Elizabeth F. Ryder

Migration patterns of clonally related granule cells and their progenitors in the developing chick cerebellum.

During cerebellar development, granule neurons and their progenitors undergo complex migrations. To define these migratory paths better, we used replication-incompetent retroviruses to label dividing cells early in cerebellar development. Clonally related granule cells were widely dispersed in both rostrocaudal and mediolateral planes; clones often spanned the midline. The data suggest that granule cell progenitors originate from the ventricular zone along the entire mediolateral extent of the caudal edge of the cerebellum. After reaching the cerebellar surface, progenitors move primarily rostrally and proliferate in the superficial external granule layer. Postmitotic granule cells then migrate long distances medially and laterally in the transverse plane in the deep external granule layer, where previously they had been thought simply to extend transverse processes.

Lineage analysis using retrovirus vectors.

Publisher Summary This chapter describes the lineage analysis using retrovirus vectors. The complexity and inaccessibility of many types of vertebrate embryos have made lineage analysis through direct approaches, such as time-lapse microscopy and injection of tracers, extremely difficult or impossible. A genetic and clonal solution to lineage mapping in some species is by infection with retrovirus vectors. The chapter summarizes the basis for this technique and details the strategies and current methods in use in the laboratory. A retrovirus vector is an infectious virus that transduces a nonviral gene into mitotic cells in vivo or in vitro. These vectors utilize the same efficient and precise integration machinery of naturally occurring retroviruses to produce a single copy of the viral genome stably integrated into the host chromosome. For lineage applications it is usually necessary to concentrate the virus to achieve sufficient titer. This is typically due to a limitation in the volume that can be injected at any one site. Viruses can be concentrated fairly easily by a relatively short centrifugation step. Virions also can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate is collected by centrifugation. The viral supernatant can be concentrated by centrifugation through a filter that allows only small molecules, to pass (for example, Centricon filters). Regardless of the protocols that are used, it must be kept in mind that retroviral particles are fragile, with short half-lives even under optimum conditions.

UNC-6/netrin and SLT-1/slit guidance cues orient axon outgrowth mediated by MIG-10/RIAM/lamellipodin

BACKGROUND Axon migrations are guided by extracellular cues that can act as repellants or attractants. However, the logic underlying the manner through which attractive and repulsive responses are determined is unclear. Many extracellular guidance cues, and the cellular components that mediate their signals, have been implicated in both types of responses. RESULTS Genetic analyses indicate that MIG-10/RIAM/lamellipodin, a cytoplasmic adaptor protein, functions downstream of the attractive guidance cue UNC-6/netrin and the repulsive guidance cue SLT-1/slit to direct the ventral migration of the AVM and PVM axons in C. elegans. Furthermore, overexpression of MIG-10 in the absence of UNC-6 and SLT-1 induces a multipolar phenotype with undirected outgrowths. Addition of either UNC-6 or SLT-1 causes the neurons to become monopolar. Moreover, the ability of UNC-6 or SLT-1 to direct the axon ventrally is enhanced by the MIG-10 overexpression. We also demonstrate that an interaction between MIG-10 and UNC-34, a protein that promotes actin-filament extension, is important in the response to guidance cues and that MIG-10 colocalizes with actin in cultured cells, where it can induce the formation of lamellipodia. CONCLUSIONS We conclude that MIG-10 mediates the guidance of AVM and PVM axons in response to the extracellular UNC-6 and SLT-1 guidance cues. The attractive and repulsive guidance cues orient MIG-10-dependant axon outgrowth to cause a directional response.

3 Lineage Analysis Using Retroviral Vectors

Knowledge of the genealogical relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Genealogical relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. The use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues is described. An overview of the relevant aspects of the retroviral life cycle is given, and the strategies and current methods in use in our laboratory are described.

FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

BackgroundAdult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence.ResultsWe identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment.ConclusionsTranscriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential.

Lineage analysis with retroviral vectors.

