The green alga Volvox carteri has a level of complexity representing an ideal model system for studies of multicellularity and cellular differentiation [1,2]; each wild-type Volvox spheroid contains only two cell types, somatic cells and reproductive cells (gonidia) (Fig. 1A). Both cell types arise through a sequence of rapid symmetric and asymmetric cleavage divisions of a single gonidium. The two cell types are arranged in a simple, well-defined pattern and are different from each other with respect to physiology, developmental potential, morphology, and size [3]. Not only is the simplicity of Volvox auspicious for developmental biologists, but its phylogenetic relationships are also promising: Volvox and its simpler, but closely related, unicellular and colonial relatives, the volvocine algae Chlamydomonas, Gonium, Pandorina, Eudorina and Pleodorina, provide a coherent family of organisms for studying the molecular evolution of multicellularity and cellular differentiation [4]. Another outstanding advantage of volvocine algae is that there are ongoing genome projects both for the multicellular alga Volvox carteri and for the unicellular alga Chlamydomonas reinhardtii: Shotgun sequencing of both nuclear genomes was performed in each case at approximate 8× coverage by the Joint Genome Institute (JGI, Walnut Creek, CA). For Chlamydomonas, extensive cDNA and genomic sequence information has already become publicly available [5], with approximately 90% of the ~120 Mb nuclear genome sequenced; genomic data and data from ~300 k ESTs have been assembled into over 12,000 'unique' cDNAs, and annotation proceeds. Regarding the Volvox genome, which is about the same size as the Chlamydomonas genome, only shotgun sequences with 1× coverage are publicly available at the moment on the JGI sites, but the completed 8× coverage genomic data will be released before long; also ~80 k ESTs have already been sequenced at JGI and will be released shortly.
Although determination of the sequence of every gene in Volvox or any other species allows a better understanding of the organism's physiological potential, it is just the first step of a complete description of how the organism works. One of the next steps should be the determination of mRNA expression levels. Because it is known from many species that much of the transcriptome is compartmentalized and Volvox is particularly suitable for studies of multicellularity and cellular differentiation, it is logical to start with an analysis of cell-type specific gene expression, i.e. somatic cells versus gonidia, in order to provide a basis for disclosing cell-specific functions.
In earlier studies, 19 gonidia-specific and 12 somatic-cell-specific cDNAs have already been identified in wild-type Volvox by a differential screen of cDNA libraries, and abundance of the transcripts has been analyzed in each of the cell types by Northern blots using radiolabeled restriction-digested DNA as probes [6]; two of these cDNAs/genes have been added to our study as a reference (gon30, gon167). Furthermore, a couple of interesting developmentally-controlled or cell-type specific genes and their gene products have been identified by generating and analyzing mutants or by Mendelian analysis, e.g. the lag gene product (late gonidia), which acts in large pregonidial cells to repress somatic development [4,7,8], and the regA gene product (somatic regenerator), which acts on somatic cells to suppress gonidial development [9]. The latter gene was also used as a control gene in our study. Previously, it has also been shown that somatic cells and gonidia display substantially different patterns of both newly synthesized and accumulated major polypeptides [10], but at that time, it was not possible to obtain discrete sequences of these polypeptides, so their identity remained unknown.
A different approach, which is used in this study, is to investigate the different developmental programs in the two cell types by characterizing the abundance of novel or previously known mRNAs separately for each cell-type by real-time RT-PCR studies. This method should allow a considerable expansion of the number of genes investigated, studies done under different physiological conditions, and repeated experiments using mutant strains.
Here we show a quantitative analysis of a diversified pool of about forty target genes with regard to cell-type specific gene expression and relative expression rate in wild-type individuals of the green alga Volvox carteri. The investigated gene pool contains previously known genes with unknown localization, novel Volvox genes, which are described in this study for the first time, and a few previously characterized genes with known cell-type specific localization as controls. The corresponding gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes.
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