Adenylate cyclase A amplification and functional diversification during <i>Polyspondylium pallidum</i> development

BACKGROUND: In Dictyostelium discoideum (Ddis), adenylate cyclase A (ACA) critically generates the cAMP oscillations that coordinate aggregation and morphogenesis. Unlike group 4 species like Ddis, other groups do not use extracellular cAMP to aggregate. However, deletion of cAMP receptors (cARs) or extracellular phosphodiesterase (PdsA) in Polyspondylium pallidum (Ppal, group 2) blocks fruiting body formation, suggesting that cAMP oscillations ancestrally control post-aggregative morphogenesis. In group 2, the acaA gene underwent several duplications. We deleted the three Ppal aca genes to identify roles for either gene and tested whether Ppal shows transient cAMP-induced cAMP accumulation, which underpins oscillatory cAMP signalling. RESULTS: In contrast to Ddis, pre-aggregative Ppal cells did not produce a pulse of cAMP upon stimulation with the cAR agonist 2′H-cAMP, but acquired this ability after aggregation. Deletion of Ppal aca1, aca2 and aca3 yielded different phenotypes. aca1ˉ cells showed relatively thin stalks, aca2ˉ showed delayed secondary sorogen formation and aca3ˉ formed less aggregation centers. The aca1ˉaca2ˉ and aca1ˉaca3ˉ mutants combined individual defects, while aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ additionally showed > 24 h delay in aggregation, with only few aggregates with fragmenting streams being formed. The fragments developed into small fruiting bodies with stalk and spore cells. Aggregation was restored in aca2ˉaca3ˉ and aca1ˉaca3ˉaca2ˉ by 2.5 mM 8Br-cAMP, a membrane-permeant activator of cAMP-dependent protein kinase (PKA). Like Ddis, Ppal sorogens also express the adenylate cyclases ACR and ACG. We found that prior to aggregation, Ddis acaˉ/ACG cells produced a pulse of cAMP upon stimulation with 2′H-cAMP, indicating that cAMP oscillations may not be dependent on ACA alone. CONCLUSIONS: The three Ppal replicates of acaA perform different roles in stalk morphogenesis, secondary branch formation and aggregation, but act together to enable development by activating PKA. While even an aca1ˉaca3ˉaca2ˉ mutant still forms (some) fruiting bodies, suggesting little need for ACA-induced cAMP oscillations in this process, we found that ACG also mediated transient cAMP-induced cAMP accumulation. It, therefore, remains likely that post-aggregative Ppal morphogenesis is organized by cAMP oscillations, favouring a previously proposed model, where cAR-regulated cAMP hydrolysis rather than its synthesis dominates oscillatory behaviour. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13227-022-00203-7

Autophagy of the somatic stalk cells likely nurses the propagating spores of Dictyostelid social amoebas [version 2; peer review: 1 approved, 2 approved with reservations]

Background: Autophagy (self-feeding) assists survival of starving cells by partial self-digestion, while dormancy as cysts, spores or seeds enables long-term survival. Starving Dictyostelium amoebas construct multicellular fruiting bodies with spores and stalk cells, with many Dictyostelia still able to encyst individually like their single-celled ancestors. While autophagy mostly occurs in the somatic stalk cells, autophagy gene knock-outs in Dictyostelium discoideum ( D. discoideum) formed no spores and lacked cAMP induction of prespore gene expression. Methods: To investigate whether autophagy also prevents encystation, we knocked-out autophagy genes atg5 and atg7 in the dictyostelid Polysphondylium pallidum, which forms both spores and cysts. We measured spore and cyst differentiation and viability in the knock-out as well as stalk and spore gene expression and its regulation by cAMP. We tested a hypothesis that spores require materials derived from autophagy in stalk cells. Sporulation requires secreted cAMP acting on receptors and intracellular cAMP acting on PKA. We compared the morphology and viability of spores developed in fruiting bodies with spores induced from single cells by stimulation with cAMP and 8Br-cAMP, a membrane-permeant PKA agonist. Results: Loss of autophagy in P. pallidum reduced but did not prevent encystation. Stalk cells still differentiated but stalks were disorganised. However, no spores were formed at all and cAMP-induced prespore gene expression was lost. D. discoideum spores induced in vitro by cAMP and 8Br-cAMP were smaller and rounder than spores formed multicellularly and while they were not lysed by detergent they germinated not (strain Ax2) or poorly (strain NC4), unlike spores formed in fruiting bodies. Conclusions: The stringent requirement of sporulation on both multicellularity and autophagy, which occurs mostly in stalk cells, suggests that stalk cells nurse the spores through autophagy. This highlights autophagy as a major cause for somatic cell evolution in early multicellularity

Evolution of Multicellular Complexity in The Dictyostelid Social Amoebas

This article belongs to the Special Issue Evolution of Multicellularity. Academic Editor: J. Mark Cock.Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as “proto-cell types” for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the “proto-cell type” gene regulatory networks.This work was supported by the European Research Council [742288], The Wellcome Trust [100293/Z/12/Z], the European Molecular Biology Organisation [ALTF 295-2015], and the Japanese Organisation for the Promotion of Science [H28-1002].Peer reviewe

A core phylogeny of Dictyostelia inferred from genomes representative of the eight major and minor taxonomic divisions of the group

BACKGROUND: Dictyostelia are a well-studied group of organisms with colonial multicellularity, which are members of the mostly unicellular Amoebozoa. A phylogeny based on SSU rDNA data subdivided all Dictyostelia into four major groups, but left the position of the root and of six group-intermediate taxa unresolved. Recent phylogenies inferred from 30 or 213 proteins from sequenced genomes, positioned the root between two branches, each containing two major groups, but lacked data to position the group-intermediate taxa. Since the positions of these early diverging taxa are crucial for understanding the evolution of phenotypic complexity in Dictyostelia, we sequenced six representative genomes of early diverging taxa. RESULTS: We retrieved orthologs of 47 housekeeping proteins with an average size of 890 amino acids from six newly sequenced and eight published genomes of Dictyostelia and unicellular Amoebozoa and inferred phylogenies from single and concatenated protein sequence alignments. Concatenated alignments of all 47 proteins, and four out of five subsets of nine concatenated proteins all produced the same consensus phylogeny with 100% statistical support. Trees inferred from just two out of the 47 proteins, individually reproduced the consensus phylogeny, highlighting that single gene phylogenies will rarely reflect correct species relationships. However, sets of two or three concatenated proteins again reproduced the consensus phylogeny, indicating that a small selection of genes suffices for low cost classification of as yet unincorporated or newly discovered dictyostelid and amoebozoan taxa by gene amplification. CONCLUSIONS: The multi-locus consensus phylogeny shows that groups 1 and 2 are sister clades in branch I, with the group-intermediate taxon D. polycarpum positioned as outgroup to group 2. Branch II consists of groups 3 and 4, with the group-intermediate taxon Polysphondylium violaceum positioned as sister to group 4, and the group-intermediate taxon Dictyostelium polycephalum branching at the base of that whole clade. Given the data, the approximately unbiased test rejects all alternative topologies favoured by SSU rDNA and individual proteins with high statistical support. The test also rejects monophyletic origins for the genera Acytostelium, Polysphondylium and Dictyostelium. The current position of Acytostelium ellipticum in the consensus phylogeny indicates that somatic cells were lost twice in Dictyostelia. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12862-016-0825-7) contains supplementary material, which is available to authorized users