Cell Type-Specific Promoters of Volvox carteri for Molecular Cell Biology Studies.

The multicellular green alga Volvox carteri has emerged as a valuable model organism for investigating various aspects of multicellularity and cellular differentiation, photoreception and phototaxis, cell division, biogenesis of the extracellular matrix and morphogenetic movements. While a range of molecular tools and bioinformatics resources have been made available for exploring these topics, the establishment of cell type-specific promoters in V. carteri has not been achieved so far. Therefore, here, we conducted a thorough screening of transcriptome data from RNA sequencing analyses of V. carteri in order to identify potential cell type-specific promoters. Eventually, we chose two putative strong and cell type-specific promoters, with one exhibiting specific expression in reproductive cells (gonidia), the PCY1 promoter, and the other in somatic cells, the PFP promoter. After cloning both promoter regions, they were introduced upstream of a luciferase reporter gene. By using particle bombardment, the DNA constructs were stably integrated into the genome of V. carteri. The results of the expression analyses, which were conducted at both the transcript and protein levels, demonstrated that the two promoters drive cell type-specific expression in their respective target cell types. Transformants with considerably diverse expression levels of the chimeric genes were identifiable. In conclusion, the screening and analysis of transcriptome data from RNA sequencing allowed for the identification of potential cell type-specific promoters in V. carteri. Reporter gene constructs demonstrated the actual usability of two promoters. The investigated PCY1 and PFP promoters were proven to be potent molecular tools for genetic engineering in V. carteri.

Molecular and cellular dynamics of early embryonic cell divisions in Volvox carteri.

Cell division is fundamental to all organisms and the green alga used here exhibits both key animal and plant functions. Specifically, we analyzed the molecular and cellular dynamics of early embryonic divisions of the multicellular green alga Volvox carteri (Chlamydomonadales). Relevant proteins related to mitosis and cytokinesis were identified in silico, the corresponding genes were cloned, fused to yfp, and stably expressed in Volvox, and the tagged proteins were studied by live-cell imaging. We reveal rearrangements of the microtubule cytoskeleton during centrosome separation, spindle formation, establishment of the phycoplast, and generation of previously unknown structures. The centrosomes participate in initiation of spindle formation and determination of spindle orientation. Although the nuclear envelope does not break down during early mitosis, intermixing of cytoplasm and nucleoplasm results in loss of nuclear identity. Finally, we present a model for mitosis in Volvox. Our study reveals enormous dynamics, clarifies spatio-temporal relationships of subcellular structures, and provides insight into the evolution of cell division.

Cell Type-Specific Pherophorins of Volvox carteri Reveal Interplay of Both Cell Types in ECM Biosynthesis.

The spheroidal green algae Volvox carteri serves as a model system to investigate the formation of a complex, multifunctional extracellular matrix (ECM) in a relatively simple, multicellular organism with cell differentiation. The V. carteri ECM is mainly composed of hydroxyproline-rich glycoproteins (HRGPs) and there are diverse region-specific, anatomically distinct structures in the ECM. One large protein family with importance for ECM biosynthesis stands out: the pherophorins. The few pherophorins previously extracted from the ECM and characterized, were specifically expressed by somatic cells. However, the localization and function of most pherophorins is unknown. Here, we provide a phylogenetic analysis of 153 pherophorins of V. carteri and its unicellular relative Chlamydomonas reinhardtii. Our analysis of cell type-specific mRNA expression of pherophorins in V. carteri revealed that, contrary to previous assumptions, only about half (52%) of the 102 investigated pherophorin-related genes show stronger expression in somatic cells, whereas about one-third (34%) of the genes show significant higher expression in reproductive cells (gonidia). We fused two pherophorin genes that are expressed by different cell types to yfp, stably expressed them in Volvox and studied the tagged proteins by live-cell imaging. In contrast to earlier biochemical approaches, this genetic approach also allows the in vivo analysis of non-extractable, covalently cross-linked ECM proteins. We demonstrate that the soma-specific pherophorin SSG185 is localized in the outermost ECM structures of the spheroid, the boundary zone and at the flagellar hillocks. SSG185:YFP is detectable as early as 1.5 h after completion of embryogenesis. It is then present for the rest of the life cycle. The gonidia-specific pherophorin PhG is localized in the gonidial cellular zone 1 ("gonidial vesicle") suggesting its involvement in the protection of gonidia and developing embryos until hatching. Even if somatic cells produce the main portion of the ECM of the spheroids, ECM components produced by gonidia are also required to cooperatively assemble the total ECM. Our results provide insights into the evolution of the pherophorin protein family and convey a more detailed picture of Volvox ECM synthesis.

