Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells.

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.

Phenotypic engineering of sperm-production rate confirms evolutionary predictions of sperm competition theory.

Sperm production is a key male reproductive trait and an important parameter in sperm competition theory. Under sperm competition, paternity success is predicted to depend strongly on male allocation to sperm production. Furthermore, because sperm production is inherently costly, individuals should economize in sperm expenditure, and conditional adjustment of the copulation frequency according to their sperm availability may be expected. However, experimental studies showing effects of sperm production on mating behaviour and paternity success have so far been scarce, mainly because sperm production is difficult to manipulate directly in animals. Here, we used phenotypic engineering to manipulate sperm-production rate, by employing dose-dependent RNA interference (RNAi) of a spermatogenesis-specific gene, macbol1, in the free-living flatworm Macrostomum lignano. We demonstrate (i) that our novel dose-dependent RNAi approach allows us to induce high variability in sperm-production rate; (ii) that a reduced sperm-production rate is associated with a decreased copulation frequency, suggesting conditional adjustment of mating behaviour; and (iii) that both sperm production and copulation frequency are important determinants of paternity success in a competitive situation, as predicted by sperm competition theory. Our study clearly documents the potential of phenotypic engineering via dose-dependent RNAi to test quantitative predictions of evolutionary theory.

Boule-like genes regulate male and female gametogenesis in the flatworm Macrostomum lignano.

Members of the DAZ (Deleted in AZoospermia) gene family are important players in the process of gametogenesis and their dysregulation accounts for 10% of human male infertility. Boule, the ancestor of the family, is mainly involved in male meiosis in most organisms. With the exception of Drosophila and C. elegans, nothing is known on the function of boule in non-vertebrate animals. In the present study, we report on three boule orthologues in the flatworm Macrostomum lignano. We demonstrate that macbol1 and macbol2 are expressed in testes whilst macbol3 is expressed in ovaries and developing eggs. Macbol1 RNAi blocked spermatocyte differentiation whereas macbol2 showed no effect upon RNAi treatment. Macbol3 RNAi resulted in aberrant egg maturation and led to female sterility. We further demonstrated the evolutionary functional conservation of macbol1 by introducing this gene into Drosophila bol(1) mutants. Macbol1 was able to rescue the progression of fly meiotic divisions. In summary, our findings provide evidence for an involvement of boule genes in male and female gamete development in one organism. Furthermore, boule gene function is shown here for the first time in a lophotrochozoan. Our results point to a more diverse functional assignment of boule genes. Therefore, a better understanding of boule function in flatworms can help to elucidate the molecular mechanisms of and concomitant infertility in higher organisms including humans.

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms.

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.

Potential of Macrostomum lignano to recover from gamma-ray irradiation.

Stem cells are the only proliferating cells in flatworms and can be eliminated by irradiation with no damage to differentiated cells. We investigated the effect of fractionated irradiation schemes on Macrostomum lignano, namely, on survival, gene expression, morphology and regeneration. Proliferating cells were almost undetectable during the first week post-treatment. Cell proliferation and gene expression were restored within 1 month in a dose-dependent manner following exposure to up to 150 Gy irradiation. During recovery, stem cells did not cross the midline but were restricted within lateral compartments. An accumulated dose of 210 Gy resulted in a lethal phenotype. Our findings demonstrate that M. lignano represents a suitable model system for elucidating the effect of irradiation on the stem cell system in flatworms and for improving our understanding of the recovery potential of severely damaged stem-cell systems.

Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano.

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.

Characterization of the stem cell system of the acoel Isodiametra pulchra.

BACKGROUND: Tissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration. RESULTS: We established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation. CONCLUSIONS: We introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.

The exceptional stem cell system of Macrostomum lignano: screening for gene expression and studying cell proliferation by hydroxyurea treatment and irradiation.

