New directions in craniofacial morphogenesis

The vertebrate head is an extremely complicated structure: development of the head requires tissue-tissue interactions between derivates of all the germ layers and coordinated morphogenetic movements in three dimensions. In this review, we highlight a number of recent embryological studies, using chicken, frog, zebrafish and mouse, which have identified crucial signaling centers in the embryonic face. These studies demonstrate how small variations in growth factor signaling can lead to a diversity of phenotypic outcomes. We also discuss novel genetic studies, in human, mouse and zebrafish, which describe cell biological mechanisms fundamental to the growth and morphogenesis of the craniofacial skeleton. Together, these findings underscore the complex interactions leading to species-specific morphology. These and future studies will improve our understanding of the genetic and environmental influences underlying human craniofacial anomalies.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.     

Differential contribution of direct-developing and stem cell-derived melanocytes to the zebrafish larval pigment pattern

The extent of adult stem cell involvement in embryonic growth is often unclear, as reliable markers or assays for whether a cell is derived from an adult stem cell, such as the melanocyte stem cell (MSC), are typically not available. We have previously shown that two lineages of melanocytes can contribute to the larval zebrafish pigment pattern. The embryo first develops an ontogenetic pattern that is largely composed of ErbB-independent, direct-developing melanocytes. This population can be replaced during regeneration by an ErbB-dependent MSC-derived population following melanocyte ablation. In this study, we developed a melanocyte differentiation assay used together with drugs that ablate the MSC to investigate whether MSC-derived melanocytes contribute to the ontogenetic pattern. We found that essentially all melanocytes that develop before 3 dpf arise from the ErbB-independent, direct-developing population. Similarly, late-developing (after 3 dpf) melanocytes of the head are also ErbB independent. In contrast, the melanocytes that develop after 3 days postfertilization in the lateral and dorsal stripe are sensitive to ErbB inhibitor, indicating that they are derived from the MSC. We show that melanocyte regeneration mutants kit^j1e99 and skiv2l2^j24e1 that are grossly normal for the overall ontogenetic pattern also lack the MSC-derived contribution to the lateral stripe. This result suggests that the underlying regeneration defect of these mutations is a defect in MSC regulation. We suggest that the regulative functions of the MSC may serve quality control roles during larval development, in addition to its established roles in larval regeneration and growth and homeostasis in the adult.     

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue,Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK AFM. Specifically, in this protocol we measure the apparent Young’s modulus of cell walls at subcellular resolutions across regions of up to 100 µm x 100 µm in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.However, several limitations remain: the method can only be used on fairly small samples (around 100 µm in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue’s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.     

Microscopic investigation of three species of Diophrys (Ciliophora,Euplotida, Uronychiidae) from Japan,including Diophrys peculiaris nov. spec

Three species of Diophrys, D. peculiaris nov. spec., D. cf. scutum and D. oligothrix, isolated from the New Nagasaki Fishing Port, Nagasaki, Japan, were investigated using live observation and protargol impregnation. Diophrys peculiaris nov. spec. can be recognized by having two characteristic clusters of rod-like structures and two groups of dikinetids located on anterior dorsal portion of cell. Morphogenetic data show that this part of the life cycle basically proceeds as in congeners, except for the formation of dikinetids under the rod-like structures. In the opisthe, the origin of dikinetids under the rod-like structures is still unknown, but the old dikinetids under the rod-like structures may be retained by the proter. The Japanese population of Diophrys cf. scutum resembles other populations of D. scutum well except for moniliform macronuclear segments. Our populations of D. oligothrix correspond well with other populations in terms of general morphology and ciliary pattern, in particular the continuous dorsal kineties with loosely arranged cilia.     

Inactivation of chitin synthase in Saccharomyces cerevisiae

The activity of chitin synthase extracted from whole cells of Saccharomyces cerevisiae shows reproducible changes during the course of batch cultivation. During exponential growth 5–10% of the enzyme occurs in the active form, whereas in the stationary phase no active enzyme can be detected. Of three yeast proteinases, A, B and C, only B is able to activate pre-chitin synthase and inactivate chitin synthase. A new model of the regulation is presented which accounts for the specific location as well as for termination of chitin synthesis during the budding cycle.These results were reported at the 4th International Symposium on Yeasts in Vienna, July 1974, and are part of doctoral thesis by A.H., University Freiburg (1974).     

Origin and function of fluctuations in cell behaviour and the emergence of patterns

Morphogenesis is the process whereby cells assemble into tissues and organs. Recent studies of this process have revealed heterogeneity of individual cell behaviours that contrasts with the deterministic activity of tissues as a whole. Here we review these observations and suggest that fluctuations and heterogeneities are a central substrate for morphogenesis and that there might exist mechanisms dedicated to the averaging of these fluctuations to ensure robust and reproducible behaviours at the tissue level.     

Morphogenesis by coupled regulatory networks: Reliable control of positional information and proportion regulation

Based on a non-equilibrium mechanism for spatial pattern formation we study how position information can be controlled by locally coupled discrete dynamical networks, similar to gene regulation networks of cells in a developing multicellular organism. As an example we study the developmental problems of domain formation and proportion regulation in the presence of noise, as well as in the presence of cell flow. We find that networks that solve this task exhibit a hierarchical structure of information processing and are of similar complexity as developmental circuits of living cells. Proportion regulation is scalable with system size and leads to sharp, precisely localized boundaries of gene expression domains, even for large numbers of cells. A detailed analysis of noise-induced dynamics, using a mean-field approximation, shows that noise in gene expression states stabilizes (rather than disrupts) the spatial pattern in the presence of cell movements, both for stationary as well as growing systems. Finally, we discuss how this mechanism could be realized in the highly dynamic environment of growing tissues in multicellular organisms.     

Evaluation of novel beta-lactam antibiotics in comparison to cefotaxime on plant regeneration of <Emphasis Type="Italic">Citrus sinensis</Emphasis> L. Osb.

Identification of beta-lactam antibiotics that have negligible effects on plant regeneration is a critical step towards the establishment of a reliable Agrobacterium-mediated transformation protocol for perennial trees. In the present report, we have evaluated the effects of the novel beta-lactam antibiotics meropenem and timentin on plant regeneration of a perennial woody fruit plant, Citrus sinensis, in comparison with the commonly used beta-lactam cefotaxime. It was observed that, in contrast to cefotaxime, meropenem and timentin had a positive or no detrimental effect on the shoot regeneration from epicotyl explants. Residual effects of the beta-lactams from shoot regeneration medium also affected the subsequent ability of the roots to elongate. The addition of meropenem and/or timentin in the rooting medium mostly improved or did not affect the rooting ability of the adventitious shoots. These data indicated that meropenem and timentin can positively replace cefotaxime in Agrobacterium-mediated transformation of C. sinensis.