Jpn. J. Protozool. Vol. 34, No. 1. (2001)

7Review

Volvox carteri: Molecular Genetics of Cell differentiation
Rüdiger SCHMITTInstitut für Biochemie, Genetik und Mikrobiologie
Universität Regensburg, D-93040 Regensburg, Germany

Evolving multicellularity and differentiation
Two remarkable properties render Volvox, thespheroidal green alga, an outstanding model forinvestigating cellular differentiation and its molecular-genetic origins: First, Volvox is a multicellular organism showing the simplest kind of cytodifferentiation, the division of labor between onlytwo types of cells, somatic and germ cells (gonidia).
Second, Volvox and its simpler unicellular and colonial relatives, the volvocine algae Chlamydomonas, Gonium, Pandorina, Eudorina andPleodorina, form an extant, coherent family oforganisms with excellent promise for studies of themolecular evolution of multicellularity and cellulardifferentiation (Kirk, 1998; Schmitt et al. 1992).
Sequence comparisons for 18S rRNA gene locisuggest that, although the green algae in the order
Volvocales shared a common ancestor with higher
plants, they constitute a separate lineage - with
Volvox at its apex - that has long been separated
from the lines leading to vascular plants and animals (Rausch et al. 1989). This is illustrated by aschematic phylogenetic tree shown in Fig. 1. On thebasis of cytochrome sequence comparisons, Amati
et al. (1988) estimated that lines leading to themodern green algae and to the higher plants diverged about 700-750 million years ago, about 500
million years after plants and animals had lastshared a common ancestor. 'Molecular clock'methods applied to the rRNA data, and the numberof silent exchanges observed in tubulin genes, led toan estimate that the evolution of Volvox carteri, amulticellular organism with complete differentiation of soma and germ line, from a Chlamydomonas-like unicellular ancestor may have occurred asrecently as 35 million years ago (Rausch et al.
1989). This relatively late emergence of Volvox as a"modern" organism raises the possibility of tracing

Fig. 1 Eukaryotic phylogenetic tree (not toscale) based on 18S rRNA sequencecomparisons of selected organisms(modified from Rausch et al. 1989). Thevolvocine algae, Chlamydomonas andVolvox, appear at the tip of the green
algal branch (Chlorophyta) that diverged
long ago from the higher plant lineage.

8Jpn. J. Protozool. Vol. 34, No. 1. (2001)

the molecular-genetic pathway in the volvocine
lineage by which that program of cell differentiation evolved.
Embryogenesis: asymmetric cell division andgerm-soma dichotomy
A V. carteri gonidium may be viewed as immortal stem cell that divides mitotically to produce
more gonidia and a multitude of mortal somaticcells (Starr, 1969). Under a 16-hour light/8-hourdark regime and at optimum conditions themorphogenetic program of the V.carteri life cycle isregular and reliably yields predictable numbers of
the two entirely different cell types. Each cell division requires ca. 35min, the entire cleavage period
lasting six to seven hours. Prior to each cell division, the nuclear genome undergoes one round ofreplication (Kobl et al. 1998). Within individual
embryos, divisions are completely synchronous;
between different embryos in one parental spheroid, cleavages may deviate from synchrony by one
to three minutes. The first five divisions are symmetric and divide the embryo into 32 equal-sized
daughter cells. The sixth division is symmetric in
the two posterior tiers of cells, but it is asymmetricin the 16 anterior cells, which leads to 16 pairs oflarge and small sister cells. The large cells are gonidial initials (for the next generation), whereas thesmall equivalents and the remaining cells (after
another 5 to 6 cell divisions) will produce the somatic cells. After the final cleavage division and apause of up to one hour, inversion commences witha wave of contraction. The phialopore widens andthe entire embryo turns "inside out". This moves the16 large gonidia to the interior and the flagellar
ends of somatic cells to the exterior. About 45
minutes after the onset of inversion, the juvenile
spheroid is completed. Cytodifferentiation begins
and extracellular matrix deposited between the cells
causes an expansion of the spheroid. Four hours
after embryogenesis somatic cells and gonidia arefully separated, but it takes another 20 hours, until

