Aldehyde-driven transcriptional stress triggers an anorexic DNA damage response

Nature

2021 Dec 01

Mulderrig, L;Garaycoechea, JI;Tuong, ZK;Millington, CL;Dingler, FA;Ferdinand, JR;Gaul, L;Tadross, JA;Arends, MJ;O'Rahilly, S;Crossan, GP;Clatworthy, MR;Patel, KJ;
PMID: 34819667 | DOI: 10.1038/s41586-021-04133-7

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.

Smad4 controls signaling robustness and morphogenesis by differentially contributing to the Nodal and BMP pathways

Nature communications

2021 Nov 04

Guglielmi, L;Heliot, C;Kumar, S;Alexandrov, Y;Gori, I;Papaleonidopoulou, F;Barrington, C;East, P;Economou, AD;French, PMW;McGinty, J;Hill, CS;
PMID: 34737283 | DOI: 10.1038/s41467-021-26486-3

The transcriptional effector SMAD4 is a core component of the TGF-β family signaling pathways. However, its role in vertebrate embryo development remains unresolved. To address this, we deleted Smad4 in zebrafish and investigated the consequences of this on signaling by the TGF-β family morphogens, BMPs and Nodal. We demonstrate that in the absence of Smad4, dorsal/ventral embryo patterning is disrupted due to the loss of BMP signaling. However, unexpectedly, Nodal signaling is maintained, but lacks robustness. This Smad4-independent Nodal signaling is sufficient for mesoderm specification, but not for optimal endoderm specification. Furthermore, using Optical Projection Tomography in combination with 3D embryo morphometry, we have generated a BMP morphospace and demonstrate that Smad4 mutants are morphologically indistinguishable from embryos in which BMP signaling has been genetically/pharmacologically perturbed. Smad4 is thus differentially required for signaling by different TGF-β family ligands, which has implications for diseases where Smad4 is mutated or deleted.

Corticospinal neuron subpopulation-specific developmental genes prospectively indicate mature segmentally specific axon projection targeting

Cell reports

2021 Oct 19

Sahni, V;Shnider, SJ;Jabaudon, D;Song, JHT;Itoh, Y;Greig, LC;Macklis, JD;
PMID: 34686320 | DOI: 10.1016/j.celrep.2021.109843

For precise motor control, distinct subpopulations of corticospinal neurons (CSN) must extend axons to distinct spinal segments, from proximal targets in the brainstem and cervical cord to distal targets in thoracic and lumbar spinal segments. We find that developing CSN subpopulations exhibit striking axon targeting specificity in spinal white matter, which establishes the foundation for durable specificity of adult corticospinal circuitry. Employing developmental retrograde and anterograde labeling, and their distinct neocortical locations, we purified developing CSN subpopulations using fluorescence-activated cell sorting to identify genes differentially expressed between bulbar-cervical and thoracolumbar-projecting CSN subpopulations at critical developmental times. These segmentally distinct CSN subpopulations are molecularly distinct from the earliest stages of axon extension, enabling prospective identification even before eventual axon targeting decisions are evident in the spinal cord. This molecular delineation extends beyond simple spatial separation of these subpopulations in the cortex. Together, these results identify candidate molecular controls over segmentally specific corticospinal axon projection targeting.

3D-cardiomics: A spatial transcriptional atlas of the mammalian heart

Journal of molecular and cellular cardiology

2021 Oct 05

Mohenska, M;Tan, NM;Tokolyi, A;Furtado, MB;Costa, MW;Perry, AJ;Hatwell-Humble, J;van Duijvenboden, K;Nim, HT;Ji, YMM;Charitakis, N;Bienroth, D;Bolk, F;Vivien, C;Knaupp, AS;Powell, DR;Elliott, DA;Porrello, ER;Nilsson, SK;Del Monte-Nieto, G;Rosenthal, NA;Rossello, FJ;Polo, JM;Ramialison, M;
PMID: 34624332 | DOI: 10.1016/j.yjmcc.2021.09.011

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/.

