Ichiro Nishii

Genomic Analysis of Organismal Complexity in the Multicellular Green Alga Volvox carteri

Going Multicellular The volvocine algae include both the unicellular Chlamydomonas and the multicellular Volvox, which diverged from one another 50 to 200 million years ago. Prochnik et al. (p. 223) compared the Volvox genome with that of Chlamydomonas to identify any genomic innovations that might have been associated with the transition to multicellularity. Size changes were observed in several protein families in Volvox, but, overall, the Volvox genome and predicted proteome were highly similar to those of Chlamydomonas. Thus, biological complexity can arise without major changes in genome content or protein domains. Comparison of the Chlamydomonas and Volvox genomes show few differences, despite their divergent life histories. The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138–mega–base pair genome of V. carteri and compared its ~14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal–specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

Evolution of an Expanded Sex-Determining Locus in Volvox

Revealing Volvox Female and male gametes of the green alga, Volvox, significantly differ in size. Those of Chlamydomonas, another green algae from a lineage that separated from Volvox some 200 million years ago, are the same size. We know sex in Chlamydomonas is governed by a sex-determining locus called MT. In a detailed comparison of the MT loci of Volvox and Chlamydomonas, Ferris et al. (p. 351) found that although MT has retained some similarity in gene order, its composition has greatly changed between the two species. In Volvox, new genes have been coopted into this locus and show sex-specific expression. Mating loci among green algae show conserved gene order, but also have many unique features that may explain gamete size differences. Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non–sex-related genes.

Identification of the Minus-Dominance Gene Ortholog in the Mating-Type Locus of Gonium pectorale

The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT− locus. 3′- and 5′-RACE RT–PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT–PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.

Volvox: Simple steps to developmental complexity?

Volvox, Chlamydomonas, and their close relatives - collectively the volvocine green algae - comprise an excellent system for investigating the origins of developmental complexity. Over a relatively short period of time Volvox evolved an impressive suite of developmental traits, including asymmetric cell division, multicellularity with germ-soma division of labor, embryonic morphogenesis, and oogamy. Recent molecular genetic analyses of important developmental genes and comparative analyses of the fully sequenced Volvox and Chlamydomonas genomes have provided important insights into how these and other traits came to be. Surprisingly, the acquisition of much of the developmental innovation in this family seems to have involved relatively minor tinkering with the ancestral unicellular blueprint.

Controlled Enlargement of the Glycoprotein Vesicle Surrounding a Volvox Embryo Requires the InvB Nucleotide-Sugar Transporter and Is Required for Normal Morphogenesis

Here, we report our analysis of a mutant of Volvox carteri, InvB, whose embryos fail to execute inversion, the process in which each Volvox embryo normally turns itself inside-out at the end of embryogenesis, thereby achieving the adult configuration. The invB gene encodes a nucleotide-sugar transporter that exhibits GDP-mannose transport activity when expressed in yeast. In wild-type embryos, the invB transcript is maximally abundant before and during inversion. A mannoside probe (fluorescent concanavalin A) stains the glycoprotein-rich gonidial vesicle (GV) surrounding wild-type embryos much more strongly than it stains the GV surrounding InvB embryos. Direct measurements revealed that throughout embryogenesis the GV surrounding a wild-type embryo increases in size much more than the GV surrounding an InvB embryo does, and the fully cleaved InvB embryo is much more tightly packed within its GV than a wild-type embryo is. To test the hypothesis that the restraint imposed by a smaller than normal GV directly causes the inversion defect in the mutant, we released InvB embryos from their GVs microsurgically. The resulting embryos inverted normally, demonstrating that controlled enlargement of the GV, by a process in which requires the InvB nucleotide-sugar transporter, is essential to provide the embryo sufficient space to complete inversion.

The VARL Gene Family and the Evolutionary Origins of the Master Cell-Type Regulatory Gene, regA, in Volvox carteri

Chlamydomonas reinhardtii, Volvox carteri, and their relatives in the family Volvocaceae provide an excellent opportunity for studying how multicellular organisms with differentiated cell types evolved from unicellular ancestors. While C. reinhardtii is unicellular, V. carteri is multicellular with two cell types, one of which resembles C. reinhardtii cytologically but is terminally differentiated. Maintenance of this “somatic cell” fate is controlled by RegA, a putative transcription factor. We recently showed that RegA shares a conserved region with several predicted V. carteri and C. reinhardtii proteins and that this region, the VARL domain, is likely to include a DNA-binding SAND domain. As the next step toward understanding the evolutionary origins of the regA gene, we analyzed the genome sequences of C. reinhardtii and V. carteri to identify additional genes with the potential to encode VARL domain proteins. Here we report that the VARL gene family, which consists of 12 members in C. reinhardtii and 14 in V. carteri, has experienced a complex evolutionary history in which members of the family have been both gained and lost over time, although several pairs of potentially orthologous genes can still be identified. We find that regA is part of a tandem array of four VARL genes in V. carteri but that a similar array is absent in C. reinhardtii. Most importantly, our phylogenetic analysis suggests that a proto-regA gene was present in a common unicellular ancestor of V. carteri and C. reinhardtii and that this gene was lost in the latter lineage.

