The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.
Animals locate and track chemoattractive gradients in the environment to find food. With its small nervous system, Caenorhabditis elegans is a good model system in which to understand how the dynamics of neural activity control this search behaviour. Extensive work on the nematode has identified the neurons that are necessary for the different locomotory behaviours underlying chemotaxis through the use of laser ablation, activity recording in immobilized animals and the study of mutants. However, we do not know the neural activity patterns in C. elegans that are sufficient to control its complex chemotactic behaviour. To understand how the activity in its interneurons coordinate different motor programs to lead the animal to food, here we used optogenetics and new optical tools to manipulate neural activity directly in freely moving animals to evoke chemotactic behaviour. By deducing the classes of activity patterns triggered during chemotaxis and exciting individual neurons with these patterns, we identified interneurons that control the essential locomotory programs for this behaviour. Notably, we discovered that controlling the dynamics of activity in just one interneuron pair (AIY) was sufficient to force the animal to locate, turn towards and track virtual light gradients. Two distinct activity patterns triggered in AIY as the animal moved through the gradient controlled reversals and gradual turns to drive chemotactic behaviour. Because AIY neurons are post-synaptic to most chemosensory and thermosensory neurons, it is probable that these activity patterns in AIY have an important role in controlling and coordinating different taxis behaviours of the animal.
Biological systems involving short-range activators and long-range inhibitors can generate complex patterns. Reaction-diffusion models postulate that differences in signaling range are caused by differential diffusivity of inhibitor and activator. Other models suggest that differential clearance underlies different signaling ranges. To test these models, we measured the biophysical properties of the Nodal/Lefty activator/inhibitor system during zebrafish embryogenesis. Analysis of Nodal and Lefty gradients revealed that Nodals have a shorter range than Lefty proteins. Pulse-labeling analysis indicated that Nodals and Leftys have similar clearance kinetics, whereas fluorescence recovery assays revealed that Leftys have a higher effective diffusion coefficient than Nodals. These results indicate that differential diffusivity is the major determinant of the differences in Nodal/Lefty range and provide biophysical support for reaction-diffusion models of activator/inhibitor-mediated patterning.
Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosmotic stress [the High Osmolarity Glycerol (HOG) pathway]. Our method requires stimulating cells with square waves of oscillatory hyperosmotic input (1 M sorbitol) over a range of frequencies and measuring the activity of the HOG pathway in response to this oscillatory input. The input frequency at which the pathway's steady-state activity drops precipitously because the stimulus is changing too rapidly for the pathway to respond faithfully is defined as the pathway bandwidth. In this chapter, we provide details of the techniques required to measure pathway bandwidth in the HOG pathway. These methods are generally useful and can be applied to signaling pathways in S. cerevisiae and other organisms whenever a rapid reporter of pathway activity is available.
Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells.
Genetic and biochemical studies yield information about the component proteins and interactions involved in a cellular signaling pathway. However this parts inventory often does not immediately reveal the in vivo signal processing capabilities and function of the pathway. Signaling pathways are complex systems with dynamic behavior and a systems level approach is needed to understand the physiological roles they play within the cell. We recently used such an approach to measure the signal processing behavior of the budding yeast HOG MAP kinase pathway in response to precisely varied temporal stimuli controlled with a microfluidic device. Despite being a well-studied pathway with well-known components the signaling dynamics and biochemical parameters of this pathway were not known. Our approach allowed us to characterize the pathway's in vivo signal processing and put bounds on all of the in vivo reaction rates. The experimental and theoretical techniques used in our study are general and can be applied to understanding other signaling pathways in a range of biological systems.
Eukaryotic protein kinase pathways have both grown in number and changed their network architecture during evolution. We wondered if there are pivotal proteins in these pathways that have been repeatedly responsible for forming new connections through evolution, thus changing the topology of the network; and if so, whether the underlying properties of these proteins could be exploited to re-engineer and rewire these pathways. We addressed these questions in the context of the mitogen-activated protein kinase (MAPK) pathways. MAPK proteins were found to have repeatedly acquired new specificities and interaction partners during evolution, suggesting that these proteins are pivotal in the kinase network. Using the MAPKs Fus3 and Hog1 of the Saccharomyces cerevisiae mating and hyper-osmolar pathways, respectively, we show that these pivotal proteins can be re-designed to achieve a wide variety of changes in the input-output properties of the MAPK network. Through an analysis of our experimental results and of the sequence and structure of these proteins, we show that rewiring of the network is possible due to the underlying modular design of the MAPKs. We discuss the implications of our findings on the radiation of MAPKs through evolution and on how these proteins achieve their specificity.
The nematode Caenorhabditis elegans has a compact nervous system with only 302 neurons. Whereas most of the synaptic connections between these neurons have been identified by electron microscopy serial reconstructions, functional connections have been inferred between only a few neurons through combinations of electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis. To map functional connections between neurons, we combined in vivo optical stimulation with simultaneous calcium imaging. We analyzed the connections from the ASH sensory neurons and RIM interneurons to the command interneurons AVA and AVD. Stimulation of ASH or RIM neurons using channelrhodopsin-2 (ChR2) resulted in activation of AVA neurons, evoking an avoidance behavior. Our results demonstrate that we can excite specific neurons expressing ChR2 while simultaneously monitoring G-CaMP fluorescence in several other neurons, making it possible to rapidly decipher functional connections in C. elegans neural circuits.