Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation

Tissue-specific Cre recombinase often drives 'off-target' reporter activity in the male reproductive tract, suggesting the potential for inter-tissue RNA or protein trafficking in control of sperm maturation or other aspects of reproductive physiology.

Tissue-specific Cre recombinase often drives 'off-target' reporter activity in the male reproductive tract, suggesting the potential for inter-tissue RNA or protein trafficking in control of sperm maturation or other aspects of reproductive physiology.

BackgroundThe Drosophila central nervous system (CNS) is a convenient model system for the study of the molecular mechanisms of conserved neurobiological processes. The manipulation of gene activity in specific cell types and subtypes of the Drosophila CNS is frequently achieved by employing the binary Gal4/UAS system. However, many Gal4 driver lines available from the Bloomington Drosophila Stock Center (BDSC) and commonly used in Drosophila neurobiology are still not well characterized. Among these are three lines with Gal4 driven by the elav promoter (BDSC #8760, #8765, and #458), one line with Gal4 driven by the repo promoter (BDSC #7415), and the 69B-Gal4 line (BDSC #1774). For most of these lines, the exact insertion sites of the transgenes and the detailed expression patterns of Gal4 are not known. This study is aimed at filling these gaps.ResultsWe have mapped the genomic location of the Gal4-bearing P-elements carried by the BDSC lines #8760, #8765, #458, #7415, and #1774. In addition, for each of these lines, we have analyzed the Gal4-driven GFP expression pattern in the third instar larval CNS and eye-antennal imaginal discs. Localizations of the endogenous Elav and Repo proteins were used as markers of neuronal and glial cells, respectively.ConclusionsWe provide a mini-atlas of the spatial activity of Gal4 drivers that are widely used for the expression of UAS–target genes in the Drosophila CNS. The data will be helpful for planning experiments with these drivers and for the correct interpretation of the results.

On Target: New Envelopes for Lentiviral Vectors

MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.

Description Investigations of human immune responses typically focus on adults, resulting in limited knowledge of the pediatric immune system and how it responds to distinct challenges early in life. To bridge this gap, we conducted a 3-year longitudinal multi-omic profiling study of 46 healthy children aged 11-13 as they received flu, HPV, DTaP, and COVID vaccines. This dataset includes scRNAseq of > 3.5 million PBMCs classified into 70 immune cell types based on our Immune Health Atlas, as well as plasma proteomics and high-dimensional flow cytometry. We are utilizing this dataset to characterize the pediatric immune system at baseline and in response to acute and chronic challenges as it compares to the adult immune system, with the ability to compare neoantigen and recall responses within the same children. This analysis revealed age-independent and age-dependent response patterns, with similar cellular compositions and gene-expression changes characterizing response to chronic CMV infection between children and adults but heightened activity in specific cell types responding to flu vaccination in children. We also identified increased activity in specific cell types in response to vaccines that elicit recall as opposed to neoantigen responses, providing a means to understand how immune memory is established and re-engaged in children. These initial findings lay the groundwork upon which we can build and deepen our understanding of immune dynamics in early adolescence. Topic Categories Vaccines and Immunotherapy (VAC)

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.

Tissue-specific Cre driver mice to study vascular diseases

135-OR: Mapping Cis-Regulatory Programs Affecting Diabetes Risk in Pancreatic Islet Cell Types Using Single-Cell Multimodal Profiling

Symptomatic and asymptomatic domoic acid exposure in zebrafish (Danio rerio) revealed distinct non-overlapping gene expression patterns in the brain

Correct gene expression patterns are central for cellular function and the development of organisms. This is controlled by regulatory elements such as enhancers and promoters. In this thesis, I present work from two projects with the goal to identify design principles of promoter and enhancer activity in mammalian genomes. In the first part of the thesis, I focused on CpG island promoters. This promoter type represents the majority of mammalian promoters and is characterised by a high density of the CpG dinucleotide. However, to what extent and how this characteristic dinucleotide contributes to promoter activity is still unclear and is one central question of this project. By monitoring binding of transcription factors (TFs) assumed to play a role in CpG island activity and quantifying the activity of promoter mutants and artificial promoters, we gained insight into the role of CpGs in transcriptional activity. The generated data suggests that high CpG density is not sufficient for transcriptional activity, yet necessary when combined with more complex TF binding motifs. We could further show that DNA methylation decreases activity of promoter mutants with low CpG density. Our experiments led us to hypothesise that high CpG density is required to generate a chromatin environment permissive for transcriptional activity. In the second part of the thesis, I focused on cell type and tissue specific regulatory elements. To illustrate an experimental workflow to identify and test regulatory elements for transcriptional activity in specific cell types, we used the mouse retina, a very specialised tissue comprised of ~50 cell types. To identify regulatory elements, we combined transcriptome and epigenome profiling to map the regulatory landscape of four distinct cell types isolated from mouse retinas (rods, cones, horizontal and starburst amacrine cells). This data also revealed sequence determinants and candidate TFs that control cellular specialisation. We tested previously identified regulatory regions using a parallelised reporter assay for their ability to autonomously control transcriptional activity in the four cell types. We were able to generate a catalogue of cis-regulatory regions active in retinal cell types and further demonstrate their utility as a potential resource for cellular tagging and manipulation. Taken together, the work presented here advances our knowledge about location and regulation of regulatory regions that function in specialised cell types and also provides insight into the regulation of CpG island promoters that tend to be ubiquitously expressed.

