Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche

Cell stem cell

2023 Apr 06

Kraiczy, J;McCarthy, N;Malagola, E;Tie, G;Madha, S;Boffelli, D;Wagner, DE;Wang, TC;Shivdasani, RA;
PMID: 37028407 | DOI: 10.1016/j.stem.2023.03.004

Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.

Emergence of CRISPR/Cas9-mediated bioimaging: A new dawn of in-situ detection

Biosensors & bioelectronics

2023 Jul 15

Tian, M;Zhang, R;Li, J;
PMID: 37086563 | DOI: 10.1016/j.bios.2023.115302

In-situ detection provides deep insights into the function of genes and their relationship with diseases by directly visualizing their spatiotemporal behavior. As an emerging in-situ imaging tool, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bioimaging can localize targets in living and fixed cells. CRISPR-mediated bioimaging has inherent advantages over the gold standard of fluorescent in-situ hybridization (FISH), including fast imaging, cost-effectiveness, and ease of preparation. Existing reviews have provided a detailed classification and overview of the principles of CRISPR-mediated bioimaging. However, the exploitation of potential clinical applicability of this bioimaging technique is still limited. Therefore, analyzing the potential value of CRISPR-mediated in-situ imaging is of great significance to the development of bioimaging. In this review, we initially discuss the available CRISPR-mediated imaging systems from the following aspects: summary of imaging substances, the design and optimization of bioimaging strategies, and factors influencing CRISPR-mediated in-situ detection. Subsequently, we highlight the potential of CRISPR-mediated bioimaging for application in biomedical research and clinical practice. Furthermore, we outline the current bottlenecks and future perspectives of CRISPR-based bioimaging. We believe that this review will facilitate the potential integration of bioimaging-related research with current clinical workflow.

EphB2-dependent prefrontal cortex activation promotes long-range social approach and partner responsiveness

Proceedings of the National Academy of Sciences of the United States of America

2023 Feb 28

He, LN;Chen, S;Yang, Q;Wu, Z;Lao, ZK;Tang, CF;Song, JJ;Liu, XD;Lu, J;Xu, XH;Chen, JJ;Xu, TL;Sun, S;Xu, NJ;
PMID: 36802416 | DOI: 10.1073/pnas.2219952120

Social behavior starts with dynamic approach prior to the final consummation. The flexible processes ensure mutual feedback across social brains to transmit signals. However, how the brain responds to the initial social stimuli precisely to elicit timed behaviors remains elusive. Here, by using real-time calcium recording, we identify the abnormalities of EphB2 mutant with autism-associated Q858X mutation in processing long-range approach and accurate activity of prefrontal cortex (dmPFC). The EphB2-dependent dmPFC activation precedes the behavioral onset and is actively associated with subsequent social action with the partner. Furthermore, we find that partner dmPFC activity is responsive coordinately to the approaching WT mouse rather than Q858X mutant mouse, and the social defects caused by the mutation are rescued by synchro-optogenetic activation in dmPFC of paired social partners. These results thus reveal that EphB2 sustains neuronal activation in the dmPFC that is essential for the proactive modulation of social approach to initial social interaction.

Inhibitory top-down projections from zona incerta mediate neocortical memory

Neuron

2023 Jan 04

Schroeder, A;Pardi, MB;Keijser, J;Dalmay, T;Groisman, AI;Schuman, EM;Sprekeler, H;Letzkus, JJ;
PMID: 36610397 | DOI: 10.1016/j.neuron.2022.12.010

Top-down projections convey a family of signals encoding previous experiences and current aims to the sensory neocortex, where they converge with external bottom-up information to enable perception and memory. Whereas top-down control has been attributed to excitatory pathways, the existence, connectivity, and information content of inhibitory top-down projections remain elusive. Here, we combine synaptic two-photon calcium imaging, circuit mapping, cortex-dependent learning, and chemogenetics in mice to identify GABAergic afferents from the subthalamic zona incerta as a major source of top-down input to the neocortex. Incertocortical transmission undergoes robust plasticity during learning that improves information transfer and mediates behavioral memory. Unlike excitatory pathways, incertocortical afferents form a disinhibitory circuit that encodes learned top-down relevance in a bidirectional manner where the rapid appearance of negative responses serves as the main driver of changes in stimulus representation. Our results therefore reveal the distinctive contribution of long-range (dis)inhibitory afferents to the computational flexibility of neocortical circuits.

Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during postnatal growth

iScience

2023 Jan 01

Zanini, F;Che, X;Knutsen, C;Liu, M;Suresh, N;Domingo-Gonzalez, R;Dou, S;Zhang, D;Pryhuber, G;Jones, R;Quake, S;Cornfield, D;Alvira, C;
| DOI: 10.1016/j.isci.2023.106097

At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2), contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury which impairs angiogenesis, induced both common and unique endothelial gene signatures, dysregulated capillary EC cross-talk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.

Cortical wiring by synapse type-specific control of local protein synthesis

Science (New York, N.Y.)

2022 Nov 25

Bernard, C;Exposito-Alonso, D;Selten, M;Sanalidou, S;Hanusz-Godoy, A;Aguilera, A;Hamid, F;Oozeer, F;Maeso, P;Allison, L;Russell, M;Fleck, RA;Rico, B;Marín, O;
PMID: 36423280 | DOI: 10.1126/science.abm7466

Neurons use local protein synthesis to support their morphological complexity, which requires independent control across multiple subcellular compartments up to the level of individual synapses. We identify a signaling pathway that regulates the local synthesis of proteins required to form excitatory synapses on parvalbumin-expressing (PV+) interneurons in the mouse cerebral cortex. This process involves regulation of the TSC subunit 2 (Tsc2) by the Erb-B2 receptor tyrosine kinase 4 (ErbB4), which enables local control of messenger RNA {mRNA} translation in a cell type-specific and synapse type-specific manner. Ribosome-associated mRNA profiling reveals a molecular program of synaptic proteins downstream of ErbB4 signaling required to form excitatory inputs on PV+ interneurons. Thus, specific connections use local protein synthesis to control synapse formation in the nervous system.

