molt [1]. C. elegans and many other nematodes are eutelic, that is, as adults, they have fixed numbers of somatic cells. However,
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75 75 75 75 75 . 76 . 76
regarded in terms of an increase in organismal volume, growth in C. elegans is not bound by the strict limit on the number of somatic cells. On the contrary, a substantial proportion of the growth that occurs during development occurs after cell division has ceased [1]. In fact, the major mechanism by which C. elegans worms grow postembryonically is by an increase in the size rather than the number of cells. Between hatching and the 1st day of adulthood there is an approximately 3.5-fold increase in the total number of nuclei (including germ-line nuclei) but a 32-fold increase in organismal volume [1]. Thus the growth in terms of volume in C. elegans is largely a result of an increase in the sizes of some or all cells. While there is little variation in the sizes of C. elegans worms grown under standard laboratory conditions, size is severely reduced when nutrients become limiting [2]. This reduction is observed even when individuals are starved after the time at which cells cease to divide implying that (not surprisingly) a scarcity of nutrients can affect cell growth as well as cell division. Almost all cells are thought to need exogenously supplied nutrients in order to grow. We can, therefore, expect that the reduced organismal size of worms grown in the presence of limiting nutrients is in part the result of cell autonomous effects on cell growth. In addition, however, the genetic analysis of body size in C. elegans, has revealed another layer of control exerted systemically [3]. Specifically, the characterization of body size mutants has revealed the existence of a signaling pathway that regulates growth and body size non-autonomously in response to nutrient status [2,3]. This pathway is separate from other systemically acting pathways that regulate development or metabolism.
A TGFβ pathway regulates body size Mutants with reduced body size were first isolated in the genetic screens for morphological mutants carried out by Sydney Brenner in the 1960s and 1970s [3]. Subsequent directed screens for small (Sma) and long (Lon) mutants, and the molecular characterization of the genes affected revealed that body size is regulated by a signaling pathway activated by a ligand in the TGFβ superfamily, DBL-1. Loss-of-function mutations in dbl-1 or in other genes in the pathway cause a marked (40%) reduction in both length and width throughout postembryonic development [4,5]. Increased DBL-1 signaling activity, on the other hand, causes worms to be more than 25% longer than wild type [5,6]. DBL-1 is highly similar to Decapentaplegic (Dpp) from Drosophila melanogaster, and to two vertebrate members of the bone morphogenetic protein family, BMP2 and BMP4. DBL-1 is expressed predominantly within specific parts of the nervous system notably in motor neurons in the ventral nerve cord (Fig. 1) [4,5]. Studies of the pathway that DBL-1 activates have led to significant advances in
EX P ER I ME NTAL C E LL RE S E ARCH
gonad
ciliated sensory neurons
ciliated sensory neuron ventral cord motor neurons
Fig. 1 – The positions of cells and tissues that modulate growth and body size systemically. DBL-1, a ligand in the TGFβ superfamily that regulates growth and body size in a dosedependent manner, is expressed predominantly in ventral cord motor neurons, coloured grey. Note that only a subset of such neurons is indicated and that although the paths depicted for their processes are representative, they are not strictly anatomically accurate. The focus of the pathway DBL-1 activates to control body size is the hypodermis, a part of which is shown in Fig. 2. The gonad (different shades of blue) inhibits the growth of somatic tissues. Ciliated sensory neurons in the head and tail promote growth. A subset of the 61 ciliated sensory neurons present is indicated (coloured brown). The pharynx, the feeding organ, is shaded green.
