SUMMARYCurrent imaging technology provides an experimental platform in which complex developmental processes can be observed at cellular resolution over an extended time frame. New computational tools are essential to achieve a systems-level understanding of this highcontent information. We have devised a structured approach to systematically analyze complex in vivo phenotypes at cellular resolution, which divides the task into a panel of statistical measurements of each cell in terms of cell differentiation, proliferation and morphogenesis, followed by their spatial and temporal organization in groups and the cohesion within the whole specimen. We demonstrate the approach to C. elegans embryogenesis with in toto imaging and automated cell lineage tracing. We define statistical distributions of the wild-type developmental behaviors at single-cell resolution based on over 50 embryos, cumulating in over 4000 distinct, developmentally based measurements per embryo. These methods enable statistical quantification of abnormalities in mutant or RNAi-treated embryos and a rigorous comparison of embryos by testing each measurement for the probability that it would occur in a wild-type embryo. We demonstrate the power of this structured approach by uncovering quantitative properties including subtle phenotypes in both wild-type and perturbed embryos, transient behaviors that lead to new insights into gene function and a previously undetected source of developmental noise and its subsequent correction.

BACKGROUND Identifying the origin of gliomas carries important implications for advancing the treatment of these recalcitrant tumors. Recent research promotes the hypothesis of a subventricular zone (SVZ) origin for the stemlike gliomagenic cells identified within human glioma specimens. However, conflicting evidence suggests that SVZ-like cells are not uniquely gliomagenic but this capacity may be shared by cycling progenitors distributed throughout the subcortical white matter (SCWM). OBJECTIVE To review radiological evidence in glioblastoma multiforme (GBM) patients to provide insight into the question of glioma ontogeny. METHODS We explored whether GBMs at first diagnosis demonstrated a pattern of anatomic distribution consistent with origin at the SVZ through retrospective analysis of preoperative contrast-enhanced T1-weighted magnetic resonance images in 63 patients. We then examined the relationship of tumor volume, point of origin, and proximity to the ventricles using a computer model of glioma growth. RESULTS Fewer than half of the GBMs analyzed had contrast-enhancing portions that contacted the ventricle on preoperative imaging. A strong correlation was found between tumor volume and the distance between the contrast-enhancing edge of the tumor and the ventricle, demonstrating that tumors abutting the ventricle are significantly larger than those that do not. The lesions simulated by the computer model validated our assumption that tumors that are radiographically distant from the ventricles are unlikely to have originated in the SVZ and supported our hypothesis that as they grow, the edges of all tumors will near the ventricles, regardless of their point of origin. CONCLUSION This work offers further support for the hypothesis that the origins of GBMs are at sites distributed throughout the white matter and are not limited to the region of the SVZ.

BackgroundGastrulation is a key transition in embryogenesis; it requires self-organized cellular coordination, which has to be both robust to allow efficient development and plastic to provide adaptability. Despite the conservation of gastrulation as a key event in Metazoan embryogenesis, the morphogenetic mechanisms of self-organization (how global order or coordination can arise from local interactions) are poorly understood.ResultsWe report a modular structure of cell internalization in Caenorhabditis elegans gastrulation that reveals mechanisms of self-organization. Cells that internalize during gastrulation show apical contractile flows, which are correlated with centripetal extensions from surrounding cells. These extensions converge to seal over the internalizing cells in the form of rosettes. This process represents a distinct mode of monolayer remodeling, with gradual extrusion of the internalizing cells and simultaneous tissue closure without an actin purse-string. We further report that this self-organizing module can adapt to severe topological alterations, providing evidence of scalability and plasticity of actomyosin-based patterning. Finally, we show that globally, the surface cell layer undergoes coplanar division to thin out and spread over the internalizing mass, which resembles epiboly.ConclusionsThe combination of coplanar division-based spreading and recurrent local modules for piecemeal internalization constitutes a system-level solution of gradual volume rearrangement under spatial constraint. Our results suggest that the mode of C. elegans gastrulation can be unified with the general notions of monolayer remodeling and with distinct cellular mechanisms of actomyosin-based morphogenesis.

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser 128 3 Ala does not, whereas the double CRP mutant Thr 127 3 Leu/ Ser 128 3 Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (⌬H b ) ranging from ؊23.4 ؎ 0.9 kJ mol ؊1 for cAMP binding to S128A at 39°C to ؊4.1 ؎ 0.6 kJ mol ؊1 for cAMP binding to T127L/S128A at 24°C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser 179 , Glu 181 , and Thr 182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.