Pooled genetic screens with image-based profiling

Molecular systems biology

2022 Nov 01

Walton, RT;Singh, A;Blainey, PC;
PMID: 36366905 | DOI: 10.15252/msb.202110768

Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Journal of visualized experiments : JoVE

2022 Oct 20

Kerloch, T;Lepko, T;Shkura, K;Guillemot, F;Gillotin, S;
PMID: 36342175 | DOI: 10.3791/64369

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Implementing a multi-colour genetic marker analysis technique for embryology education

Anatomia, histologia, embryologia

2022 Sep 30

Yahya, I;Omer, EAM;Gellisch, M;Brand-Saberi, B;Morosan-Puopolo, G;
PMID: 36177714 | DOI: 10.1111/ahe.12868

Embryology belongs to the basic sciences and is usually an integral part of the anatomy. The subject is traditionally taught by visual inspection of embryonic tissue slides stained with Haematoxylin and Eosin (H&E) to expose the dynamics of tissue histology as development proceeds. While combining in situ hybridization for gene expression analysis and immunostaining for protein expression analysis is an established technique for embryology research, the implementation of this tool in embryology teaching has not been described. The present study was conducted to assess the use of an online multi-colour gene expression analysis technique, alongside histological sections and diagrams, to improve students' understanding of embryology. The participants of this study were bachelor's students of Veterinary Medicine at the University of Khartoum. The method was also evaluated by distributing questionnaire items to Veterinary students via Google forms; subsequently, their responses were analysed qualitatively. The majority of students stated that the new technique was beneficial for their learning of embryology. The multi-colour images proved a more effective means for learning embryology than the traditional H&E image. Results from the students strengthen the belief in applying the multi-colour technique for better embryology course learning.

A red nucleus-VTA glutamate pathway underlies exercise reward and the therapeutic effect of exercise on cocaine use

Science advances

2022 Sep 02

He, Y;Madeo, G;Liang, Y;Zhang, C;Hempel, B;Liu, X;Mu, L;Liu, S;Bi, GH;Galaj, E;Zhang, HY;Shen, H;McDevitt, RA;Gardner, EL;Liu, QS;Xi, ZX;
PMID: 36054363 | DOI: 10.1126/sciadv.abo1440

Physical exercise is rewarding and protective against drug abuse and addiction. However, the neural mechanisms underlying these actions remain unclear. Here, we report that long-term wheel-running produced a more robust increase in c-fos expression in the red nucleus (RN) than in other brain regions. Anatomic and functional assays demonstrated that most RN magnocellular portion (RNm) neurons are glutamatergic. Wheel-running activates a subset of RNm glutamate neurons that project to ventral tegmental area (VTA) dopamine neurons. Optogenetic stimulation of this pathway was rewarding, as assessed by intracranial self-stimulation and conditioned place preference, whereas optical inhibition blocked wheel-running behavior. Running wheel access decreased cocaine self-administration and cocaine seeking during extinction. Last, optogenetic stimulation of the RNm-to-VTA glutamate pathway inhibited responding to cocaine. Together, these findings indicate that physical exercise activates a specific RNm-to-VTA glutamatergic pathway, producing exercise reward and reducing cocaine intake.

Lentiviral Vectors for Ocular Gene Therapy

Pharmaceutics

2022 Jul 31

Arsenijevic, Y;Berger, A;Udry, F;Kostic, C;
PMID: 36015231 | DOI: 10.3390/pharmaceutics14081605

This review offers the basics of lentiviral vector technologies, their advantages and pitfalls, and an overview of their use in the field of ophthalmology. First, the description of the global challenges encountered to develop safe and efficient lentiviral recombinant vectors for clinical application is provided. The risks and the measures taken to minimize secondary effects as well as new strategies using these vectors are also discussed. This review then focuses on lentiviral vectors specifically designed for ocular therapy and goes over preclinical and clinical studies describing their safety and efficacy. A therapeutic approach using lentiviral vector-mediated gene therapy is currently being developed for many ocular diseases, e.g., aged-related macular degeneration, retinopathy of prematurity, inherited retinal dystrophies (Leber congenital amaurosis type 2, Stargardt disease, Usher syndrome), glaucoma, and corneal fibrosis or engraftment rejection. In summary, this review shows how lentiviral vectors offer an interesting alternative for gene therapy in all ocular compartments.

Repair of the Murine Tympanic Membrane Displays Hallmarks of Regeneration

The FASEB Journal

2022 May 01

Scaria, S;Frumm, S;Sheth, A;Tward, A;
| DOI: 10.1096/fasebj.2022.36.S1.R3190

RNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time-dependent transcriptional shifts in each layer of the TM. From both cross-sectional and whole-mount views, the TM shows a rapid, proliferative response to injury by 18 hours post-injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi-layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar-free structure. We identified a regeneration-induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A _K5Cre-ERT2;Confetti_ mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co-factors. When EGFR is deleted _in vivo_, using a _K5-CreERT2_;_Egfrfl/fl; R26mTmG/mTmG_ mouse model, TMs no longer display proliferation post-injury and cannot repair perforations.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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