321 (2014) 71 –76
73
for lon-2 null mutations are more than 25% longer than wild type, a phenotype that is entirely dependent upon BMP pathway activity [13]. lon-2 encodes plasma-membrane anchored proteoglycan in the glypican family that can bind BMPs directly [6]. Recent intriguing results with LON-2 have shown that the protein contains two separate domains that both, independently, are able to inhibit DBL-1 activity [13]. drag-1 encodes the sole member of the repulsive guidance molecule (RGM) family of proteins in C. elegans [14]. Like part of LON-2, RGM family proteins are GPI-linked membraneassociated proteins. DRAG-1 is expressed in the hypodermis, which is also its focus of activity [14]. kin-29 encodes a serine/threonine kinase that may have more than one function in regulating body size [15,16]. Genetically, kin-29 acts downstream of dbl-1. However expression of kin-29 either under the control of dbl-1 promoter, which is principally active in neurons, or under the control of a promoter active in the cells in which DAF-4 and SMA-6 function can rescue the kin-29 body size defect [15], suggesting that kin-29 acts in both signaling and receiving cells. In yeast cells and in vitro, KIN-29 can bind TAX-6, the catalytic subunit of calcineurin [16].
The BMP pathway regulates endoreduplication of hypodermal nuclei the understanding of TGFβ–mediated signaling. For example, Smad proteins were first shown to be the downstream effectors of BMP receptors from studies of DBL-1 and Dpp signaling in C. elegans and Drosophila [3]. In target tissues, DBL-1 activates type I and type II coreceptors, SMA-6 and DAF-4, which are serine kinases [3]. Transduction of the signal to the nucleus requires SMA-2, SMA-3 and SMA-4, which are thought to function together as a complex to regulate the transcription of target genes [3]. SMA-2 and SMA-3 are homologous to receptorregulated Smad (R-Smad) proteins whereas SMA-4 is homologous to common-mediator Smads (Co-Smads). At least for SMA-3, receptor activity promotes accumulation of the protein in the nucleus [7]. Besides, sma-2, sma-3 and sma-4, sma-9 also functions genetically downstream of the BMP (DBL-1) pathway to regulate body size [8]. At least eight different transcripts are generated from the sma-9 locus most of which encode proteins homologous to Schnurri, a zinc-finger transcription factor that acts in the Dpp signaling pathway in the fly [8–10]. The SMAD complex may act together with a RUNX-family transcription factor, RNT-1. rnt-1 mutants are small and RNT-1 protein can bind SMA-4 in vitro [11].
Proteins modulating the DBL-1 signaling pathway Analyses of sma-10, lon-2, drag-1 and kin-29 have revealed that the proteins they encode act by modulating the BMP pathway regulating body size. sma-10 null mutations cause a Sma phenotype very similar to that caused by null mutations in dbl-1 [12]. SMA-10 encodes a widely-expressed protein with leucine-rich repeats and immunoglobulin-like domains (LRIG) that is found in puncta both at the plasma membrane and within cells [12]. In a heterologous system, SMA-10 binds directly to the receptors, SMA-6 and DAF-4, and stimulates their activity [12]. The Drosophila orthologue, lambik, rescues sma-10 phenotypes showing that sma-10 function has been conserved in evolution [12]. Worms homozygous
Consistent with the observation that a large proportion of the increase in volume that occurs during wild-type C. elegans development is the result of increases in cell size rather than cell number, mutations that cause a decrease in the activity of the BMP pathway appear to reduce body size by reducing the sizes of some or all cells rather than their number [4,5]. Although the pathway does have a function in the control of development in the male tail [17], only a small number of cells are affected and the mutants have almost wild-type numbers of cells. Likewise, lon-2 mutants or those with elevated dbl-1 gene copy number are longer than wild type but do not have more cells [6]. Genetic mosaic analysis indicates that a major focus of activity of the receptor with respect to body size (at least in adult worms) is in syncytial hypodermal cells that surround the internal tissues (and which secrete the cuticle) (Fig. 2) [3]. The pathway acts within these cells in part by affecting the process of endoreduplication [18]. In wild type, many of the nuclei in the hypodermal syncytia undergo two or three rounds of endoreduplication so that in 3-day old adults these nuclei have an average ploidy of 12. In mutants with reduced BMP signaling, however, the hypodermal nuclei undergo fewer rounds of endoreduplication with the result that their average ploidy is only 2/3 that in wild type [18]. Body size and hypodermal ploidy is also reduced in worms mutant for CRM-1, a C. elegans homologue of vertebrate Crim1, a transmembrane protein with multiple cysteine rich domains [19]. Blocking endoreduplication in adult wild-type worms by treatment with hydroxyurea (which blocks S phase) leads to a reduction in body size [20]. However, dbl-1 mutants treated with hydroxyurea are not further reduced in size strongly suggesting that the reduced body size of BMP mutants is the result of reduced endoreduplication rather than vice versa [20]. Thus, while in many species growth is controlled by regulation of cell division, which entails replication of chromosomes, nuclear division and then finally cytokinesis, growth in adult C. elegans worms is associated with rounds of chromosomal duplication within certain cells that occurs in the absence of nuclear division or cytokinesis.