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.

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri.

In photosynthetic organisms many processes are light dependent and sensing of light requires light-sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1-related proteins from a wide range of species. The detailed subcellular localization of fluorescence-tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four-celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d-roots. The unequal distribution of Babo1 in four-celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior-posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal-binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.

Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases.

BACKGROUND: The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics. RESULTS: Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery. CONCLUSIONS: Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

Whole transcriptome RNA-Seq analysis reveals extensive cell type-specific compartmentalization in Volvox carteri.

BACKGROUND: One of evolution's most important achievements is the development and radiation of multicellular organisms with different types of cells. Complex multicellularity has evolved several times in eukaryotes; yet, in most lineages, an investigation of its molecular background is considerably challenging since the transition occurred too far in the past and, in addition, these lineages evolved a large number of cell types. However, for volvocine green algae, such as Volvox carteri, multicellularity is a relatively recent innovation. Furthermore, V. carteri shows a complete division of labor between only two cell types - small, flagellated somatic cells and large, immotile reproductive cells. Thus, V. carteri provides a unique opportunity to study multicellularity and cellular differentiation at the molecular level. RESULTS: This study provides a whole transcriptome RNA-Seq analysis of separated cell types of the multicellular green alga V. carteri f. nagariensis to reveal cell type-specific components and functions. To this end, 246 million quality filtered reads were mapped to the genome and valid expression data were obtained for 93% of the 14,247 gene loci. In the subsequent search for protein domains with assigned molecular function, we identified 9435 previously classified domains in 44% of all gene loci. Furthermore, in 43% of all gene loci we identified 15,254 domains that are involved in biological processes. All identified domains were investigated regarding cell type-specific expression. Moreover, we provide further insight into the expression pattern of previously described gene families (e.g., pherophorin, extracellular matrix metalloprotease, and VARL families). Our results demonstrate an extensive compartmentalization of the transcriptome between cell types: More than half of all genes show a clear difference in expression between somatic and reproductive cells. CONCLUSIONS: This study constitutes the first transcriptome-wide RNA-Seq analysis of separated cell types of V. carteri focusing on gene expression. The high degree of differential expression indicates a strong differentiation of cell types despite the fact that V. carteri diverged relatively recently from its unicellular relatives. Our expression dataset and the bioinformatic analyses provide the opportunity to further investigate and understand the mechanisms of cell type-specific expression and its transcriptional regulation.

Distinct shape-shifting regimes of bowl-shaped cell sheets - embryonic inversion in the multicellular green alga Pleodorina.

BACKGROUND: The multicellular volvocine alga Pleodorina is intermediate in organismal complexity between its unicellular relative, Chlamydomonas, and its multicellular relative, Volvox, which shows complete division of labor between different cell types. The volvocine green microalgae form a group of genera closely related to the genus Volvox within the order Volvocales (Chlorophyta). Embryos of multicellular volvocine algae consist of a cellular monolayer that, depending on the species, is either bowl-shaped or comprises a sphere. During embryogenesis, multicellular volvocine embryos turn their cellular monolayer right-side out to expose their flagella. This process is called 'inversion' and serves as simple model for epithelial folding in metazoa. While the development of spherical Volvox embryos has been the subject of detailed studies, the inversion process of bowl-shaped embryos is less well understood. Therefore, it has been unclear how the inversion of a sphere might have evolved from less complicated processes. RESULTS: In this study we characterized the inversion of initially bowl-shaped embryos of the 64- to 128-celled volvocine species Pleodorina californica. We focused on the movement patterns of the cell sheet, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. The development of living embryos was recorded using time-lapse light microscopy. Moreover, fixed and sectioned embryos throughout inversion and at successive stages of development were analyzed by light and transmission electron microscopy. We generated three-dimensional models of the identified cell shapes including the localization of CBs. CONCLUSIONS: In contrast to descriptions concerning volvocine embryos with lower cell numbers, the embryonic cells of P. californica undergo non-simultaneous and non-uniform cell shape changes. In P. californica, cell wedging in combination with a relocation of the CBs to the basal cell tips explains the curling of the cell sheet during inversion. In volvocine genera with lower organismal complexity, the cell shape changes and relocation of CBs are less pronounced in comparison to P. californica, while they are more pronounced in all members of the genus Volvox. This finding supports an increasing significance of the temporal and spatial regulation of cell shape changes and CB relocations with both increasing cell number and organismal complexity during evolution of differentiated multicellularity.

