During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.One-Sentence Summary Gaining sequential genetic access to vertebrate cell lineages.Competing Interest StatementThe authors have declared no competing interest.

A complex nervous system requires precise numbers of various neuronal types produced with exquisite spatiotemporal control. This striking diversity is generated by a limited number of neural stem cells (NSC), where spatial and temporal patterning intersect. Drosophila is a genetically tractable model system that has significant advantages for studying stem cell biology and neuronal fate specification. Here we review the latest findings in the rich literature of temporal patterning of neuronal identity in the Drosophila central nervous system. Rapidly changing consecutive transcription factors expressed in NSCs specify short series of neurons with considerable differences. More slowly progressing changes are orchestrated by NSC intrinsic temporal factor gradients which integrate extrinsic signals to coordinate nervous system and organismal development.

During the development of the central nervous system (CNS) of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, ’secondary’ neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Theoretical neuroscientists often try to understand how the structure of a neural network relates to its function by focusing on structural features that would either follow from optimization or occur consistently across possible implementations. Both optimization theories and ensemble modeling approaches have repeatedly proven their worth, and it would simplify theory building considerably if predictions from both theory types could be derived and tested simultaneously. Here we show how tensor formalism from theoretical physics can be used to unify and solve many optimization and ensemble modeling approaches to predicting synaptic connectivity from neuronal responses. We specifically focus on analyzing the solution space of synaptic weights that allow a thresholdlinear neural network to respond in a prescribed way to a limited number of input conditions. For optimization purposes, we compute the synaptic weight vector that minimizes an arbitrary quadratic loss function. For ensemble modeling, we identify synaptic weight features that occur consistently across all solutions bounded by an arbitrary quadratic function. We derive a common solution to this suite of nonlinear problems by showing how each of them reduces to an equivalent linear problem that can be solved analytically. Although identifying the equivalent linear problem is nontrivial, our tensor formalism provides an elegant geometrical perspective that allows us to solve the problem numerically. The final algorithm is applicable to a wide range of interesting neuroscience problems, and the associated geometric insights may carry over to other scientific problems that require constrained optimization.

BACKGROUND: Conditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation. RESULTS: To investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons. CONCLUSIONS: The finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.

The subcellular locations of synapses on pyramidal neurons strongly influences dendritic integration and synaptic plasticity. Despite this, there is little quantitative data on spatial distributions of specific types of synaptic input. Here we use array tomography (AT), a high-resolution optical microscopy method, to examine thalamocortical (TC) input onto layer 5 pyramidal neurons. We first verified the ability of AT to identify synapses using parallel electron microscopic analysis of TC synapses in layer 4. We then use large-scale array tomography (LSAT) to measure TC synapse distribution on L5 pyramidal neurons in a 1.00 × 0.83 × 0.21 mm(3) volume of mouse somatosensory cortex. We found that TC synapses primarily target basal dendrites in layer 5, but also make a considerable input to proximal apical dendrites in L4, consistent with previous work. Our analysis further suggests that TC inputs are biased toward certain branches and, within branches, synapses show significant clustering with an excess of TC synapse nearest neighbors within 5-15 μm compared to a random distribution. Thus, we show that AT is a sensitive and quantitative method to map specific types of synaptic input on the dendrites of entire neurons. We anticipate that this technique will be of wide utility for mapping functionally-relevant anatomical connectivity in neural circuits.

Understanding the functions of a brain region requires knowing the neural representations of its myriad inputs, local neurons and outputs. Primary visual cortex (V1) has long been thought to compute visual orientation from untuned thalamic inputs, but very few thalamic inputs have been measured in any mammal. We determined the response properties of ~28,000 thalamic boutons and ~4,000 cortical neurons in layers 1–5 of awake mouse V1. Using adaptive optics that allows accurate measurement of bouton activity deep in cortex, we found that around half of the boutons in the main thalamorecipient L4 carried orientation-tuned information and that their orientation and direction biases were also dominant in the L4 neuron population, suggesting that these neurons may inherit their selectivity from tuned thalamic inputs. Cortical neurons in all layers exhibited sharper tuning than thalamic boutons and a greater diversity of preferred orientations. Our results provide data-rich constraints for refining mechanistic models of cortical computation.