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cuticle
annuli
hypodermis
seam cells
ventral nerve cord
furrows
Fig. 2 – Diagram showing part of the hypodermis and cuticle. The hypodermis, shaded yellow in cross-section, is made up of a 12 syncytial hypodermal cells, which together contain 157 nuclei in the adult. These hypodermal cells secrete the cuticle (shaded grey). Circumferential rings called annuli run almost the entire length of the cuticle. Between adjacent annuli are furrows, which form immediately above circumferential bundles of actin (not shown) present in hypodermal cells during embryogenesis and molting. The annuli are discontinuous and contain gaps above two rows of cells termed seam cells (coloured orange) that run along the left and right sides. The position of ventral nerve cord (coloured green) is indicated.
Studies on other species of nematode have shown that a number are not eutelic. In particular, different individuals of the same species can have different numbers of hypodermal nuclei [21]. However, between different species, no strong correlation exists between the average number of hypodermal nuclei and body size. It is possible, therefore, that within these species, body size is determined in part by the ploidy of hypodermal nuclei rather than their number. However, other mechanisms very likely also influence growth. Within C. elegans, while effects on ploidy of hypodermal cells undoubtedly account for part of the reduced size of the BMP mutants, they do not account for all of it: the mutants are reduced in size even during larval stages before most hypodermal endoreduplication has occurred.
Gonad and nutrient signals modulate body size Fundamental studies within evolutionary biology have shown that trade-offs exists between the germ line and the soma that can affect different aspects of the soma including its size. In almost all organisms tested to date (including humans), reducing the size of the germ line by surgical or genetic manipulation, leads to increased lifespan and fat storage, and to significantly increased body size. In keeping with the studies on other organisms, ablation of the gonad (depicted in Fig. 1) in C. elegans and some other nematodes also leads to a dramatic increase in body size [22] and lifespan. In C. elegans, the increase in size is not caused by changes in insulin signaling (which account for the increase in longevity) but by a different (as-yetunknown) mechanism. Gonadectomy of dbl-1 mutant worms also results in an increase in body size implying that the gonad must signal, at least in part, independently of BMP pathway.
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A worm subjected to dietary restriction will fail to grow to its proper size even if it is starved after the time at which somatic cell division has ceased. The reduced body size of worms subjected to dietary restriction is associated with reduced BMP signaling: dietary restriction reduces endoreduplication of hypodermal nuclei in wildtype worms but has much less effect on body size and endoreduplication in dbl-1 mutants [2]. A number of studies indicate, however, that in addition to DBL-1, other signals likely regulate body size in response to nutrient sensation. First, che-2, che-3, egl-4, osm-6, tax-2 and tax-4 gene activities have all been shown to be required in a group of sensory neurons in the head and tail (in which no dbl-1 expression has been observed) for worms to grow to the proper size (Fig. 1) [23]. che-3 and osm-6 are required for the formation of the sensory cilia in 61 ciliated sensory neurons through which worms sense chemicals in (and temperature of) their environment; che-2, egl-4, tax-2 and tax-4 encode proteins that directly mediate chemo- or thermosensation. Other proteins required for correct cilia formation such as certain members of the Bardet-Biedl syndrome (BBS) family of proteins are also required for worms to grow to their proper size [24]. ASNA-1, an ATPase is required in certain sensory neurons to promote cell growth [25]. Although ASNA-1 stimulates the secretion of insulin/insulin-like proteins, ASNA-1’s effect on body size does not appear to be mediated via insulin signaling [25]. Mutations in cnb-1 and tax-6, which encode the regulatory and catalytic subunits of calcineurin respectively can also affect sensory neuron function and body size [16,26]. Together, these studies strongly suggest that, in response to chemosensory cues, ciliated sensory neurons secrete a factor (or factors) that promote growth. Although, such a factor has yet to be identified, EGL-4, a cyclic GMP-dependent protein kinase, might negatively regulate its secretion. Whereas mutations affecting cilia structure, asna-1 or the chemosensory receptor gene, che-2, reduce body size, loss-of-function mutations in egl-4 increase body size [23,27].
