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.

The volvocine algae provide an unrivalled opportunity to explore details of an evolutionary pathway leading from a unicellular ancestor to multicellular organisms with a division of labor between different cell types. Members of this monophyletic group of green flagellates range in complexity from unicellular Chlamydomonas through a series of extant organisms of intermediate size and complexity to Volvox, a genus of spherical organisms that have thousands of cells and a germ-soma division of labor. It is estimated that these organisms all shared a common ancestor about 50 +/- 20 MYA. Here we outline twelve important ways in which the developmental repertoire of an ancestral unicell similar to modern C. reinhardtii was modified to produce first a small colonial organism like Gonium that was capable of swimming directionally, then a sequence of larger organisms (such as Pandorina, Eudorina and Pleodorina) in which there was an increasing tendency to differentiate two cell types, and eventually Volvox carteri with its complete germ-soma division of labor.

In the fruit fly, Drosophila melanogaster, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, we investigated the involvement of a candidate gene, cAMP response-element binding protein (CREB). The duration of rest was inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies did not affect the clock, but increased rest rebound. CREB mutants also had a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increased after rest deprivation and remained elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila.

Stable nuclear transformation of Volvox carten was achieved using the cloned V. carteni itAr gene (which encodes nitrate reductase) to complement a nitA mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of -2.5 x 10-5. DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. cartie becomes one ofthe simplest multicellular organisms that Is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.Volvox carteri is a multicellular organism with a complete division of labor between somatic and reproductive cells (1, 2). Genetic analysis (1-4) has led to the hypothesis that a small number of loci act to cause differentiation of these two cell types (5), and patterns of cell-type-specific gene expression in wild-type and mutant embryos are consistent with that hypothesis (6). However, detailed molecular analysis ofthese putative regulatory loci has awaited a method for transforming the organism with exogenous DNA.We repeatedly tried to transform V. carteri with various bacterial or plant selectable markers that were introduced by microinjection, electroporation, particle bombardment (7), UV-laser microbeam irradiation (8), agitation with glass beads (9), etc. As with the related unicellular alga, Chlamydomonas reinhardtii, reproducible transformation with heterologous selectable markers was not achieved, possibly because of an inability of these algae to express heterologous genes. Again in parallel with C. reinhardtii (9-12), success in transforming V. carteri has come with the availability of a homologous selectable marker: here we report use of the recently cloned nitrate reductase-encoding gene of V. carteri, nitA (13), to complement a nitA mutation. MATERIALS AND METHODSRecipient Strains. Strains used as DNA recipients were F1 female progeny of HB11A, a previously described, multiply marked strain of V. carteri f. nagariensis (4). All of these strains inherited from HB11A a stable mutant allele (reversion rate, <10-8) that confers resistance to chlorate, abolishes the ability to utilize nitrate as a nitrogen source, maps to the nitA locus, and is therefore inferred to be a stable loss-of-function mutation of nitA, the gene encoding nitrate reductase (13). Strain 153-81 was given the mnemonic Gls/ Reg ("gonidialess/regenerator") because it inherited from HB11A a regA mutation that causes somatic cells to redifferentiate as gonidia (asexual reproductive cells) and also has a spontaneous mutation at the gis ("gonidialess") locus that results in an absence of any "true" gonidia (6). GIs/Reg was used in initial studies because it has only one type of cells, all of which can reproduce; thus it provides a homogeneous populati...

Inversion is a dominant aspect of morphogenesis in Volvox. In this process, the hollow, spheroidal Volvox embryo turns inside-out through a small opening called the phialopore to bring flagella from its inner to its outer surface. Analyses of intact, sectioned, and fragmented embryos by light, scanning electron, and transmission electron microscopy, suggest that shape changes preprogrammed into the cells cause inversion. First, cells throughout the embryo change from pear to spindle shape, which causes the embryo to contract and the phialopore to open. Then cells adjacent to the phialopore become flask-shaped, with long, thin stalks at their outer ends. Simultaneously, the cytoplasmic bridges joining all adjacent cells migrate from the midpoint of the cells to the stalk tips. Together, these changes cause the lips of cells at the phialopore margin to cud outward. Now cells progressively more distal to the phialopore become flask-shaped while the more proximal cells become columnar, causing the lips to curl progressively further over the surface of the embryo until the latter has turned completely inside-out. Fine structural analysis reveals a peripheral cytoskeleton of microtubules that is apparently involved in cellular elongation. Cell clusters isolated before inversion undergo a similar program of shape changes; this suggests that the changes in cellular shape are the cause rather than an effect of the inversion process. KEY WORDS cell shape changes eytoskeleton inversion microtubules morphogenesis VolvoxEmbryonic morphogenesis, or the development of form, is frequently accompanied by specific, sequential shape changes in the various cells that participate in the elaboration of the forming structures (2-4, 15). Many of these changes in cell shape can be attributed to the action of cytoskeletal elements such as microtubules and actinlike microfilaments (1,6,17,22,23). But how are such cytoskeletal changes controlled in a cell-and stage-specific manner? Since the form of an adult individual and its component parts are heritable features, it seems obvious that many aspects of morphogenesis, as well as the cellular processes responsible for them, must be under genetic control. However, we have at present little knowledge of the mechanisms by which specific genes control the spatial and temporal coordination of cytoskeletal elements to generate characteristic form. We have chosen to study morphogenesis in Volvox because it offers unique possibilities for obtaining fresh insights into this problem.Members of the genus Volvox are simple, multicellular, eukaryotic, photoautotrophic organisms composed of only two cell types (somatic and THE JOURNAL OF CELL B~LOGu VOLUME 75,