Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding

Neuron

2021 Aug 11

Han, S;Okawa, S;Wilkinson, GA;Ghazale, H;Adnani, L;Dixit, R;Tavares, L;Faisal, I;Brooks, MJ;Cortay, V;Zinyk, D;Sivitilli, A;Li, S;Malik, F;Ilnytskyy, Y;Angarica, VE;Gao, J;Chinchalongporn, V;Oproescu, AM;Vasan, L;Touahri, Y;David, LA;Raharjo, E;Kim, JW;Wu, W;Rahmani, W;Chan, JA;Kovalchuk, I;Attisano, L;Kurrasch, D;Dehay, C;Swaroop, A;Castro, DS;Biernaskie, J;Del Sol, A;Schuurmans, C;
PMID: 34407390 | DOI: 10.1016/j.neuron.2021.07.007

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

Spatial omics and multiplexed imaging to explore cancer biology

Nature methods

2021 Aug 02

Lewis, SM;Asselin-Labat, ML;Nguyen, Q;Berthelet, J;Tan, X;Wimmer, VC;Merino, D;Rogers, KL;Naik, SH;
PMID: 34341583 | DOI: 10.1038/s41592-021-01203-6

Understanding intratumoral heterogeneity-the molecular variation among cells within a tumor-promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. Single-cell analyses, especially RNA sequencing and other genomics modalities, have been transformative in revealing novel biomarkers and molecular regulators associated with tumor growth, metastasis and drug resistance. However, these approaches fail to provide a complete picture of tumor biology, as information on cellular location within the tumor microenvironment is lost. New technologies leveraging multiplexed fluorescence, DNA, RNA and isotope labeling enable the detection of tens to thousands of cancer subclones or molecular biomarkers within their native spatial context. The expeditious growth in these techniques, along with methods for multiomics data integration, promises to yield a more comprehensive understanding of cell-to-cell variation within and between individual tumors. Here we provide the current state and future perspectives on the spatial technologies expected to drive the next generation of research and diagnostic and therapeutic strategies for cancer.

Morphological Object Localization: A Novel Image Analysis Pipeline for Quantitative Spatial Localization of Biomolecule Signal from Fluorescence Microscopy Data

Microscopy and Microanalysis

2021 Aug 01

Soltisz, A;Veeraraghavan, R;Bogdanov, V;Gyorke, S;
| DOI: 10.1017/s1431927621009181

The spatial distribution of biomolecules (BMs) within cells and tissues is often a significant determinant of biological / physiological function. Thus, its assessment is not only a ubiquitous feature in the life sciences but also a vital component in the clinical diagnosis and treatment of many pathologies. This typically entails microscopic imaging of fluorescently labeled biomolecules within biological specimens and analysis and interpretation of the resulting images. This is accomplished by methods ranging from qualitative visual assessment of representative images to colocalization analysis, which quantifies the superposition of immunosignals corresponding to co-labeled BMs. These commonly employed approaches have several key limitations. The selection and visual assessment of representative images is subjective and highly susceptible to bias. And, although quantitative, conventional colocalization analysis is susceptible to variations between fluorophores, and oversimplifies complex spatial distributions of BMs into a single numerical index of signal superposition.[1] It provides no information about nonsuperimposed signals, and thus, lacks the sensitivity and selectivity to capture intra- and inter-individual variability or discern subtle forms of biological remodeling, as occurs in the early stages of disease. Here, we present a novel, high-throughput image analysis pipeline, called Morphological Object Localization (MOL), for comprehensive, quantitative spatial localization of BM signals relative to each other as well as structural landmarks from fluorescence microscopy data. This tool offers a quick and user-friendly alternative to current approaches with unprecedented capabilities for quantitative assessment of cell / tissue structure.

Exposure of human fetal kidneys to mild analgesics interferes with early nephrogenesis

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Jul 01

Leverrier-Penna, S;Michel, A;Lecante, LL;Costet, N;Suglia, A;Desdoits-Lethimonier, C;Boulay, H;Viel, R;Chemouny, JM;Becker, E;Lavoué, V;Rolland, AD;Dejucq-Rainsford, N;Vigneau, C;Mazaud-Guittot, S;
PMID: 34105801 | DOI: 10.1096/fj.202100050R

Acetaminophen, aspirin, and ibuprofen are mild analgesics commonly used by pregnant women, the sole current recommendation being to avoid ibuprofen from the fifth month of gestation. The nephrotoxicity of these three analgesics is well documented in adults, as is their interference with prostaglandins biosynthesis. Here we investigated the effect of these analgesics on human first trimester kidneys ex vivo. We first evaluated prostaglandins biosynthesis functionality by performing a wide screening of prostaglandin expression patterns in first trimester human kidneys. We demonstrated that prostaglandins biosynthesis machinery is functional during early nephrogenesis. Human fetal kidney explants aged 7-12 developmental weeks were exposed ex vivo to ibuprofen, aspirin or acetaminophen for 7 days, and analyzed by histology, immunohistochemistry, and flow cytometry. This study has revealed that these analgesics induced a spectrum of abnormalities within early developing structures, ranging from cell death to a decline in differentiating glomeruli density. These results warrant caution for the use of these medicines during the first trimester of pregnancy.

Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1

EMBO reports

2021 Jun 29

Jia, H;Yang, Y;Li, M;Chu, Y;Song, H;Zhang, J;Zhang, D;Zhang, Q;Xu, Y;Wang, J;Xu, H;Zou, X;Peng, H;Hou, Z;
PMID: 34184805 | DOI: 10.15252/embr.202051780

Snail is a dedicated transcriptional repressor and acts as a master inducer of EMT and metastasis, yet the underlying signaling cascades triggered by Snail still remain elusive. Here, we report that Snail promotes colorectal cancer (CRC) migration by preventing non-coding RNA LOC113230-mediated degradation of argininosuccinate synthase 1 (ASS1). LOC113230 is a novel Snail target gene, and Snail binds to the functional E-boxes within its proximal promoter to repress its expression in response to TGF-β induction. Ectopic expression of LOC113230 potently suppresses CRC cell growth, migration, and lung metastasis in xenograft experiments. Mechanistically, LOC113230 acts as a scaffold to facilitate recruiting LRPPRC and the TRAF2 E3 ubiquitin ligase to ASS1, resulting in enhanced ubiquitination and degradation of ASS1 and decreased arginine synthesis. Moreover, elevated ASS1 expression is essential for CRC growth and migration. Collectively, these findings suggest that TGF-β and Snail promote arginine synthesis via inhibiting LOC113230-mediated LRPPRC/TRAF2/ASS1 complex assembly and this complex can serve as potential target for the development of new therapeutic approaches to treat CRC.

N6-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation

Cancer cell

2021 May 12

Cheng, Y;Xie, W;Pickering, BF;Chu, KL;Savino, AM;Yang, X;Luo, H;Nguyen, DT;Mo, S;Barin, E;Velleca, A;Rohwetter, TM;Patel, DJ;Jaffrey, SR;Kharas, MG;
PMID: 34048709 | DOI: 10.1016/j.ccell.2021.04.017

N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m6A dictates its diverse molecular and cellular effects in leukemias remains unknown. We found that YTHDC1 is the essential m6A reader in myeloid leukemia from a genome-wide CRISPR screen and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m6A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m6A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.

PRISM: Recovering cell type specific expression profiles from individual composite RNA-seq samples

Bioinformatics (Oxford, England)

2021 Mar 15

Häkkinen, A;Zhang, K;Alkodsi, A;Andersson, N;Pekcan Erkan, E;Dai, J;Kaipio, K;Lamminen, T;Mansuri, N;Huhtinen, K;Vähärautio, A;Carpén, O;Hynninen, J;Hietanen, S;Lehtonen, R;Hautaniemi, S;
PMID: 33720334 | DOI: 10.1093/bioinformatics/btab178

A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell type specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell type specific expression through whole-genome sequencing and RNA in situ hybridization experiments. https://bitbucket.org/anthakki/prism. Supplementary data are available at Bioinformatics online.

Single cell transcriptomic analysis of murine lung development on hyperoxia-induced damage

Nature communications

2021 Mar 10

Hurskainen, M;Mižíková, I;Cook, DP;Andersson, N;Cyr-Depauw, C;Lesage, F;Helle, E;Renesme, L;Jankov, RP;Heikinheimo, M;Vanderhyden, BC;Thébaud, B;
PMID: 33692365 | DOI: 10.1038/s41467-021-21865-2

During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.

