Malathion is an organophosphorus insecticide, which is often sprayed to control mosquitoes. When applied to aquatic habitats, malathion can also influence the embryogenesis of non-target organisms such as frogs and fish. We modified the frog embryo teratogen assay in Xenopus (FETAX), a standard toxicological assay, into an investigative undergraduate laboratory exercise. This exercise provided students with experience in developmental toxicology, experimental design, quantitative morphology, digital imaging, and presentation of research results. Their results demonstrated that Xenopus embryos exposed to malathion on the first or second day of development were indistinguishable from controls, while embryos exposed to malathion on the third day of development exhibited significantly bent tails and shorter body lengths. Similarly, sublethal malathion exposures also compromised early zebrafish development. To determine if this investigative laboratory exercise met its goal of fostering conceptual understanding of developmental toxicology, we compared student performance on a questionnaire before and after the laboratory exercise, which demonstrated significant improvement in conceptual understanding. Moreover, all (45/45) students successfully completed a modified FETAX and prepared posters of their results, indicating that students learned quantitative morphology and imaging skills while also gaining valuable experience in designing, executing, and communicating an experiment.

Laminin-1 is a glycoprotein found in the basement membrane of many tissues. In the cerebellum of rodents, it has also been localized along Bergmann glial fibers, where it is thought to be involved in promoting granule cell migration by enhancing adhesion and neurite outgrowth along these fibers. Recent reports, however, indicate that laminin-1 is not present on Bergmann fibers, but instead is associated with blood vessels and meninges. Furthermore, attempts to block granule cell migration using antibodies against laminin-1 have yielded conflicting results. In this report, we provide further evidence that laminin-1 is associated exclusively with blood vessels and meninges in the cerebellum of postnatal rats. In addition, we show that adhesion and neurite outgrowth of granule cells was impeded on laminin-coated surfaces. In fact, cerebellar cells dramatically and consistently avoided laminin-1 regions of patterned surfaces. Cells did adhere to laminin regions if it was coadsorbed with polylysine or tested in serum-containing medium. Avoidance of laminin-1 regions in culture was not, however, blocked by pretreatment with laminin-1 antibodies. By comparison, mouse neuroblastoma cells adhered preferentially to laminin-1 regions in serum-free medium, a response which was blocked by laminin-1 antibodies. These results indicate that laminin-1 is not involved in granule cell migration along Bergmann glial fibers. Instead, they suggest that laminin-1 may function as a repulsive guidance cue preventing granule cells from following inappropriate pathways during development.

Materials with spatially resolved chemistries (i.e. patterned surfaces) have been used to guide and organize the position of mammalian cells in vitro. A common theme in guiding the spatial distribution of cells has been the use of patterned alkylsiloxanes, where one region contains an aminosilane and the other an alkylsilane. The regions of the aminosilane served as preferential sites for cell attachment and spreading, presumably dependent on the association between cell surface proteoglycans the positively charged amine. In this study, experiments were conducted with patterns of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS) to determine the kinetics of spatial organization of bone-derived cells, and whether initial attachment and spreading affected the rate of matrix mineralization (i.e. bone formation) in extended cultures. The bone cells required the presence of serum or preadsorption of serum proteins to the patterned EDS/DMS surface to organize according to the lithographically defined surface chemistry. Time-lapse video microscopy indicated that cells were randomly distributed over the EDS/DMS surface at the time of plating, but organized on the EDS regions within 30 min. When cultures were extended for 15 and 25 days, the matrix synthesized by the cells was preferentially mineralized on the EDS chemistry. These results demonstrate the ability of surface chemistry modifications to organize cells and form mineralized tissue in vitro. The methods employed should have general value to the engineering of tissues in vitro.

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n ‐octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N ‐(2‐aminoethyl)‐3‐aminopropyl‐trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time‐dependent process. Angle dependent x‐ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was imobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.

Pathfinding is a fundamental behavior of migrating neuroblasts and advancing growth cones. We have analyzed this behavior in culture using mouse neuroblastoma (N1E-115) cells grown on a chemically patterned surface. The patterned surface was defined photolithographically and consisted of intersecting 10-μm-wide pathways. The pathways were coated with positively charged amines and separated by regions bound with uncharged alkanes. Cells and growth cones were guided along the pathways and made choices at intersections. Whereas migrating cells made random choices at intersections, growth cones displayed a preference for advancing straight ahead. Interference reflection microscopy (IRM) revealed that pathfinding by cells and growth cones was correlated with greater overall attachment to aminated regions, although cell bodies and appendages also attached to adjacent alkanated regions. Thus guidance was not simply due to contact inhibition by alkanes; rather, it was due to "preferential" adhesion to aminated surfaces. Gray level analysis of IRM images demonstrated that focal and close contacts were made on both surfaces, indicating that preferential adhesion was not the result of tighter attachment to aminated surfaces. Fluorescent labeling of F-actin and microtubules indicated that preferential adhesion was not due to compartmentalization of these cytoskeletal structures on aminated regions. We propose that preferential adhesion involved a signal transduction mechanism that discriminated between positively charged and uncharged molecules. Such a mechanism could contribute to pathfinding by neuroblasts and growth cones along extracellular matrix proteins in vivo.

A fast, inexpensive, and versatile technique for patterning the surface of glass coverslips with molecules of biological interest is described. The technique combines photolithographic, silane-coupling, and protein adsorption procedures to pattern coverslips with amines, alkanes, and proteins with micrometer spatial resolution. The attachment of amines and alkanes was verified using contact angle and X-ray photoelectron spectroscopic (XPS) measurements. XPS results showed that amines and alkanes were attached in 1-4 nm thickness covering approximately 20% and 45%, respectively, of the surface. Patterns of amines were visualized using fluorescent staining, and patterns of proteins were detected immunochemically. Patterned coverslips were used to investigate adhesion and neurite outgrowth of mouse neuroblastoma (N1E-115) cells. Cells were examined on the following patterns: alkane-glass, protein-glass, amine-alkane, and amine-protein. Cell attachment and neurite outgrowth on patterned coverslips displayed the following preferences: laminin, fibronectin, or collagen IV > amine or glass > alkane or bovine serum albumin. This patterning method should be useful for studies of cell-surface interactions, cell migration, nerve regeneration, and the formation of neural networks in vitro.