Reproductive cell specification during Volvox obversus development

Asexual spheroids of the genus Volvox contain only two cell types: flagellated somatic cells and immotile asexual reproductive cells known as gonidia. During each round of embryogenesis in Volvox obversus, eight large gonidial precursors are produced at the anterior extremity of the embryo. These cells arise as a consequence of polarized, asymmetric divisions of the anteriormost blastomeres at the fourth through nine cleavage cycles, while all other blastomeres cleave symmetrically to yield somatic cell precursors. Blastomeres isolated from embryos at any point between the 2-cell and the 32-cell stage cleaved in the normal pattern and produced the same complement and spatial distribution of cell types as they would have in an intact embryo. This result indicates that intrinsic features control the cleavage patterns and developmental potentials of blastomeres, and rules out any significant role for cell-cell interactions in gonidial specification. When substantial quantities of anterolateral cytoplasm were deleted from uncleaved gonidia or 4-cell stage blastomeres, the cell fragments frequently regulated and embryos were produced with the expected number of asymmetrically cleaving cells and gonidial precursors at their anterior ends. However, when anterior cytoplasm was deleted from 8-cell stage blastomeres, the depleted cells frequently failed to cleave asymmetrically and produced no gonidial precursors. Furthermore, when compression was used to reorient cleavage planes at the fourth division cycle, so that anterior cytoplasm was transmitted to more than the normal number of cells, those cells receiving a significant amount of such cytoplasm cleaved asymmetrically to produce supernumerary gonidial precursors. Together, these last two experiments indicate that blastomeres in the V. obversus embryo acquire (at least by the end of the third cleavage cycle) a polarized organization in which anterior cytoplasm plays a causal role in the process of reproductive-cell specification.     

A kinesin,invA, plays an essential role in volvox morphogenesis

In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

Germ-soma differentiation in volvox.

Volvox carteri is a spherical green alga with a predominantly asexual mode of reproduction and a complete germ-soma division of labor. Its somatic cells are specialized for motility, incapable of dividing, and programmed to die when only a few days old, whereas its gonidia (asexual reproductive cells) are nonmotile, specialized for growth and reproduction, and potentially immortal. When a gonidium is less than 2 days old it divides to produce a juvenile spheroid containing all of the somatic cells and gonidia that will be present in an adult of the next generation. The first visible step in germ-soma differentiation is a set of asymmetric cleavage divisions in the embryo that set apart small somatic initials from their large gonidial-initial sister cells. Three types of genes have been found to play key roles in germ-soma specification. First a set of gls genes act in the embryos to shift cell-division planes, resulting in the asymmetric divisions that set apart the large-small sister-cell pairs. Then a set of lag genes act in the large cells to prevent somatic differentiation, while the regA gene acts in the small cells to prevent reproductive development. An inducible transposon was used to tag and recover some of these and other developmentally important genes. The glsA gene encodes a chaperone-like protein that, like another chaperone that is one of its putative binding partners, is associated with the cell division apparatus, although how this leads to asymmetric division remains to be elucidated. The regA gene encodes a somatic-cell-specific nuclear protein that appears to function by repressing genes required for chloroplast biogenesis, thereby preventing somatic cells from growing enough to reproduce. Somatic-cell-specific expression of regA is controlled by three intronic enhancers.     

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri

The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.     

A NEW SPECIES OF VOLVOX SECT. MERRILLOSPHAERA (VOLVOCACEAE,CHLOROPHYCEAE) FROM TEXAS1

Smith (1944) divided the familiar genus Volvox L. into four sections, placing seven species that lacked cytoplasmic bridges between adult cells in the section Merrillosphaera. Herein, we describe a new member of the section Merrillosphaera originating from Texas (USA): Volvox ovalis Pocock ex Nozaki et A. W. Coleman sp. nov. Asexual spheroids of V. ovalis are ovoid or elliptical, with a monolayer of 1,000–2,000 somatic cells that are not linked by cytoplasmic bridges, an expanded anterior region, and 8–12 gonidia in the posterior region. Visibly asymmetric cleavage divisions do not occur in V. ovalis embryos as they do Volvox carteri F. Stein, Volvox obversus (W. Shaw) Printz, and Volvox africanus G. S. West, so the gonidia of the next generation are not yet recognizable in V. ovalis embryos prior to inversion. Molecular phylogenetic analyses of the five chloroplast genes and the internal transcribed spacer (ITS) regions of nuclear rDNA indicated that V. ovalis is closely related to Volvox spermatosphaera Powers ( Powers 1908 , as “spermatosphara”) and/or Volvox tertius Art. Mey.; however, V. ovalis can be distinguished from V. spermatosphaera by its larger gonidia, and from V. tertius by visible differences in gonidial chloroplast morphology.     