Past, Present and Future of Cocaine- and Amphetamine-Regulated Transcript Peptide

Physiology & behavior

2021 Mar 08

Yosten, GLC;Haddock, CJ;Harada, CM;Almeida-Pereira, G;Kolar, GR;Stein, LM;Hayes, MR;Salvemini, D;Samson, WK;
PMID: 33705816 | DOI: 10.1016/j.physbeh.2021.113380

The existence of the peptide encoded by the cocaine- and amphetamine-regulated transcript (Cartpt) has been recognized since 1981, but it was not until 1995, that the gene encoding CART peptide (CART) was identified. With the availability of the predicted protein sequence of CART investigators were able to identify sites of peptide localization, which then led to numerous approaches attempting to clarify CART's multiple pharmacologic effects and even provide evidence of potential physiologic relevance. Although not without controversy, a picture emerged of the importance of CART in ingestive behaviors, reward behaviors and even pain sensation. Despite the wealth of data hinting at the significance of CART, in the absence of an identified receptor, the full potential for this peptide or its analogs to be developed into therapeutic agents remained unrealized. There was evidence favoring the action of CART via a G protein-coupled receptor (GPCR), but despite multiple attempts the identity of that receptor eluded investigators until recently. Now with the identification of the previously orphaned GPCR, GPR160, as a receptor for CART, focus on this pluripotent neuropeptide will in all likelihood experience a renaissance and the potential for the development of pharmcotherapies targeting GPR160 seems within reach.

Molecular correlates of muscle spindle and Golgi tendon organ afferents

Nature communications

2021 Mar 01

Oliver, KM;Florez-Paz, DM;Badea, TC;Mentis, GZ;Menon, V;de Nooij, JC;
PMID: 33649316 | DOI: 10.1038/s41467-021-21880-3

Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.

Localized EMT reprograms glial progenitors to promote spinal cord repair

Developmental cell

2021 Feb 16

Klatt Shaw, D;Saraswathy, VM;Zhou, L;McAdow, AR;Burris, B;Butka, E;Morris, SA;Dietmann, S;Mokalled, MH;
PMID: 33609461 | DOI: 10.1016/j.devcel.2021.01.017

Anti-regenerative scarring obstructs spinal cord repair in mammals and presents a major hurdle for regenerative medicine. In contrast, adult zebrafish possess specialized glial cells that spontaneously repair spinal cord injuries by forming a pro-regenerative bridge across the severed tissue. To identify the mechanisms that regulate differential regenerative capacity between mammals and zebrafish, we first defined the molecular identity of zebrafish bridging glia and then performed cross-species comparisons with mammalian glia. Our transcriptomics show that pro-regenerative zebrafish glia activate an epithelial-to-mesenchymal transition (EMT) gene program and that EMT gene expression is a major factor distinguishing mammalian and zebrafish glia. Functionally, we found that localized niches of glial progenitors undergo EMT after spinal cord injury in zebrafish and, using large-scale CRISPR-Cas9 mutagenesis, we identified the gene regulatory network that activates EMT and drives functional regeneration. Thus, non-regenerative mammalian glia lack an essential EMT-driving gene regulatory network that reprograms pro-regenerative zebrafish glia after injury.

Evolving Up‐regulation of Biliary Fibrosis–Related Extracellular Matrix Molecules After Successful Portoenterostomy

Hepatology Communications

2021 Feb 09

Kyrönlahti, A;Godbole, N;Akinrinade, O;Soini, T;Nyholm, I;Andersson, N;Hukkinen, M;Lohi, J;Wilson, D;Pihlajoki, M;Pakarinen, M;Heikinheimo, M;
| DOI: 10.1002/hep4.1684

Successful portoenterostomy (SPE) improves the short‐term outcome of patients with biliary atresia (BA) by relieving cholestasis and extending survival with native liver. Despite SPE, hepatic fibrosis progresses in most patients, leading to cirrhosis and a deterioration of liver function. The goal of this study was to characterize the effects of SPE on the BA liver transcriptome. We used messenger RNA sequencing to analyze global gene‐expression patterns in liver biopsies obtained at the time of portoenterostomy (n = 13) and 1 year after SPE (n = 8). Biopsies from pediatric (n = 2) and adult (n = 2) organ donors and other neonatal cholestatic conditions (n = 5) served as controls. SPE was accompanied by attenuation of inflammation and concomitant up‐regulation of key extracellular matrix (ECM) genes. Highly overexpressed genes promoting biliary fibrosis and bile duct integrity, such as integrin subunit beta 6 and previously unreported laminin subunit alpha 3, emerged as candidates to control liver fibrosis after SPE. At a cellular level, the relative abundance of activated hepatic stellate cells and liver macrophages decreased following SPE, whereas portal fibroblasts (PFs) and cholangiocytes persisted. Conclusion: The attenuation of inflammation following SPE coincides with emergence of an ECM molecular fingerprint, a set of profibrotic molecules mechanistically connected to biliary fibrosis. The persistence of activated PFs and cholangiocytes after SPE suggests a central role for these cell types in the progression of biliary fibrosis.

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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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