the mother spheres release 16 juvenile spheroids,
who upon further maturation start a new cleavage
division cycle. The "old" somatic cells embedded in
the matrix of the former mother spheroid haveturned light green, they senesce and die after another 30 to 40 hours.
The regA gene: key to cell differentiation
A typical asexual spheroid of V.carteri contains 16 large reproductive cells (gonidia) and asurface layer of about 2000 small, biflagellate somatic cells. Gonidia are immotile and specialized
for reproduction. Somatic cells, by contrast, arespecialized for motility but have no reproductivepotential. Once differentiated, they never divide,and they eventually undergo programmed death(Pommerville and Kochert, 1982). The regA geneplays a central role in establishing this germ-soma
dichotomy by acting as a negative regulator of reproductive development in somatic cells. Whereas
regA+ mature somatic cells have ceased to divide,regA- somatic cells regain the full reproductive
potential of gonidia including the ability to divide(Huskey and Griffin, 1979; Kirk et al. 1987; Starr,1970). Mutations in regA have no effect on "true"
gonidia indicating that the gene is not expressed ingonidia. The expression pattern of regA in the V.
carteri life cycle is schematically viewed in Fig. 2.
By using the Volvox transposon Jordan (Milleret al. 1993), we have successfully tagged andcloned the regA gene locus (Kirk et al. 1999). Rescue of a regA- mutant via biolistic cotransformation with a selectable marker(Schiedlmeier et al., 1994) was used to test theidentity and integrity of the cloned regA gene. The
12.5-kb regA transcription unit generates a 6.725nucleotide mRNA that emerges at the beginning ofsomatic cell differentiation. It encodes a 111-kDa
RegA protein with all features of a transcription
factor that is localized to the nucleus. The HAtagged protein accumulates progressively for several hours, and then it remains moderately abundantJpn. J. Protozool. Vol. 34, No. 1. (2001)

9Fig. 2 The postulated roles two types of genes in establishing and maintaining the germ-soma dichotomy of V.carteri. After a series of symmetric divisions, the gls gene acts to permit asymmetric division and the formation of large and small cells (Miller and Kirk 1999). After thetermination of embryogenesis, the regA gene is expressed selectively in the small cells, whichleads to repression of reproductive genes, notably those encoding essential chloroplast proteins, and causes these cells to undergo terminal differentiation (Kirk et al. 1999).

throughout the life of the somatic cells, but it isnever detected in gonidia. These features of RegA
are consistent with its role as transcriptional repressor in somatic cells of functions required forgerm cell development.
What are the targets of RegA?
Previously, Tam and Kirk (1991) used celltype and stage-specific cDNAs to define a set of 18"maturation-abundant gonidial genes", whose transcripts accumulate in developing gonidia (and in
regA- somatic cells), but not in wild-type (regA+)somatic cells. This expression pattern was highlysuggestive of genes that are negatively regulated byRegA. All 18 cDNAs have been sequenced andcharacterized (Choi et al. 1996; Meissner et al.
1999). Of these, 14 DNA sequences encode
chloroplast proteins of known function, two encode
putative chloroplast proteins of unknown function,
and another two encode polypeptides with no recognizable features. The 14 assigned nuclear genes
encode proteins involved in the most important
aspects of chloroplast biology: light harvesting,photolysis of water, transfer of reducing powerfrom photosystem II to photosystem I and fromphotosystem I to the dark reactions, starch biosynthesis, ATP generation and chloroplast gene expression.
These findings suggest that germ-soma differentiation and chloroplast biogenesis must be intimately connected in V.carteri. It has been suggested that the huge chloroplast of a pre-cleavage
gonidium becomes progressively subdivided during
cleavages, such that a small portion of it (< 0.05%)
ends up in each of the somatic cells of the progeny
embryo. If these somatic cells cease to synthesize
new chloroplast proteins after the end of embryogenesis due to repression by RegA, they would beunable to grow to any significant extent and, hence,
they could not reproduce. The contribution of RegA
to the establishment of germ-soma differentiation
thus resides in the repression of chloroplast biogenesis in somatic cells, thereby preventing thosecells to grow sufficiently to be able to reproduce(Fig. 2). This suggestion does not necessarily imply
that the nuclear-encoded chloroplast proteins are10Jpn. J. Protozool. Vol. 34, No. 1. (2001)

