Multicellularity makes somatic differentiation evolutionarily stable Mary E. Wahla,b,1 and Andrew W. Murraya,b,2 aDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; and bFAS Center for Systems Biology, Harvard University, Cambridge, MA 02138 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Contributed by Andrew W. Murray, June 15, 2016 (sent for review September 13, 2015; reviewed by Harmit S. Malik and Boris I. Shraiman) Many multicellular organisms produce two cell lineages: germ cells, through lineage sequestration (12) or reduced oxidative stress whose descendants produce the next generation, and somatic cells, (13), are thus likely accessible to unicellular species. which support, protect, and disperse the germ cells. This germ-soma We propose the alternative hypothesis that unicellular somatic demarcation has evolved independently in dozens of multicellular differentiation can offer fitness benefits in a population of ge- taxa but is absent in unicellular species. A common explanation holds netically identical cells but remains rare because it is not an that in these organisms, inefficient intercellular nutrient exchange evolutionarily stable strategy (14). Commonly occurring mutants compels the fitness cost of producing nonreproductive somatic cells that do not differentiate (“cheats”) could take advantage of somatic to outweigh any potential benefits. We propose instead that the cell products in the shared media without paying the reproductive absence of unicellular, soma-producing populations reflects their costs of differentiation, thus increasing in frequency until their ge- susceptibility to invasion by nondifferentiating mutants that ulti- notype prevails. We also posit that if multicellularity results from mately eradicate the soma-producing lineage. We argue that multi- cells of a single lineage failing to disperse (rather than cells aggre- cellularity can prevent the victory of such mutants by giving germ gating from different lineages), differentiating populations can cells preferential access to the benefits conferred by somatic cells. The outcompete cheats: although cheats initially arise through mutation absence of natural unicellular, soma-producing species previously in a group with somatic cells (which the cheats can exploit), lineage- prevented these hypotheses from being directly tested in vivo: to restricted propagation forces the cheat’s descendants to be confined overcome this obstacle, we engineered strains of the budding yeast to multicellular groups composed entirely of cheats, which thus Saccharomyces cerevisiae that differ only in the presence or absence cannot benefit from the local accumulation of somatic cell products of multicellularity and somatic differentiation, permitting direct com- (15–17). This hypothesis invokes the demonstrated ability of pop- parisons between organisms with different lifestyles. Our strains im- ulation structure to maintain altruistic traits (15, 18, 19). plement the essential features of irreversible conversion from germ To test the evolutionary stability of germ-soma differentiation, line to soma, reproductive division of labor, and clonal multicellularity we designed strains of the budding yeast Saccharomyces cerevisiae while maintaining sufficient generality to permit broad extension of that produce soma, are multicellular, or combine both traits: one our conclusions. Our somatic cells can provide fitness benefits that strain is a multicellular, differentiating organism and the other two exceed the reproductive costs of their production, even in unicellular represent both possible intermediates in its evolution from a strains. We find that nondifferentiating mutants overtake unicellular nondifferentiating, unicellular ancestor (Fig. 1A). Using synthetic populations but are outcompeted by multicellular, soma-producing strains that differ from one another at only a few, well-defined loci strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. Significance evolution | multicellularity | differentiation | synthetic biology | yeast Unicellular species lack the nonreproductive somatic cell types that characterize complex multicellular organisms. We consider omatic differentiation, a permanent change in gene expression two alternative explanations: first, that the costs of lost re- ’ Sinherited by all of a cell s descendants, produces somatic cells productive potential never exceed the benefits of somatic cells in from a totipotent germ line. Although somatic cells may divide unicellular organisms; and second, that somatic cells may profit a indefinitely, they cannot beget the complete organism and are thus unicellular population but leave it vulnerable to invasion by considered nonreproductive. Generating such sterile cells has clear common mutants. We test these hypotheses using engineered fitness costs that must be offset by somatic functions that improve yeast strains that permit direct comparisons of fitness and evo- the viability or fecundity of germ cells. The absence of a soma in lutionary stability between lifestyles. We find that the benefits of unicellular species (1), as well as the persistence of undifferentiated somatic cell production can exceed the costs in unicellular strains. multicellular groups among the volvocine algae (2) and cyano- Multicellular, soma-producing strains resist invasion by non- differentiating mutants that overtake unicellular populations, bacteria (3), has fueled speculation that multicellularity must arise – supporting the theory that somatic differentiation is stabilized by before somatic differentiation can evolve (4 7). It has been argued population structure imposed by multicellularity. that somatic differentiation is not observed in unicellular species because the fitness benefits of somatic cells can never exceed the Author contributions: M.E.W. and A.W.M. designed research; M.E.W. performed research; cost of making them (6–8): although soma can contribute motility M.E.W. analyzed data; and M.E.W. and A.W.M. wrote the paper. and protective structures to multicellular organisms, somatic cells Reviewers: H.S.M., Fred Hutchinson Cancer Research Center; and B.I.S., University of Cal- ifornia, Santa Barbara. in a unicellular species can only benefit the germ line by secreting The authors declare no conflict of interest. useful products into a shared extracellular milieu. However, nutrient exchange between members of microbial consortia (9, Freely available online through the PNAS open access option. 1Present address: Microsoft New England Research and Development Center, Cambridge, 10) demonstrates the potential for productive interactions be- MA 02142. tween cell types in the absence of physical adhesion. Benefits 2To whom correspondence should be addressed. Email: [email protected]. associated with somatic differentiation, including reproductive This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. division of labor (11) and suppression of germ-line mutations 1073/pnas.1608278113/-/DCSupplemental. 8362–8367 | PNAS | July 26, 2016 | vol. 113 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1608278113 Downloaded by guest on September 30, 2021 ensures that no undesired variables confound the direct comparison sucrose minimal media. In nature, differentiation is often achieved of fitness and evolutionary stability and permits the study of a uni- through multiply redundant gene regulatory networks that stabilize INAUGURAL ARTICLE cellular, differentiating lifestyle not found in nature. Experiments on cell fate (22): to simplify our system, we instead made differenti- living organisms also avoid the potential pitfall of biologically un- ation permanent and heritable by making the expression of somatic realistic parameter regimes in simulations or purely analytical cell-specific genes depend on a site-specific recombinase that ex- models. Our results show that production of soma can be advan- cises a gene needed for rapid cell proliferation (Fig. 1C). tageous even in unicellular populations, although these are sus- Germ and somatic cells must be present at a suitable ratio for ceptible to invasion by nondifferentiating cheats, which multicellular growth on sucrose: germ cells have the higher maximum growth populations effectively repel. We therefore speculate that multi- rate, but monosaccharides become limiting when somatic cells are cellularity facilitates the evolution of somatic differentiation prin- rare. We predicted that the ratio between cell types would reach a cipally by providing stability against common mutants that prevent steady-state value reflecting the balance between unidirectional differentiation. conversion of germ cells into somatic cells and the restricted di- vision of somatic cells. We designed tunable differentiation and Construction of a Synthetic Differentiation System division rates to allow us to regulate the ratio between cell types We mimicked somatic differentiation by engineering fast-growing and thus control the growth rate of the culture as a whole. “germ” cells that can give rise to slower dividing, differentiated Both features depend on a single, genetically engineered locus “somatic” cells that secrete invertase (Suc2), an enzyme that hy- (Fig. 1C). In germ cells, this locus expresses the fluorescent protein drolyzes sucrose (which laboratory yeast strains cannot take up mCherry and a gene that