Linking activity in specific cell types with perception, cognition, and action, requires quantitative behavioral experiments in genetic model systems such as the mouse. In head-fixed primates, the combination of precise stimulus control, monitoring of motor output, and physiological recordings over large numbers of trials are the foundation on which many conceptually rich and quantitative studies have been built. Choice-based, quantitative behavioral paradigms for head-fixed mice have not been described previously. Here, we report a somatosensory absolute object localization task for head-fixed mice. Mice actively used their mystacial vibrissae (whiskers) to sense the location of a vertical pole presented to one side of the head and reported with licking whether the pole was in a target (go) or a distracter (no-go) location. Mice performed hundreds of trials with high performance (>90% correct) and localized to <0.95 mm (<6 degrees of azimuthal angle). Learning occurred over 1-2 weeks and was observed both within and across sessions. Mice could perform object localization with single whiskers. Silencing barrel cortex abolished performance to chance levels. We measured whisker movement and shape for thousands of trials. Mice moved their whiskers in a highly directed, asymmetric manner, focusing on the target location. Translation of the base of the whiskers along the face contributed substantially to whisker movements. Mice tended to maximize contact with the go (rewarded) stimulus while minimizing contact with the no-go stimulus. We conjecture that this may amplify differences in evoked neural activity between trial types.

Matrix metalloproteinases (MMPs) are a group of zinc endopeptidases that break down the extracellular matrix. MMP-2 and MMP-9 are particularly abundant in the brain and are essential for synaptic plasticity. When studying synaptic plasticity, it is crucial to measure their activity in both normal and pathological conditions. Although gel and in situ zymography are commonly used to assess MMP proteolytic activity, these methods are typically applied to samples and tissues ex vivo and do not allow the quantification of MMP function during a behavioral experiment. Consequently, the in vivo zymography represents the ideal activity assay to study the MMP function. Moreover, we recently developed a method to quantify MMP activity in specific cell types in rat brains. This chapter details the in vivo zymography approach used to accurately quantify the gelatinolytic activity of MMP-2 and MMP-9 in rat brains in both non-cell-specific and cell-specific manners.

Many neural disorders are associated with aberrant activity in specific cell types or neural projection pathways embedded within the densely-wired, heterogeneous matter of the brain. An ideal therapy would permit correction of activity just in specific target neurons, while leaving other neurons unaltered. Recently our lab revealed that the naturally-occurring light-activated proteins channelrhodopsin-2 (ChR2) and halorhodopsin (Halo/NpHR) can, when genetically expressed in neurons, enable them to be safely, precisely, and reversibly activated and silenced by pulses of blue and yellow light, respectively. We here describe the ability to make specific neurons in the brain light-sensitive, using a viral approach. We also reveal the design and construction of a scalable, fully-implantable optical prosthetic capable of delivering light of appropriate intensity and wavelength to targeted neurons at arbitrary 3-D locations within the brain, enabling activation and silencing of specific neuron types at multiple locations. Finally, we demonstrate control of neural activity in the cortex of the non-human primate, a key step in the translation of such technology for human clinical use. Systems for optical targeting of specific neural circuit elements may enable a new generation of high-precision therapies for brain disorders.

A decade has passed since the transcription factor, Runx2, was found to be essential for osteoblast development and chondrocyte hypertrophy. During the last 10 years, our understanding of Runx2's physiological roles and the molecular mechanisms whereby it regulates gene expression to control cell-cycle progression and cellular differentiation has increased exponentially. Runx2 is expressed in osteoblasts, prehypertrophic chondrocytes, mesenchymal cells of the perichondrium, T lymphocytes, endothelial cells, and breast and prostate epithelial cells, with increased expression observed in breast and prostate carcinomas. Although Runx2 and other mammalian Runt domain proteins were originally described as transcriptional activators, they are also transcriptional repressors and thus maintain functional similarities with their Drosophila homolog, Runt. Runx2 binds a consensus DNA sequence but does not possess any enzymatic activities that directly affect chromatin structure. It alters gene expression by recruiting cofactors to gene regulatory elements. Histone deacetylases (HDACs) are among the co-repressors that interact with Runx2. In this review, we summarize data demonstrating that several HDACs and their associated proteins interact with Runx2, regulate its activity, and affect bone formation. HDACs are components of multiprotein complexes that interact with many transcription factors and are subject to regulation by extracellular signals. The elucidation of HDAC complex components that influence Runx2 activity in specific cell types and in response to various extracellular stimuli will increase our understanding of how this crucial transcription factor functions, and how we might be able to control its activity to influence bone formation or reduce bone disease associated with cancer metastasis.