BACKGROUND: Flatworms are characterized by an outstanding stem cell system. These stem cells (neoblasts) can give rise to all cell types including germ cells and power the exceptional regenerative capacity of many flatworm species. Macrostomum lignano is an emerging model system to study stem cell biology of flatworms. It is complementary to the well-studied planarians because of its small size, transparency, simple culture maintenance, the basal taxonomic position and its less derived embryogenesis that is more closely related to spiralians. The development of cell-, tissue- and organ specific markers is necessary to further characterize the differentiation potential of flatworm stem cells. Large scale in situ hybridization is a suitable tool to identify possible markers. Distinguished genes identified in a large scale screen in combination with manipulation of neoblasts by hydroxyurea or irradiation will advance our understanding of differentiation and regulation of the flatworm stem cell system. RESULTS: We have set up a protocol for high throughput large scale whole mount in situ hybridization for the flatworm Macrostomum lignano. In the pilot screen, a number of cell-, tissue- or organ specific expression patterns were identified. We have selected two stem cell- and germ cell related genes--macvasa and macpiwi--and studied effects of hydroxyurea (HU) treatment or irradiation on gene expression. In addition, we have followed cell proliferation using a mitosis marker and bromodeoxyuridine labeling of S-phase cells after various periods of HU exposure or different irradiation levels. HU mediated depletion of cell proliferation and HU induced reduction of gene expression was used to generate a cDNA library by suppressive subtractive hybridization. 147 differentially expressed genes were sequenced and assigned to different categories. CONCLUSION: We show that Macrostomum lignano is a suitable organism to perform high throughput large scale whole mount in situ hybridization. Genes identified in such screens--together with BrdU/H3 labeling--can be used to obtain information on flatworm neoblasts.

Reprogramming to pluripotency does not require transition through a primitive streak-like state.

Pluripotency can be induced in vitro from adult somatic mammalian cells by enforced expression of defined transcription factors regulating and initiating the pluripotency network. Despite the substantial advances over the last decade to improve the efficiency of direct reprogramming, exact mechanisms underlying the conversion into the pluripotent stem cell state are still vaguely understood. Several studies suggested that induced pluripotency follows reversed embryonic development. For somatic cells of mesodermal and endodermal origin that would require the transition through a Primitive streak-like state, which would necessarily require an Eomesodermin (Eomes) expressing intermediate. We analyzed reprogramming in human and mouse cells of mesodermal as well as ectodermal origin by thorough marker gene analyses in combination with genetic reporters, conditional loss of function and stable fate-labeling for the broad primitive streak marker Eomes. We unambiguously demonstrate that induced pluripotency is not dependent on a transient primitive streak-like stage and thus does not represent reversal of mesendodermal development in vivo.

A Resource for the Transcriptional Signature of Bona Fide Trophoblast Stem Cells and Analysis of Their Embryonic Persistence.

Trophoblast stem cells (TSCs) represent the multipotent progenitors that give rise to the different cells of the embryonic portion of the placenta. Here, we analysed the expression of key TSC transcription factors Cdx2, Eomes, and Elf5 in the early developing placenta of mouse embryos and in cultured TSCs and reveal surprising heterogeneity in protein levels. We analysed persistence of TSCs in the early placenta and find that TSCs remain in the chorionic hinge until E9.5 and are lost shortly afterwards. To define the transcriptional signature of bona fide TSCs, we used inducible gain- and loss-of-function alleles of Eomes or Cdx2, and Eomes (GFP), to manipulate and monitor the core maintenance factors of TSCs, followed by genome-wide expression profiling. Combinatorial analysis of resulting expression profiles allowed for defining novel TSC marker genes that might functionally contribute to the maintenance of the TSC state. Analyses by qRT-PCR and in situ hybridisation validated novel TSC- and chorion-specific marker genes, such as Bok/Mtd, Cldn26, Duox2, Duoxa2, Nr0b1, and Sox21. Thus, these expression data provide a valuable resource for the transcriptional signature of bona fide and early differentiating TSCs and may contribute to an increased understanding of the transcriptional circuitries that maintain and/or establish stemness of TSCs.

To be or not to be a flatworm: the acoel controversy.

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa -- the Catenulida, the Acoelomorpha and the Rhabditophora -- have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.