Stick around: Cell-Cell Adhesion Molecules during Neocortical Development.

The neocortex is an exquisitely organized structure achieved through complex cellular processes from the generation of neural cells to their integration into cortical circuits after complex migration processes. During this long journey, neural cells need to establish and release adhesive interactions through cell surface receptors known as cell adhesion molecules (CAMs). Several types of CAMs have been described regulating different aspects of neurodevelopment. Whereas some of them mediate interactions with the extracellular matrix, others allow contact with additional cells. In this review, we will focus on the role of two important families of cell-cell adhesion molecules (C-CAMs), classical cadherins and nectins, as well as in their effectors, in the control of fundamental processes related with corticogenesis, with special attention in the cooperative actions among the two families of C-CAMs.

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations.

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro.

A comprehensive understanding of the mechanisms controlling the behavior of cell populations with regenerative potential is the first step to design effective therapeutic strategies for many diseases. However, a precise description of the biological events involved, such as proliferation, differentiation, cell fate decisions, migration, or viability, may be hampered by the classical use of experiments based on end-point analysis. By contrast, live imaging and single cell tracking provides researchers with an accurate readout of these features in cells throughout an experiment. Here, we describe a protocol to apply time-lapse video microscopy and post-processing of the data to study critical aspects of the biology and the lineage progression of multiple neural populations.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.