The effect of cuticle structure on body size One way in which the BMP pathway affects growth in terms of length and morphology is by directly or indirectly regulating components of the cuticle (part of which is depicted in Fig. 2). Microarray analysis has revealed that the expression of several cuticle collagen genes is altered in mutants with elevated or reduced pathway activity [9,28,29]. Collagens make up over 50% of the cuticle by mass and the C. elegans genome contains 158 cuticle collagen genes [30]. In keeping with its vital function in acting as a barrier to the exterior, and with this surprisingly large number of cuticle collagen genes, the cuticle has a very complex, multi-layered structure; different collagens are found in the different layers. A small subset of the collagens appears to be particularly important for proper organismal length and morphology. Null or reduction-of-function mutations in dpy-2, dpy-3, dpy-5, dpy-7, dpy-8, dpy-10, dpy-13, and sqt-3 all cause a Dpy phenotype (in which length is reduced but width is essentially normal) similar to some hypomorphic BMP pathway mutations [30]. On the other hand, null mutations in just one cuticle collagen, lon-3, cause a Lon phenotype strikingly similar to that caused by elevated levels of dbl-1 [31,32]. The expression of a LON-3::GFP fusion protein is reduced in worms with increased dbl-1 gene copy number [32]. Furthermore, increasing lon-3 gene copy number reduces body length [31].
EX P ER I ME NTAL C E LL RE S E ARCH
Besides LON-3, two other lon proteins, LON-1 and LON-8, likely affect body size by directly or indirectly affecting the cuticle. LON-1 is a cysteine-rich secretory protein that is expressed in hypodermal cells [3]. lon-1 gene expression is inhibited by the BMP pathway [15]. lon-8 encodes a novel secreted protein that is highly conserved throughout nematode evolution [33]. LON-8 is expressed in hypodermal cells, and lon-8 mutations interact strongly genetically with those in dpy-11 and dpy-18, which encode collagen-modifying enzymes. LON-8 is thought to affect body length independently of the BMP pathway [33]. Mutations in adt-2 cause a Sma phenotype but are also thought to affect body size by affecting the cuticle. ADT-2 is a member of the disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted metalloproteases [34]. adt-2 mutations also affect dbl-1 gene transcription suggesting that the cuticle can signal back to the BMP pathway in some way [34].