Differentiation of germinal and somatic cells in Volvox carteri

Volvox carteri is a spherical alga with a complete division of labor between around 2000 biflagellate somatic cells and 16 asexual reproductive cells (gonidia). It provides an attractive system for studying how a molecular genetic program for cell-autonomous differentiation is encoded within the genome. Three types of genes have been identified as key players in germ-soma differentiation: a set of gls genes that act in the embryo to shift cell-division planes, resulting in asymmetric divisions that set apart the large-small sister-cell pairs; a set of lag genes that act in the large gonidial initials to prevent somatic differentiation; and the regA gene, which acts in the small somatic initials to prevent reproductive development. Somatic-cell-specific expression of regA is controlled by intronic enhancer and silencer elements.     

Ultrastructure of Volvox carteri

Summary Somatic cells of mature asexual colonies of Volvox carteri do not possess a true cell wall, but are otherwise similar in ultrastructure to Chlamydomonas. Somatic cells are embedded in multilayered fibrillar material of the colonial matrix. The reproductive cells (gonidia) of Volvox carteri lie internal to the somatic cell layer of the colony matrix in an apparently structureless portion of the colony matrix. Mature gonidia are large vacuolate cells with a central nucleus and parietal chloroplasts and mitochondria. They are non-flagellated at maturity, but each contains a pair of kinetosomes.     

The role of GlsA in the evolution of asymmetric cell division in the green alga<Emphasis Type="Italic"> Volvox carteri</Emphasis>

Volvox carteri, a green alga in the order Volvocales, contains two completely differentiated cell types, small motile somatic cells and large reproductive cells called gonidia, that are set apart from each other during embryogenesis by a series of visibly asymmetric cell divisions. Mutational analysis has revealed a class of genes (gonidialess, gls) that are required specifically for asymmetric divisions in V. carteri, but that are dispensable for symmetric divisions. Previously we cloned one of these genes, glsA, and showed that it encodes a chaperone-like protein (GlsA) that has close orthologs in a diverse set of eukaryotes, ranging from fungi to vertebrates and higher plants. In the present study we set out to explore the role of glsA in the evolution of asymmetric division in the volvocine algae by cloning and characterizing a glsA ortholog from one of the simplest members of the group, Chlamydomonas reinhardtii, which does not undergo asymmetric divisions. This ortholog (which we have named gar1, for glsA related) is predicted to encode a protein that is 70% identical to GlsA overall, and that is most closely related to GlsA in the same domains that are most highly conserved between GlsA and its other known orthologs. We report that a gar1 transgene fully complements the glsA mutation in V. carteri, a result that suggests that asymmetric division probably arose through the modification of a gene whose product interacts with GlsA, but not through a modification of glsA itself.     

Hsp70A and GlsA interact as partner chaperones to regulate asymmetric division in Volvox

GlsA, a J-protein chaperone, is required for the asymmetric divisions that set aside germ and somatic cell precursors during embryogenesis in Volvox carteri, and previous evidence indicated that this function requires an intact Hsp70-binding site. To determine if Hsp70A, the only known cytoplasmic Hsp70 in V. carteri, is the chaperone partner of GlsA, we investigated the localization of the two proteins during critical stages of embryogenesis and tested their capacity to interact. We found that a substantial fraction of Hsp70A co-localizes with GlsA, both in interphase and mitotic blastomeres. In addition, Hsp70A coimmunoprecipitated with GlsA, and co-expression of GlsA and Hsp70A variants partially rescued the Gls phenotype of a glsA mutant, whereas neither variant by itself rescued the mutant phenotype. Immunofluorescence analysis demonstrated that GlsA is about equally abundant in all blastomeres at all cleavage stages examined but that Hsp70A is more abundant in anterior (asymmetrically dividing) blastomeres than in posterior (symmetrically dividing) blastomeres during the period of asymmetric division. We conclude that Hsp70A and GlsA function as chaperone partners that regulate asymmetric division and that the relative abundance of Hsp70A in asymmetrically dividing embryos may determine which blastomeres divide asymmetrically and which do not.     

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.     

VOLVOX POCOCKIAE,A NEW SPECIES WITH DWARF MALES1

Volvox pocockiae is described as the second species in the section Janetosphaera. The somatic protoplasts are connected by cytoplasmic strands approximately the same diameter as flagella, and the construction of the spheroid is identical to that of V. aureus. Asexual reproduction by the division of gonidia differs from that in V. aureus in the enlargement of the gonidium prior to its division to form the embryo. Sexual reproduction is very similar to that in V. spermatosphaera, a species in the section Merrillosphaera without cytoplasmic connections. Dwarf males are formed in the posterior end of the parental spheroid, and, as in V. spermatosphaera, the dwarf males are composed exclusively of androgonidia with no sterile somatic cells. Females are facultatively asexual spheroids, the gonidia of which function as eggs. The single biflagellate zoospore produced by the germinating zygote undergoes cleavage to form a germling spheroid. The differentiation of gonidia in the asexual embryo and in the germling spheroid is evident only after inversion and enlargement of the spheroid have begun.     

Cadherin-mediated cell interaction regulates germ cell determination in mice

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.