Idaten is a New Cold-inducible Transposon of Volvox carteri that can be Used for Tagging Developmentally Important Genes

A cold-inducible transposon called Jordan has previously been used to tag and recover genes controlling key aspects of Volvox development, including the process called inversion. In a search for additional genes, we isolated 17 new inversionless mutants from cultures grown at 24° (the temperature that activates Jordan transposition). These mutants were stable at 32°, but generated revertants at 24°. DNA blots revealed that one mutant had a transposon unrelated to Jordan inserted in invA (“inversionless A”). This new transposon, which we named Idaten, has terminal inverted repeats (TIRs) beginning with CCCTA, and upon insertion it creates a 3-bp target-site duplication. It appears to belong to the CACTA superfamily of class II DNA transposons, which includes En/Spm. No significant open reading frames were in the Idaten sequence, but we retrieved another element with Idaten-type TIRs encoding a protein similar to the En/Spm transposase as a candidate for an Idaten-specific transposase. We found that in five of the new inversionless strains we could not find any Jordan insertions causing the phenotype to possess insertions of an Idaten family member in a single locus (invC). This clearly indicates that Idaten is a potentially powerful alternative to Jordan for tagging developmentally important genes in Volvox.

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga, Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.

Mitochondrial and plastid genomes of the colonial green alga Gonium pectorale give insights into the origins of organelle DNA architecture within the Volvocales

Volvocalean green algae have among the most diverse mitochondrial and plastid DNAs (mtDNAs and ptDNAs) from the eukaryotic domain. However, nearly all of the organelle genome data from this group are restricted to unicellular species, like Chlamydomonas reinhardtii, and presently only one multicellular species, the ∼4,000-celled Volvox carteri, has had its organelle DNAs sequenced. The V. carteri organelle genomes are repeat rich, and the ptDNA is the largest plastome ever sequenced. Here, we present the complete mtDNA and ptDNA of the colonial volvocalean Gonium pectorale, which is comprised of ∼16 cells and occupies a phylogenetic position closer to that of V. carteri than C. reinhardtii within the volvocine line. The mtDNA and ptDNA of G. pectorale are circular-mapping AT-rich molecules with respective lengths and coding densities of 16 and 222.6 kilobases and 73 and 44%. They share some features with the organelle DNAs of V. carteri, including palindromic repeats within the plastid compartment, but show more similarities with those of C. reinhardtii, such as a compact mtDNA architecture and relatively low organelle DNA intron contents. Overall, the G. pectorale organelle genomes raise several interesting questions about the origin of linear mitochondrial chromosomes within the Volvocales and the relationship between multicellularity and organelle genome expansion.

Distribution of the Sex-Determining Gene MID and Molecular Correspondence of Mating Types within the Isogamous Genus Gonium (Volvocales, Chlorophyta)

Background Isogamous organisms lack obvious cytological differences in the gametes of the two complementary mating types. Consequently, it is difficult to ascertain which of the two mating types are homologous when comparing related but sexual isolated strains or species. The colonial volvocalean algal genus Gonium consists of such isogamous organisms with heterothallic mating types designated arbitrarily as plus or minus in addition to homothallic strains. Homologous molecular markers among lineages may provide an “objective” framework to assign heterothallic mating types. Methodology/Principal Findings Using degenerate primers designed based on previously reported MID orthologs, the “master regulator” of mating types/sexes in the colonial Volvocales, MID homologs were identified and their presence/absence was examined in nine strains of four species of Gonium. Only one of the two complementary mating types in each of the four heterothallic species has a MID homolog. In addition to heterothallic strains, a homothallic strain of G. multicoccum has MID. Molecular evolutionary analysis suggests that MID of this homothallic strain retains functional constraint comparable to that of the heterothallic strains. Conclusion/Significance We coordinated mating genotypes based on presence or absence of a MID homolog, respectively, in heterothallic species. This scheme should be applicable to heterothallic species of other isogamous colonial Volvocales including Pandorina and Yamagishiella. Homothallism emerged polyphyletically in the colonial Volvocales, although its mechanism remains unknown. Our identification of a MID homolog for a homothallic strain of G. multicoccum suggests a MID-dependent mechanism is involved in the sexual developmental program of this homothallic species.