Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.Choanoflagellates have long fascinated evolutionary biologists for their marked similarity to the 'feeding cells' (choanocytes) of sponges and the possibility that they might represent the closest living relatives of metazoans 1,2 . Over the past decade or so, evidence supporting this relationship has accumulated from phylogenetic analyses of nuclear and mitochondrial genes [3][4][5][6] , comparative genomics between the mitochondrial genomes of choanoflagellates, sponges and other metazoans 7,8 , and the finding that choanoflagellates express homologues of metazoan signalling and adhesion genes 9-12 . Furthermore, species-rich phylogenetic analyses demonstrate that choanoflagellates are not derived from metazoans, but instead represent a distinct lineage that evolved before the origin and diversification of metazoans (Fig. 1a, Supplementary Fig. 1 and Supplementary Note 3.1) 8,13 . By virtue of their position on the tree of life, studies of choanoflagellates provide an unparallelled window into the nature of the unicellular and colonial progenitors of metazoans 14 .Choanoflagellates are abundant and globally distributed microbial eukaryotes found in marine and freshwater environments 15,16 . Like sponge choanocytes, each cell bears an apical flagellum surrounded by a distinctive collar of actin-filled microvilli, with which choanoflagellates trap bacteria and detritus (Fig. 1b). Using this highly effective means of prey capture, choanoflagellates link bacteria to higher trophic levels and thus have critical roles in oceanic carbon cycling and in the microbial food web 17,18 .More than 125 choanoflagellate species have been identified, and all species have a unicellular life-history stage. Some can also form simple colonies of equipotent cells, although these differ substantially from the obligate associations of differentiated cells in metazoans 19 . Studies of basal metazoans indicate that the ancestral metazoan was multicellular and had differentiated cell types, an epithelium, a body plan and regulated development including gastrulation. In contrast, the last common ancestor of choanoflagellates and metazoans was unicellular or possibly capable of forming simple colonies, underscoring the abundant biologi...
SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling.
Cadherin-mediated cell adhesion and signaling is essential for metazoan development and yet is absent from all other multicellular organisms. We found cadherin genes at numbers similar to those observed in complex metazoans in one of the closest single-celled relatives of metazoans, the choanoflagellate Monosiga brevicollis. Because the evolution of metazoans from a single-celled ancestor required novel cell adhesion and signaling mechanisms, the discovery of diverse cadherins in choanoflagellates suggests that cadherins may have contributed to metazoan origins.
The morphological and physiological diversity of animals, fungi, plants, and other multicellular organisms stems from the fact that each lineage acquired multicellularity independently during the course of evolution. A prerequisite for the origin of multicellularity in each lineage was the evolution of mechanisms for stable cell-cell adhesion or attachment. Recent advances in genomics and phylogenetics allow comparative studies that provide critical insights into the evolutionary foundations of cell adhesion. Reconstructing the evolution of cell junction proteins in animals and their unicellular relatives exemplifies the roles of co-option and innovation during evolutionary transitions to multicellularity. Comparative studies of volvocine algae reveal specific molecular changes that accompanied the evolution of multicellularity in Volvox. Finally, comparisons between animals and Dictyostelium indicate the extent to which commonalities and differences in the biology of their unicellular ancestors influenced the evolution of adhesive mechanisms in each lineage. Understanding the unicellular ancestry of cell adhesion helps illuminate the basic cell biology of multicellular development in modern organisms.
INTRODUCTIONChoanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Most, if not all, choanoflagellates are heterotrophs that prey on bacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumed by the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplemented with a specific bacterial strain that has been grown separately. On the other hand, if a culture already contains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. In either case, it is common to enrich the medium with an organic extract, such as liver extract, cereal grass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to start cultures of the marine choanoflagellate species, Monosiga brevicollis, from frozen stocks. These cultures can then be maintained and expanded in preparation for DNA or RNA isolation or cell biological assays.
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