Environmentally induced responses co-opted for reproductive altruism

Reproductive altruism is an extreme form of altruism best typified by sterile castes in social insects and somatic cells in multicellular organisms. Although reproductive altruism is central to the evolution of multicellularity and eusociality, the mechanistic basis for the evolution of this behaviour is yet to be deciphered. Here, we report that the gene responsible for the permanent suppression of reproduction in the somatic cells of the multicellular green alga, Volvox carteri, evolved from a gene that in its unicellular relative, Chlamydomonas reinhardtii, is part of the general acclimation response to various environmental stress factors, which includes the temporary suppression of reproduction. Furthermore, we propose a model for the evolution of soma, in which by simulating the acclimation signal (i.e. a change in cellular redox status) in a developmental rather than environmental context, responses beneficial to a unicellular individual can be co-opted into an altruistic behaviour at the group level. The co-option of environmentally induced responses for reproductive altruism can contribute to the stability of this behaviour, as the loss of such responses would be costly for the individual. This hypothesis also predicts that temporally varying environments, which will select for more efficient acclimation responses, are likely to be more conducive to the evolution of reproductive altruism.     

EARLY EVOLUTION OF THE GENETIC BASIS FOR SOMA IN THE VOLVOCACEAE

To understand the hierarchy of life in evolutionary terms, we must explain why groups of one kind of individual, say cells, evolve into a new higher level individual, a multicellular organism. A fundamental step in this process is the division of labor into nonreproductive altruistic soma. The regA gene is critical for somatic differentiation in Volvox carteri, a multicellular species of volvocine algae. We report the sequence of regA‐like genes and several syntenic markers from divergent species of Volvox. We show that regA evolved early in the volvocines and predict that lineages with and without soma descended from a regA‐containing ancestor. We hypothesize an alternate evolutionary history of regA than the prevailing “proto‐regA” hypothesis. The variation in presence of soma may be explained by multiple lineages independently evolving soma utilizing regA or alternate genetic pathways. Our prediction that the genetic basis for soma exists in species without somatic cells raises a number of questions, most fundamentally, under what conditions would species with the genetic potential for soma, and hence greater individuality, not evolve these traits. We conclude that the evolution of individuality in the volvocine algae is more complicated and labile than previously appreciated on theoretical grounds.     

Genetic basis for soma is present in undifferentiated volvocine green algae

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co‐opted to produce soma. Determining that the reg cluster was co‐opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.     

Genetic control of somatic cell differentiation in Volvox analysis of somatic regenerator mutants

The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirty-nine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvox spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.     

Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in Dictyostelium

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2⁻ and gα4⁻ cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2⁻ mutants. The regA⁻ mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4-mediated folate chemotaxis. However, the regA gene disruption in gα4⁻ cells, but not in gα2⁻ cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA⁻ strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4⁻regA⁻ strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4^HC (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA⁻ associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.     

Genetic interactions of the E3 ubiquitin ligase component FbxA with cyclic AMP metabolism and a histidine kinase signaling pathway during Dictyostelium discoideum development

Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.     

microRNAs in a multicellular green alga Volvox carteri

microRNAs(miRNAs)have emerged as key components in the eukaryotic gene regulatory network.We and others have previously identified many miRNAs in a unicellular green alga,Chlamydomonas reinhardtii.To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage,we examined small RNAs in Volvox carteri,a multicellular species in the same family with Chlamydomonas reinhardtii.We identified 174 miRNAs in Volvox,with many of them being highly enriched in gonidia or somatic cells.The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo,suggesting that miRNAs play regulatory roles in the biology of green algae.Our catalog of miRNAs and their targets provides a resource for further studies on the evolution,biological functions,and genomic properties of miRNAs in green algae.     

