Logo: to the web site of Uppsala University

The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6::RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-yr-old at relapse, the cell line offers a practical model for the study of pediatric B-ALL. In recent years, short- and long-read DNA and RNA sequencing have emerged as a complement to karyotyping techniques in the resolution of structural variants in an oncological context. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole-genome sequencing, IsoSeq RNA sequencing, and short-read Illumina sequencing to create a detailed genomic and transcriptomic characterization of the REH cell line. Whole-genome sequencing clarified the molecular traits of disrupted ALL-associated genes including CDKN2A, PAX5, BTG1, VPREB1, and TBL1XR1, as well as the glucocorticoid receptor NR3C1. Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.

Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27 327 cells are freely available to extend to future scRNA-seq methodological comparisons.

Objectives

The aim of this data paper is to describe a collection of 33 genomic, transcriptomic and epigenomic sequencing datasets of the B-cell acute lymphoblastic leukemia (ALL) cell line REH. REH is one of the most frequently used cell lines for functional studies of pediatric ALL, and these data provide a multi-faceted characterization of its molecular features. The datasets described herein, generated with short- and long-read sequencing technologies, can both provide insights into the complex aberrant karyotype of REH, and be used as reference datasets for sequencing data quality assessment or for methods development.

Data description

This paper describes 33 datasets corresponding to 867 gigabases of raw sequencing data generated from the REH cell line. These datasets include five different approaches for whole genome sequencing (WGS) on four sequencing platforms, two RNA sequencing (RNA-seq) techniques on two different sequencing platforms, DNA methylation sequencing, and single-cell ATAC-sequencing.

Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.

The mammalian brain's somatosensory cortex is a topographic map of the body's sensory experience. In mice, cortical barrels reflect whisker input. We asked whether these cortical structures require sensory input to develop or are driven by intrinsic activity. Thalamocortical columns, connecting the thalamus to the cortex, emerge before sensory input and concur with calcium waves in the embryonic thalamus. We show that the columnar organization of the thalamocortical somatotopic map exists in the mouse embryo before sensory input, thus linking spontaneous embryonic thalamic activity to somatosensory map formation. Without thalamic calcium waves, cortical circuits become hyperexcitable, columnar and barrel organization does not emerge, and the somatosensory map lacks anatomical and functional structure. Thus, a self-organized protomap in the embryonic thalamus drives the functional assembly of murine thalamocortical sensory circuits.

When activating muscles, motor neurons in the spinal cord also activate Renshaw cells, which provide recurrent inhibitory feedback to the motor neurons. The tight coupling with motor neurons suggests that Renshaw cells have an integral role in movement, a role that is yet to be elucidated. Here we used the selective expression of the nicotinic cholinergic receptor α2 (Chrna2) in mice to genetically target the vesicular inhibitory amino acid transporter (VIAAT) in Renshaw cells. Loss of VIAAT from Chrna2^Cre-expressing Renshaw cells did not impact any aspect of drug-induced fictive locomotion in the neonatal mouse or change gait, motor coordination, or grip strength in adult mice of both sexes. However, motor neurons from neonatal mice lacking VIAAT in Renshaw cells received spontaneous inhibitory synaptic input with a reduced frequency, showed lower input resistance, and had an increased number of proprioceptive glutamatergic and calbindin-labeled putative Renshaw cell synapses on their soma and proximal dendrites. Concomitantly, Renshaw cells developed with increased excitability and a normal number of cholinergic motor neuron synapses, indicating a compensatory mechanism within the recurrent inhibitory feedback circuit. Our data suggest an integral role for Renshaw cell signaling in shaping the excitability and synaptic input to motor neurons.

The thalamus is a central brain structure with topographically ordered long-range axonal projections that convey sensory information to the cortex via distinct nuclei. Although there is an increasing knowledge about genes important for thalamocortical (TC) development, the identification of genetic landmarks of the distinct thalamic nuclei during the embryonic development has not been addressed systematically. Indeed, a more comprehensive understanding of how the axons from the individual nuclei find their way and connect to their corresponding cortical area is called for. Here, we used a genetic dual labeling strategy in mice to purify distinct principal sensory thalamic neurons. Subsequent genome-wide transcriptome profiling revealed genes specifically expressed in each nucleus during embryonic development. Analysis of regulatory regions of the identified genes revealed key transcription factors and networks that likely underlie the specification of individual sensory-modality TC connections. Finally, the importance of correct axon targeting for the specific sensory-modality population transcriptome was evidenced in a Sema6A mutant, in which visual TC axons are derailed at embryonic life. In sum, our data determined the developmental transcriptional profile of the TC neurons that will eventually support sensory processing.

The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorβ upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.

The thalamus is a central structure of the brain, primarily recognized for the relay of incoming sensory and motor information to the cerebral cortex but also key in high order intracortical communication. It consists of glutamatergic projection neurons organized in several distinct nuclei, each having a stereotype connectivity pattern and functional roles. In the adult, these nuclei can be appreciated by architectural boundaries, although their developmental origin and specification is only recently beginning to be revealed. Here, we summarize the current knowledge on the specification of the distinct thalamic neurons and nuclei, starting from early embryonic patterning until the postnatal days when active sensory experience is initiated and the overall system connectivity is already established. We also include an overview of the guidance processes important for establishing thalamocortical connections, with emphasis on the early topographical specification. The extensively studied thalamocortical axon branching in the cortex is briefly mentioned; however, the maturation and plasticity of this connection are beyond the scope of this review. In separate chapters, additional mechanisms and/or features that influence the specification and development of thalamic neurons and their circuits are also discussed. Finally, an outlook of future directions is given. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 830-843, 2017.

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCs2) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCs2 properties confirmed that neurons genetically marked by the Chrna2-Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole-cell patch-clamp recordings revealed that RCs2 constitute an electrophysiologically stereotyped population with a resting membrane potential of -50.5 +/- 0.4mV and an input resistance of 233.1 +/- 11M. We identified a ZD7288-sensitive hyperpolarization-activated cation current (I-h) in all RCs2, contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCs2 to express small calcium-activated potassium currents (I-SK) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCs2 can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.