Algal Photobiology: A Rich Source of Unusual Light Sensitive Proteins for Synthetic Biology and Optogenetics.

The light absorption system in eukaryotic (micro)algae includes highly sensitive photoreceptors, which change their conformation in response to different light qualities on a subsecond time scale and induce physiological and behavioral responses. Some of the light sensitive modules are already in use to engineer and design photoswitchable tools for control of cellular and physiological activities in living organisms with various degrees of complexity. Thus, identification of new light sensitive modules will not only extend the source material for the generation of optogenetic tools but also foster the development of new light-based strategies in cell signaling research. Apart from searching for new proteins with suitable light-sensitive modules, smaller variants of existing light-sensitive modules would be helpful to simplify the construction of hybrid genes and facilitate the generation of mutated and chimerized modules. Advances in genome and transcriptome sequencing as well as functional analysis of photoreceptors and their interaction partners will help to discover new light sensitive modules.

Genome-wide identification and phylogenetic analysis of plant RNA binding proteins comprising both RNA recognition motifs and contiguous glycine residues.

This study focused on the identification and phylogenetic analysis of glycine-rich RNA binding proteins that contain an RNA recognition motif (RRM)-type RNA binding domain in addition to a region with contiguous glycine residues in representative plant species. In higher plants, glycine-rich proteins with an RRM have met considerable interest as they are responsive to environmental cues and play a role in cold tolerance, pathogen defense, flowering time control, and circadian timekeeping. To identify such RRM containing proteins in plant genomes we developed an RRM profile based on the known glycine-rich RRM containing proteins in the reference plant Arabidopsis thaliana. The application of this remodeled RRM profile that omitted sequences from non-plant species reduced the noise when searching plant genomes for RRM proteins compared to a search performed with the known RRM_1 profile. Furthermore, we developed an island scoring function to identify regions with contiguous glycine residues, using a sliding window approach. This approach tags regions in a protein sequence with a high content of the same amino acid, and repetitive structures score higher. This definition of repetitive structures in a fixed sequence length provided a new glance for characterizing patterns which cannot be easily described as regular expressions. By combining the profile-based domain search for well-conserved regions (the RRM) with a scoring technique for regions with repetitive residues we identified groups of proteins related to the A. thaliana glycine-rich RNA binding proteins in eight plant species.

Spotlighted Brains: Optogenetic Activation and Silencing of Neurons.

Optogenetics is revolutionizing cell biology and neuroscience research by allowing precise biochemical control of neuronal activity through light-activated channels. Light-induced ion transporters have been used extensively for cellular activation, and now light-gated inhibitory channels have been discovered. These represent a key new tool to elucidate the molecular mechanisms underlying neurological and neuropsychiatric disorders.

The inducible nitA promoter provides a powerful molecular switch for transgene expression in Volvox carteri.

BACKGROUND: The multicellular green alga Volvox carteri represents an attractive model system to study various aspects of multicellularity like cellular differentiation, morphogenesis, epithelial folding and ECM biogenesis. However, functional and molecular analyses of such processes require a wide array of molecular tools for genetic engineering. So far there are only a limited number of molecular tools available in Volvox. RESULTS: Here, we show that the promoter of the V. carteri nitrate reductase gene (nitA) is a powerful molecular switch for induction of transgene expression. Strong expression is triggered by simply changing the nitrogen source from ammonium to nitrate. We also show that the luciferase (g-luc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of the unicellular alga Chlamydomonas reinhardtii, is a suitable reporter gene in V. carteri. Emitted light of the chemiluminescent reaction can be easily detected and quantified with a luminometer. Long-term stability of inducible expression of the chimeric nitA/g-luc transgenes after stable nuclear transformation was demonstrated by transcription analysis and bioluminescence assays. CONCLUSION: Two novel molecular tools for genetic engineering of Volvox are now available: the nitrate-inducible nitA promoter of V. carteri and the codon-adapted luciferase reporter gene of G. princeps. These novel tools will be useful for future molecular research in V. carteri.

Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study show that Volvox photoreceptors are mostly expressed in a cell-type specific manner. This gives reason to believe that cell-type specific light-signaling pathways allow differential regulation of cellular and developmental processes in response to the environmental light cues.

Genome-wide analysis of alternative splicing in Volvox carteri.