Fig. 3. Organisation of the regA gene locus of V.carteri exhibiting eight exons, only of them (E5-E8)
being translated, and seven introns. Transcription from the promoter, Preg, starts at position+1, ATG and TGA mark translation start and stop signals. Mutants containing deletions ofeither intron 3 or intron 5 fail to express the regA gene in somatic cells and result in the RegAphenotype: somatic cells regain the full reproductive potential of gonidia. If intron 7 is deleted, the regA gene is expressed in gonidia and the gene product, RegA, suppresses reproductive functions: gonidia cease to divide and lose their photosynthetic potential, thus causing
the entire spheroid to die after two or three cycles ("fruitless" phenotype; Stark et al., 2001).
direct targets of RegA. In a network of feedback
relationships, RegA would only need to repress one
or a few key nuclear genes in order to suppress
chloroplast biogenesis completely. One of our major challenges for the immediate future, therefore, isto determine which (if any) of the genes defined
here and which of the "upstream" genes may bedirect targets of RegA regulation.
What regulates the RegA regulator?
The 12.5-kb regA gene consists of eight exons,the first four of them untranslated, seven introns
and 5' and 3' untranslated regions (Fig. 3; Stark etal. 2001). The gene is "switched on" in differentiating somatic cells, but it remains silent in the germcells (Fig. 2). If regA is inactivated by mutation,
somatic cells dedifferentiate to reproductive germ
cells. Reversely, if regA is being forced into expression by placing it under the control of the strong
constitutive promotor of a b-tubulin gene (Mages etal., 1995), gonidia cease to divide, remain small andappear blackish, wheras somatic cells differentiate
quite normally. These "fruitless" mutants die after
one or two cycles, clearly indicating that the(artificial) expression of regA in gonidia represses
the reproductive potential and thus renders suchmutants lethal. It is therefore a crucial requirementof Volvox differentiation that regA remains silent ingonidial, but that it becomes active in differentiating somatic cells (Kirk et al. 1999).
We have approached this central issue of differential regA gene expression by a systematic deletion analysis including the upstream portion andthe seven introns of this gene (Fig. 3). The minimal
promoter is limited to 42 bp upstream of transcription and contains putative TATA and initiator sequences, but no regulatory elements. However,introns 3 and 5 both contain enhancers absolutely
needed for regA gene expression in somatic cells.
On the other hand, intron 7 is essential for silencing
regA in gonidia. If deleted, it leads to the fruitlessphenotype described above indicating that intronseven less mutants express regA in germline cells.
Intron 7 bears an open reading frame contigous withthose of exons 7 and 8; the introduction of nonsense
mutations into this frame did not affect normal development suggesting that intron 7 is spliced andeliminated before translation. The most simple interpretation of these results suggests that introns 3and 5 contain enhancer elements that are activatedin differentiating somatic cells and that intron 7
contains a "silencer" active in gonidia throughout

Jpn. J. Protozool. Vol. 34, No. 1. (2001)

the life cylce (Stark et al., 2001). Cell-specific factors interacting with these elements are under current investigation.
ACKNOWLEDGEMENTS:
Many results reported here are the product of aclose collaboration with David L. Kirk, Washington
University, St. Louis, USA. Our laboratory is supported by a grant from the Deutsche Forschungsgemeinschaft (SFB521/B1).
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