Publisher Summary Knowledge of the genealogical relationships of cells during development provides an insight concerning when and where developmental decisions are being made. Hypotheses can be ruled in or out concerning the commitment of cells to particular fates. For example, when analyzing the cell types that result from the marking of a single progenitor cell, insight can be gained as to whether the progenitor is committed to the production of one or multiple cell types. If multiple cell types are found in a clone, it may be concluded that the progenitor that gave rise to these cells is not restricted to the production of only one cell type. If all of the cells that descend from a progenitor are the same type, the hypothesis is supported that the progenitor is committed to making only that cell type. The complexity and inaccessibility of many types of embryos have made lineage analysis through direct approaches, such as time-lapse microscopy and injection of tracers, almost impossible. A genetic and clonal solution to lineage mapping is the use of retrovirus vectors. The chapter summarizes the basis of this technique, and discusses the current methods and strategies used in the laboratory.

Chemosensory control of surface antigen switching in the nematode Caenorhabditis elegans

Nematodes change their surface compositions in response to environmental signals, which may allow them to survive attacks from microbial pathogens or host immune systems. In the free‐living species Caenorhabditis elegans, wild‐type worms are induced to display an L1 (first larval stage) surface epitope at later larval stages when grown on an extract of spent culture medium (Inducible Larval Display or ILD). Before this study, it was not known whether ILD was regulated by the well‐characterized, neurologically based chemical senses of C. elegans, which mediate other behavioural and developmental responses to environmental signals such as chemotaxis and formation of the facultatively arrested dauer larva stage. We show here that ILD requires the activities of three genes that are essential for the function of the C. elegans chemosensory neurons. ILD was abolished in chemotaxis‐defective che‐3, osm‐3 and tax‐4 mutants. In contrast, chemotaxis‐defective mutants altered in a different gene, srf‐6, show constitutive display of the L1 epitope on all four larval stages. The ILD‐defective che‐3, osm‐3 and tax‐4 mutations blocked the constitutive larval display of an srf‐6 mutant. Combining srf‐6 and certain dauer‐constitutive mutations in double mutants enhanced constitutive dauer formation, consistent with the idea that srf‐6 acts in parallel with specific components of the dauer formation pathway. These results taken together are consistent with the hypothesis that ILD is triggered by environmental signals detected by the nematode‘s chemosensory neurons.

Statistics for the molecular biologist: group comparisons.

In this appendix, some of the statistical tests most commonly used (and misused) in biological research are discussed. These tests are used for comparisons among groups (e.g., t test and ANOVA). A number of other important areas (e.g., linear regression, correlation, and goodness‐of‐fit testing) are not covered. The purpose is to enable the reader to determine rapidly the most appropriate way to analyze data, and to point out some of the most common errors to avoid.

Graphical simulation of early development of the cerebral cortex

Much experimental data exists concerning the development of the cerebral cortex. There is a need for a common vehicle to integrate this data and to allow the testing of hypotheses concerning development. Computer simulation and visualization are powerful mechanisms for hypothesis testing. Our long-term goal is to create a robust, extensible, portable tool for simulation and visualization of cortical development to serve both research and educational purposes. This paper describes a simulation program, SimCortex, which models the early stages of development of the cerebral cortex of the mouse. Version 1.0 of SimCortex models the proliferation of progenitor cells in the pseudostratified ventricular epithelium (PVE), the generation of young neurons and their migration into the cortical plate, which is the forerunner of cell layers II through VI of the adult cortex. We present an overview of the design and implementation of SimCortex, sample output of the system and a comparison of our results with experimental data. We conclude with a brief overview of proposed future enhancements of the system.

Computational methods for single-point and multipoint analysis of genetic variants associated with a simulated complex disorder in a general population.

Several techniques for association analysis have been applied to simulated genetic data for a general population. We describe and compare the performance of three single‐point methods and two multipoint approaches rooted in machine learning and data mining.