BACKGROUND: Alternative splicing is an essential mechanism for increasing transcriptome and proteome diversity in eukaryotes. Particularly in multicellular eukaryotes, this mechanism is involved in the regulation of developmental and physiological processes like growth, differentiation and signal transduction. RESULTS: Here we report the genome-wide analysis of alternative splicing in the multicellular green alga Volvox carteri. The bioinformatic analysis of 132,038 expressed sequence tags (ESTs) identified 580 alternative splicing events in a total of 426 genes. The predominant type of alternative splicing in Volvox is intron retention (46.5%) followed by alternative 5' (17.9%) and 3' (21.9%) splice sites and exon skipping (9.5%). Our analysis shows that in Volvox at least ~2.9% of the intron-containing genes are subject to alternative splicing. Considering the total number of sequenced ESTs, the Volvox genome seems to provide more favorable conditions (e.g., regarding length and GC content of introns) for the occurrence of alternative splicing than the genome of its close unicellular relative Chlamydomonas. Moreover, many randomly chosen alternatively spliced genes of Volvox do not show alternative splicing in Chlamydomonas. Since the Volvox genome contains about the same number of protein-coding genes as the Chlamydomonas genome (~14,500 protein-coding genes), we assumed that alternative splicing may play a key role in generation of genomic diversity, which is required to evolve from a simple one-cell ancestor to a multicellular organism with differentiated cell types (Mol Biol Evol 31:1402-1413, 2014). To confirm the alternative splicing events identified by bioinformatic analysis, several genes with different types of alternatively splicing have been selected followed by experimental verification of the predicted splice variants by RT-PCR. CONCLUSIONS: The results show that our approach for prediction of alternative splicing events in Volvox was accurate and reliable. Moreover, quantitative real-time RT-PCR appears to be useful in Volvox for analyses of relationships between the appearance of specific alternative splicing variants and different kinds of physiological, metabolic and developmental processes as well as responses to environmental changes.

Stable nuclear transformation of Pandorina morum.

BACKGROUND: Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8-16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest. RESULTS: Stable nuclear transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3'-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain.The heterologous luciferase (gluc) gene of Gaussia princeps, which was previously genetically engineered to match the nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays. CONCLUSION: Stable nuclear transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii. The aphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for transformation experiments in the green alga P. morum. The gluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.

Algal photoreceptors: in vivo functions and potential applications.

Many algae, particularly microalgae, possess a sophisticated light-sensing system including photoreceptors and light-modulated signaling pathways to sense environmental information and secure the survival in a rapidly changing environment. Over the last couple of years, the multifaceted world of algal photobiology has enriched our understanding of the light absorption mechanisms and in vivo function of photoreceptors. Moreover, specific light-sensitive modules have already paved the way for the development of optogenetic tools to generate light switches for precise and spatial control of signaling pathways in individual cells and even in complex biological systems.

Validation of reference genes for quantitative gene expression studies in Volvox carteri using real-time RT-PCR.

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for analysis of gene expression under a wide diversity of biological conditions. However, the identification of suitable reference genes is a critical factor for analysis of gene expression data. To determine potential reference genes for normalization of qRT-PCR data in the green alga Volvox carteri, the transcript levels of ten candidate reference genes were measured by qRT-PCR in three experimental sample pools containing different developmental stages, cell types and stress treatments. The expression stability of the candidate reference genes was then calculated using the algorithms geNorm, NormFinder and BestKeeper. The genes for 18S ribosomal RNA (18S) and eukaryotic translation elongation factor 1α2 (eef1) turned out to have the most stable expression levels among the samples both from different developmental stages and different stress treatments. The genes for the ribosomal protein L23 (rpl23) and the TATA-box binding protein (tbpA) showed equivalent transcript levels in the comparison of different cell types, and therefore, can be used as reference genes for cell-type specific gene expression analysis. Our results indicate that more than one reference gene is required for accurate normalization of qRT-PCRs in V. carteri. The reference genes in our study show a much better performance than the housekeeping genes used as a reference in previous studies.

Stable nuclear transformation of Eudorina elegans.

BACKGROUND: A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16-32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available. RESULTS: Here, we describe the stable nuclear transformation of E. elegans by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3'-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded gluc gene was demonstrated by transcription analysis and bioluminescence assays. CONCLUSIONS: Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable nuclear transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.

There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator.

BACKGROUND: Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult configuration; this process is called 'inversion.' There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out? RESULTS: In this study, we investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri. CONCLUSIONS: Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.

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.