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

Organizational requirements for multicellular autonomy: insights from a comparative case study

In this paper we explore the organizational conditions underlying the emergence of organisms at the multicellular level. More specifically, we shall propose a general theoretical scheme according to which a multicellular organism is an ensemble of cells that effectively regulates its own development through collective (meta-cellular) mechanisms of control of cell differentiation and cell division processes. This theoretical result derives from the detailed study of the ontogenetic development of three multicellular systems (Nostoc punctiforme, Volvox carteri and Strongylocentrotus purpuratus) and, in particular, of their corresponding cell-to-cell signaling networks. The case study supports our claim that a specific type of functional integration among the cells of a multicellular ensemble (namely, a regulatory control system consisting in several inter-cellular mechanisms that modulate epigenesis and whose operation gets decoupled from the intra-cellular metabolic machinery), is required for it to qualify as a proper organism. Finally, we argue why a multicellular system exhibiting this type of functionally differentiated and integrated developmental organization becomes a self-determining collective entity and, therefore, should be considered as a second-order autonomous system.     

Evolution of reproductive development in the volvocine algae

The evolution of multicellularity, the separation of germline cells from sterile somatic cells, and the generation of a male–female dichotomy are certainly among the greatest innovations of eukaryotes. Remarkably, phylogenetic analysis suggests that the shift from simple to complex, differentiated multicellularity was not a unique progression in the evolution of life, but in fact a quite frequent event. The spheroidal green alga Volvox and its close relatives, the volvocine algae, span the full range of organizational complexity, from unicellular and colonial genera to multicellular genera with a full germ–soma division of labor and male–female dichotomy; thus, these algae are ideal model organisms for addressing fundamental issues related to the transition to multicellularity and for discovering universal rules that characterize this transition. Of all living species, Volvox carteri represents the simplest version of an immortal germline producing specialized somatic cells. This cellular specialization involved the emergence of mortality and the production of the first dead ancestors in the evolution of this lineage. Volvocine algae therefore exemplify the evolution of cellular cooperation from cellular autonomy. They also serve as a prime example of the evolution of complex traits by a few successive, small steps. Thus, we learn from volvocine algae that the evolutionary transition to complex, multicellular life is probably much easier to achieve than is commonly believed.     

Two Different Rickettsial Bacteria Invading Volvox carteri

BackgroundBacteria of the family Rickettsiaceae are principally associated with arthropods. Recently, endosymbionts of the Rickettsiaceae have been found in non-phagotrophic cells of the volvocalean green algae Carteria cerasiformis, Pleodorina japonica, and Volvox carteri. Such endosymbionts were present in only C. cerasiformis strain NIES-425 and V. carteri strain UTEX 2180, of various strains of Carteria and V. carteri examined, suggesting that rickettsial endosymbionts may have been transmitted to only a few algal strains very recently. However, in preliminary work, we detected a sequence similar to that of a rickettsial gene in the nuclear genome of V. carteri strain EVE.Conclusion/SignificanceAt least two different rickettsial organisms may have invaded the V. carteri lineage, one of which may be the direct ancestor of the endosymbiont of V. carteri strain UTEX 2180, whereas the other may be closely related to the endosymbiont of P. japonica. Endosymbiotic gene transfer from the latter rickettsial organism may have occurred in an ancestor of V. carteri. Thus, the rickettsiae may be widely associated with V. carteri, and likely have often been lost during host evolution.     

Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

Spontaneous frequency of a developmental mutant in Volvox

Two types of mutants, those resistant to the base analog 5-bromo-2′-deoxyuridine (BrdU) and somatic regenerator (SR) mutants, have been analyzed in Volvox carteri. In somatic regenerator mutants, the somatic cells which are normally terminally differentiated dedifferentiate and regenerate gonidia [Sessoms, A., and Huskey, R. J. (1973). Proc. Nat. Acad. Sci. USA70, 1335–1338; Starr, R. C. (1970). Develop. Biol. Suppl.4, 59–100]. The SR phenotype allows recovery of SR mutations arising in somatic cells, since such somatic cells would regenerate gonidia and give rise to mutant clones. Mutants of any phenotype other than SR can only be recovered if the mutation first appears in a gonidium. Since the somatic cells are 100-fold more numerous than reproductive cells (gonidia), we have determined the spontaneous frequency of both somatic regenerator mutants and mutations to BrdU resistance in order to determine if the SR mutation exerts its effect in the gonidium or in the somatic cell. The two frequencies were found to be nearly identical, suggesting that the SR mutation must first appear in a gonidium in order to be expressed.