The actin cytoskeleton in hypodermal cells has profound effects on organismal shape sma-1 encodes the βΗ-spectrin orthologue and is found on the apical surfaces of polarized epithelial cells including hypodermal cells. SMA-1 protein is required to maintain the association between the actin cytoskeleton and apical membranes [35]. During embryogenesis in wild-type larvae, following enclosure, cortical arrays of actin within epidermal cells, dramatically reorganize to form parallel circumferential bundles close to the apical surfaces. The transition from the stocky, so-called comma-stage embryo resulting from enclosure to the lithe first larval (L1) hatchling is driven by dramatic changes in shape of epithelial cells. The cells elongate along the anteriorposterior axis lengthways while at the same time the circumferential bundles of actin within them constrict; the vermiform shape is thus the result of the cells in the middle of the embryo being squeezed out at either end by the epithelial cells tightening around them. In sma-1 mutants, the actin cytoskeleton dissociates from the apical membrane, and, as a result, the extension of the epithelial cells is incomplete. The resulting larva is then considerably shorter than normal [35]. Interestingly, the function of the circumferential rings of actin in maintaining organismal morphology appears not to be restricted to embryonic development but may also be important during larval development. During postembryonic stages, circumferential ridges in the cuticle termed annuli are present along the entire length of the worm (Fig. 2). The troughs (furrows) between these ridges coincide precisely with the circumferential actin bundles that reform periodically during molting [30]. In animals homozygous for mutations in dpy-2, dpy-3, dpy-7, dpy-8 or dpy-10 the annuli are absent suggesting perhaps that the cuticle collagens they encode are required for linking parts of the cuticle to the apical membranes of hypodermal cells adjacent to the positions of the circumferential actin bundles. DPY-7 and DPY-10 are known to be located in the furrows of the annuli [30].
TFG-1 and CED-4 have antagonistic effects on cell growth and apoptosis Mutations in tgf-1 have been described that profoundly affect body size by reducing cell growth through a BMP-independent mechanism. tfg-1 encodes the C. elegans orthologue of the human
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proto-oncogene, TRK-fused gene. Reducing the function of tfg-1 in worms by RNAi results in a 70% reduction in body size caused by a reduction in cell size rather than cell number [36]. Complete loss of tfg-1 activity leads to ectopic activation of the programmed cell death pathway. Remarkably, inactivation of a gene in this pathway, ced-4 efficiently suppresses the tfg-1 size defects. Thus, tfg-1 and ced-4 link pathways regulating cell growth and cell death.
Other genes required for growth sma-5 encodes an ERK5/MAPK7/BMK1 MAP kinase homologue that is required for many cells in the worm to grow to their proper sizes [37]. Within the hypodermis, sma-5 functions cell autonomously but the gene can also affect body size non-autonomously [37]. Mutants lacking the activity of RICT-1, a component of the TORC2 complex, are also smaller [38]. ncl-1, which encodes a homologue of Brain tumour, a Drosophila tumour suppressor, acts cell autonomously to control cell and nucleolar size by negatively regulating the transcription of ribosomal and 5S RNAs [39]. Finally, mutants lacking bec-1 or unc-51, which are required for autophagy, are smaller than wild type [40] implying that autophagy is required for normal growth.
Future studies Considerable progress has been made over the past 15 years in understanding how cell growth and body size are regulated in C. elegans. Many interesting questions remain, however. How is the synthesis, secretion or activity of DBL-1 regulated in response to nutritional cues? How does the BMP pathway regulate endoreduplication of hypodermal nuclei? Does the hypodermis promote the growth of other tissues and, if so, how? The mechanism by which the germ line can affect body size is presently not well understood. Since the trade-offs that exist between the germ line and the soma is known to be of fundamental importance for evolutionary fitness, it is important to understand how the germ line influences the size of the soma. Among other outstanding questions are how ciliated sensory neurons modulate body size, how the actin cytoskeleton within hypodermal cells is regulated and how, in turn, it affects the elasticity of the cuticle. Lastly, it should not be forgotten that an organism's size is of central importance to its ecology. With the exception of the air, nematodes are ubiquitous in most ecological niches. In terms of numbers of individuals, they are by far the most abundant type of animal on the planet. Nematode species (of which around 24,000 have been described to date) vary in length from fractions of a millimeter to greater than 8 m. Research with C. elegans has provided important mechanistic insights into how growth is regulated, and likely will continue to do so in the future. In addition, the results obtained with C. elegans provide a platform for studies aimed at understanding how growth and size in other members of this important and varied group of animals is controlled.
Conflict of interest None.
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Acknowledgments Work in my laboratory is supported by grants from Cancerfonden and Vetenskapsrådet. I apologize to authors whose work on growth and body size, for lack of space, I was unable to cite directly.
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