Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expression by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. Since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction.
- Naama Barkai, Weizmann Institute of Science, Israel
© 2015, Peláez et al.
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Epifluorescence miniature microscopes ('miniscopes') are widely used for in vivo calcium imaging of neural population activity. Imaging data is typically collected during a behavioral task and stored for later offline analysis, but emerging techniques for online imaging can support novel closed-loop experiments in which neural population activity is decoded in real time to trigger neurostimulation or sensory feedback. To achieve short feedback latencies, online imaging systems must be optimally designed to maximize computational speed and efficiency while minimizing errors in population decoding. Here we introduce DeCalciOn, an open-source device for real-time imaging and population decoding of in vivo calcium signals that is hardware compatible with all miniscopes that use the UCLA Data Acquisition (DAQ) interface. DeCalciOn performs online motion stabilization, neural enhancement, calcium trace extraction, and decoding of up to 1024 traces per frame at latencies of <50 ms after fluorescence photons arrive at the miniscope image sensor. We show that DeCalciOn can accurately decode the position of rats (n=12) running on a linear track from calcium fluorescence in the hippocampal CA1 layer, and can categorically classify behaviors performed by rats (n=2) during an instrumental task from calcium fluorescence in orbitofrontal cortex (OFC). DeCalciOn achieves high decoding accuracy at short latencies using innovations such as field-programmable gate array (FPGA) hardware for real time image processing and contour-free methods to efficiently extract calcium traces from sensor images. In summary, our system offers an affordable plug-and-play solution for real-time calcium imaging experiments in behaving animals.
High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies is still challenging. Here we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.