A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies

Nature neuroscience

2021 Aug 19

Kang, Y;Zhou, Y;Li, Y;Han, Y;Xu, J;Niu, W;Li, Z;Liu, S;Feng, H;Huang, W;Duan, R;Xu, T;Raj, N;Zhang, F;Dou, J;Xu, C;Wu, H;Bassell, GJ;Warren, ST;Allen, EG;Jin, P;Wen, Z;
PMID: 34413513 | DOI: 10.1038/s41593-021-00913-6

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that can regulate the translation of specific mRNAs. In this study, we developed an FXS human forebrain organoid model and observed that the loss of FMRP led to dysregulated neurogenesis, neuronal maturation and neuronal excitability. Bulk and single-cell gene expression analyses of FXS forebrain organoids revealed that the loss of FMRP altered gene expression in a cell-type-specific manner. The developmental deficits in FXS forebrain organoids could be rescued by inhibiting the phosphoinositide 3-kinase pathway but not the metabotropic glutamate pathway disrupted in the FXS mouse model. We identified a large number of human-specific mRNAs bound by FMRP. One of these human-specific FMRP targets, CHD2, contributed to the altered gene expression in FXS organoids. Collectively, our study revealed molecular, cellular and electrophysiological abnormalities associated with the loss of FMRP during human brain development.

Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence

Nature

2021 Apr 01

Choi, S;Zhang, B;Ma, S;Gonzalez-Celeiro, M;Stein, D;Jin, X;Kim, ST;Kang, YL;Besnard, A;Rezza, A;Grisanti, L;Buenrostro, JD;Rendl, M;Nahrendorf, M;Sahay, A;Hsu, YC;
PMID: 33790465 | DOI: 10.1038/s41586-021-03417-2

Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here we report that the stress hormone corticosterone-which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans-regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Scientific reports

2021 Mar 24

Ma, J;Tran, G;Wan, AMD;Young, EWK;Kumacheva, E;Iscove, NN;Zandstra, PW;
PMID: 33762663 | DOI: 10.1038/s41598-021-86087-4

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

ASH2L Controls Ureteric Bud Morphogenesis via Regulation of RET/GFRA1 Signaling Activity in a Mouse Model

Journal of the American Society of Nephrology : JASN

2023 Feb 09

Zhao, Z;Dai, X;Jiang, G;Lin, F;
PMID: 36758123 | DOI: 10.1681/ASN.0000000000000099

Ureteric bud induction and branching morphogenesis is fundamental to the establishment of the renal architecture and is a key determinant of nephron number. Defective ureteric bud morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor RET and coreceptor GFRA1 appears to be particularly important in ureteric bud development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT.To investigate whether and how inactivation of Ash2l, which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the ureteric bud lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with CUT&Tag-seq.Ureteric bud-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the ureteric bud tip, delaying budding and impairing ureteric bud branching morphogenesis. These effects were associated with downregulation of Ret, Gfra1, and Wnt11, which participate in RET/GFRA1 signaling.These experiments identify ASH2L-dependent H3K4 methylation in the ureteric bud lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in ureteric bud morphogenesis, which, if deficient, may lead to CAKUT.

Transcriptomic mapping of the metzincin landscape in human trophoblasts

Gene expression patterns : GEP

2022 Oct 25

Wächter, J;Shannon, MJ;Beristain, AG;
PMID: 36307023 | DOI: 10.1016/j.gep.2022.119283

The metzincin family of metalloproteases coordinates tissue developmental processes through regulation of growth factor availability, receptor signaling, and cell-cell/cell-matrix adhesion. While roles for select metzincins in controlling trophoblast functions in human placental development have been described, a comprehensive understanding of metzincin dynamics during trophoblast differentiation is lacking. To address this knowledge gap, single cell transcriptomic datasets derived from first trimester chorionic villi and decidua were used to decipher metzincin expression profiles and kinetics in diverse cell types within the utero-placental interface. Further, specific protease-substrate interactions within progenitor trophoblasts were examined to better define the progenitor niche. Within the uterine-placental compartment, 43 metzincin proteases were expressed across 15 cell-type clusters. Metzincin subgroups expressed in placental trophoblasts, placental mesenchymal cells, uterine stromal, and immune cells included multiple matrix metalloproteases (MMPs), a disintegrin and metalloproteases (ADAMs), a disintegrin and metalloproteases with thrombospondin repeats (ADAMTSs), pappalysins, and astacins. Within the trophoblast compartment, eight distinct trophoblasts states were identified: four cytotrophoblast (CTB), one syncytiotrophoblast precursor (SCTp), two column CTB (cCTB), and one extravillous trophoblast (EVT). Within these states 7 MMP, 8 ADAM, 4 ADAMTS, 2 pappalysin, and 3 astacin proteases were expressed. Cell trajectory modeling shows that expression of most (19/24) metzincins increase during EVT differentiation, though expression of select metalloproteases increase along the villous pathway. Eleven metzincins (ADAM10, -17, MMP14, -15, -19, -23B, ADAMTS1, -6, -19, TLL-1, -2) showed enrichment within CTB progenitors, and analysis of metzincin-substrate interactions identified ∼150 substrates and binding partners, including FBN2 as an ADAMTS6-specific substrate. Together, this work characterizes the metzincin landscape in human first trimester trophoblasts and establishes insight into the roles specific proteases perform within distinct trophoblast niches and across trophoblast differentiation. This resource serves as a guide for future investigations into the roles of metzincin proteases in human placental development.

Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome

American journal of human genetics

2021 Oct 07

Mahyari, E;Guo, J;Lima, AC;Lewinsohn, DP;Stendahl, AM;Vigh-Conrad, KA;Nie, X;Nagirnaja, L;Rockweiler, NB;Carrell, DT;Hotaling, JM;Aston, KI;Conrad, DF;
PMID: 34626582 | DOI: 10.1016/j.ajhg.2021.09.001

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.

Transcriptional profiling of equine endometrium before, during and after capsule disintegration during normal pregnancy and after oxytocin-induced luteostasis in non-pregnant mares

PloS one

2021 Oct 06

Klein, C;Bruce, P;Hammermueller, J;Hayes, T;Lillie, B;Betteridge, K;
PMID: 34614002 | DOI: 10.1371/journal.pone.0257161

The current study used RNA sequencing to determine transcriptional profiles of equine endometrium collected 14, 22, and 28 days after ovulation from pregnant mares. In addition, the transcriptomes of endometrial samples obtained 20 days after ovulation from pregnant mares, and from non-pregnant mares which displayed and failed to display extended luteal function following the administration of oxytocin, were determined and compared in order to delineate genes whose expressions depend on the presence of the conceptus as opposed to elevated progesterone alone. A mere fifty-five transcripts were differentially expressed between samples collected from mares at Day 22 and Day 28 of pregnancy. This likely reflects the longer-term exposure to a relatively constant, progesterone-dominated environment with little change in factors secreted by the conceptus that would affect endometrial gene expression. The complement system was amongst the canonical pathways significantly enriched in transcripts differentially expressed between Day 14 and Day 22/28 of pregnancy. The expression of complement components 7 and 8 was confirmed using in situ hybridization. The expression of SERPING1, an inhibitor of the complement system, was confirmed by immunohistochemistry. In line with the resumed capacity of the endometrium to produce prostaglandin, prostaglandin G/H synthase 1 was expressed at higher levels at Days 22 and 28 than at Day 14 of pregnancy. Our data suggest that this up-regulation is enhanced by the presence of the conceptus; samples obtained from mares at Day 20 of pregnancy had significantly higher levels of prostaglandin G/H synthase 1 transcript than mares with extended luteal function.

Human Adult Fibroblast-like Synoviocytes and Articular Chondrocytes Exhibit Prominent Overlap in Their Transcriptomic Signatures

ACR open rheumatology

2021 May 01

Jones, K;Angelozzi, M;Gangishetti, U;Haseeb, A;de Charleroy, C;Lefebvre, V;Bhattaram, P;
PMID: 33931959 | DOI: 10.1002/acr2.11255

Fibroblast-like synoviocytes (FLS) and articular chondrocytes (AC) derive from a common pool of embryonic precursor cells. They are currently believed to engage in largely distinct differentiation programs to build synovium and articular cartilage and maintain healthy tissues throughout life. We tested this hypothesis by deeply characterizing and comparing their transcriptomic attributes. We profiled the transcriptomes of freshly isolated AC, synovium, primary FLS, and dermal fibroblasts from healthy adult humans using bulk RNA sequencing assays and downloaded published single-cell RNA sequencing data from freshly isolated human FLS. We integrated all data to define cell-specific signatures and validated findings with quantitative reverse transcription PCR of human samples and RNA hybridization of mouse joint sections. We identified 212 AC and 168 FLS markers on the basis of exclusive or enriched expression in either cell and 294 AC/FLS markers on the basis of similar expression in both cells. AC markers included joint-specific and pan-cartilaginous genes. FLS and AC/FLS markers featured 37 and 55 joint-specific genes, respectively, and 131 and 239 pan-fibroblastic genes, respectively. These signatures included many previously unrecognized markers with potentially important joint-specific roles. AC/FLS markers overlapped in their expression patterns among all FLS and AC subpopulations, suggesting that they fulfill joint-specific properties in all, rather than in discrete, AC and FLS subpopulations. This study broadens knowledge and identifies a prominent overlap of the human adult AC and FLS transcriptomic signatures. It also provides data resources to help further decipher mechanisms underlying joint homeostasis and degeneration and to improve the quality control of tissues engineered for regenerative treatments.

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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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