Cell movement during chick primitive streak formation

Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI-XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6-7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell-cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell-cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell-cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.     

Key role played by RhoA in the balance between planar and apico-basal cell divisions in the chick neuroepithelium

The cell division axis determines the position of daughter cells and is therefore critical for cell fate. During vertebrate neurogenesis, most cell divisions take place within the plane of the neuroepithelium (Das, T., Payer, B., Cayouette, M., and Harris, W.A. (2003). In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. Neuron 37, 597-609. Haydar, T.F., Ang, E., Jr., and Rakic, P. (2003). Mitotic spindle rotation and mode of cell division in the developing telencephalon. Proc Natl Acad Sci U S A 100, 2890-5. Kosodo, Y., Roper, K., Haubensak, W., Marzesco, A. M., Corbeil, D., and Huttner, W. B. (2004). Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J. 23, 2314-24). The cellular constraints responsible for this preferential orientation are poorly understood. Combining electroporation and time-lapse confocal imaging of chick neural progenitors, the events responsible for positioning the mitotic spindle and their dependence on RhoA were investigated. The results indicate that the spindle forms with a random orientation. However, the final orientation of cell divisions is dependent on two main factors: (i) an early rotation of the spindle that aligns it within the plane of the neuroepithelium, and (ii) a specific limitation of spindle oscillations, despite free rotation around the apico-basal axis. Expressing a dominant-negative RhoA leads to apico-basal cell divisions after a correct initial rotation of the spindle. Our data reveal a specific role for RhoA in the maintenance of spindle orientation, prior to anaphase. Thus, RhoA could be a key player potentially regulated by the neurogenic program or by the neural stem cell environment to control the balance between planar and apico-basal divisions, during normal or pathological development.     

THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.     

Premature centromere division of the X chromosome in neurons in Alzheimer's disease

Premature centromere division (PCD) represents a loss of control over the sequential separation and segregation of chromosome centromeres. Although first described in aging women, PCD on the X chromosome (PCD,X) is markedly elevated in peripheral blood lymphocytes of individuals suffering from Alzheimer disease (AD). The present study evaluated PCD,X, using a fluorescent in situ hybridization method, in interphase nuclei of frontal cerebral cortex neurons from sporadic AD patients and age-matched controls. The average frequency of PCD,X in AD patients (8.60 ± 1.20%) was almost three times higher ( p  < 0.01) than in the control group (2.96 ± 1.20). However, consistent with previous studies, no mitotic cells were found in neurons in either AD or control brain, suggesting an intrinsic inability of post-mitotic neurons to divide. In view of the fact that it has been well-documented that neurons in AD can re-enter into the cell division cycle, the findings presented here of increased PCD advance the hypothesis that deregulation of the cell cycle may contribute to neuronal degeneration and subsequent cognitive deficits in AD.     

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, we detect independent movement of both apical and basal spindle poles, suggesting that forces act on both poles. We show that reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. We propose that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation.     

Hormone mediated regulation of the shoot apical meristem

Recent work on hormone mediated regulation of the SAM is reviewed, emphasizing how combinations of genetic, molecular and modelling approaches have refined models based on classic experimental and physiological work. Special emphasis is given to newly described mechanisms that modulate the responsiveness of specific tissues to hormones and their potential to direct position dependent determination processes.     

Organism size promotes the evolution of specialized cells in multicellular digital organisms

Specialized cells are the essence of complex multicellular life. Fossils allow us to study the modification of specialized, multicellular features such as jaws, scales, and muscular appendages. But it is still unclear what organismal properties contributed to the transition from undifferentiated organisms, which contain only a single cell type, to multicellular organisms with specialized cells. Using digital organisms I studied this transition. My simulations show that the transition to specialized cells happens faster in organism composed of many cells than in organisms composed of few cells. Large organisms suffer less from temporarily unsuccessful evolutionary experiments with individual cells, allowing them to evolve specialized cells via evolutionary trajectories that are unavailable to smaller organisms. This demonstrates that the evolution of simple multicellular organisms which are composed of many functionally identical cells accelerates the evolution of more complex organisms with specialized cells.