Muscle injury induces a transient senescence-like state that is required for myofiber growth during muscle regeneration

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

2022 Nov 01

Young, LV;Wakelin, G;Cameron, AWR;Springer, SA;Ross, JP;Wolters, G;Murphy, JP;Arsenault, MG;Ng, S;Collao, N;De Lisio, M;Ljubicic, V;Johnston, APW;
PMID: 36190443 | DOI: 10.1096/fj.202200289RR

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-β-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.

Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFβ to rotate the gut

Science (New York, N.Y.)

2022 Sep 23

Sanketi, BD;Zuela-Sopilniak, N;Bundschuh, E;Gopal, S;Hu, S;Long, J;Lammerding, J;Hopyan, S;Kurpios, NA;
PMID: 36137018 | DOI: 10.1126/science.abl3921

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-β (TGFβ), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.

Origin and function of activated fibroblast states during zebrafish heart regeneration

Nature genetics

2022 Jul 21

Hu, B;Lelek, S;Spanjaard, B;El-Sammak, H;Simões, MG;Mintcheva, J;Aliee, H;Schäfer, R;Meyer, AM;Theis, F;Stainier, DYR;Panáková, D;Junker, JP;
PMID: 35864193 | DOI: 10.1038/s41588-022-01129-5

The adult zebrafish heart has a high capacity for regeneration following injury. However, the composition of the regenerative niche has remained largely elusive. Here, we dissected the diversity of activated cell states in the regenerating zebrafish heart based on single-cell transcriptomics and spatiotemporal analysis. We observed the emergence of several transient cell states with fibroblast characteristics following injury, and we outlined the proregenerative function of collagen-12-expressing fibroblasts. To understand the cascade of events leading to heart regeneration, we determined the origin of these cell states by high-throughput lineage tracing. We found that activated fibroblasts were derived from two separate sources: the epicardium and the endocardium. Mechanistically, we determined Wnt signalling as a regulator of the endocardial fibroblast response. In summary, our work identifies specialized activated fibroblast cell states that contribute to heart regeneration, thereby opening up possible approaches to modulating the regenerative capacity of the vertebrate heart.

Differential Etv2 threshold requirement for endothelial and erythropoietic development

Cell reports

2022 May 31

Sinha, T;Lammerts van Bueren, K;Dickel, DE;Zlatanova, I;Thomas, R;Lizama, CO;Xu, SM;Zovein, AC;Ikegami, K;Moskowitz, IP;Pollard, KS;Pennacchio, LA;Black, BL;
PMID: 35649376 | DOI: 10.1016/j.celrep.2022.110881

Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage

Developmental cell

2021 May 17

Thomas, K;Henley, T;Rossi, S;Costello, MJ;Polacheck, W;Griffith, BE;Bressan, M;
PMID: 33891897 | DOI: 10.1016/j.devcel.2021.04.004

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

Cell

2022 Jan 20

Guilliams, M;Bonnardel, J;Haest, B;Vanderborght, B;Wagner, C;Remmerie, A;Bujko, A;Martens, L;Thoné, T;Browaeys, R;De Ponti, FF;Vanneste, B;Zwicker, C;Svedberg, FR;Vanhalewyn, T;Gonçalves, A;Lippens, S;Devriendt, B;Cox, E;Ferrero, G;Wittamer, V;Willaert, A;Kaptein, SJF;Neyts, J;Dallmeier, K;Geldhof, P;Casaert, S;Deplancke, B;Ten Dijke, P;Hoorens, A;Vanlander, A;Berrevoet, F;Van Nieuwenhove, Y;Saeys, Y;Saelens, W;Van Vlierberghe, H;Devisscher, L;Scott, CL;
PMID: 35021063 | DOI: 10.1016/j.cell.2021.12.018

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Integration of single-cell transcriptomes and chromatin landscapes reveals regulatory programs driving pharyngeal organ development

Nature communications

2022 Jan 24

Magaletta, ME;Lobo, M;Kernfeld, EM;Aliee, H;Huey, JD;Parsons, TJ;Theis, FJ;Maehr, R;
PMID: 35075189 | DOI: 10.1038/s41467-022-28067-4

Maldevelopment of the pharyngeal endoderm, an embryonic tissue critical for patterning of the pharyngeal region and ensuing organogenesis, ultimately contributes to several classes of human developmental syndromes and disorders. Such syndromes are characterized by a spectrum of phenotypes that currently cannot be fully explained by known mutations or genetic variants due to gaps in characterization of critical drivers of normal and dysfunctional development. Despite the disease-relevance of pharyngeal endoderm, we still lack a comprehensive and integrative view of the molecular basis and gene regulatory networks driving pharyngeal endoderm development. To close this gap, we apply transcriptomic and chromatin accessibility single-cell sequencing technologies to generate a multi-omic developmental resource spanning pharyngeal endoderm patterning to the emergence of organ-specific epithelia in the developing mouse embryo. We identify cell-type specific gene regulation, distill GRN models that define developing organ domains, and characterize the role of an immunodeficiency-associated forkhead box transcription factor.

The assembly of caprine Y chromosome sequence reveals a unique paternal phylogenetic pattern and improves our understanding of the origin of domestic goat

Ecology and evolution

2021 Jun 01

Xiao, C;Li, J;Xie, T;Chen, J;Zhang, S;Elaksher, SH;Jiang, F;Jiang, Y;Zhang, L;Zhang, W;Xiang, Y;Wu, Z;Zhao, S;Du, X;
PMID: 34188851 | DOI: 10.1002/ece3.7611

The mammalian Y chromosome offers a unique perspective on the male reproduction and paternal evolutionary histories. However, further understanding of the Y chromosome biology for most mammals is hindered by the lack of a Y chromosome assembly. This study presents an integrated in silico strategy for identifying and assembling the goat Y-linked scaffolds using existing data. A total of 11.5 Mb Y-linked sequences were clustered into 33 scaffolds, and 187 protein-coding genes were annotated. We also identified high abundance of repetitive elements. A 5.84 Mb subset was further ordered into an assembly with the evidence from the goat radiation hybrid map (RH map). The existing whole-genome resequencing data of 96 goats (worldwide distribution) were utilized to exploit the paternal relationships among bezoars and domestic goats. Goat paternal lineages were clearly divided into two clades (Y1 and Y2), predating the goat domestication. Demographic history analyses indicated that maternal lineages experienced a bottleneck effect around 2,000 YBP (years before present), after which goats belonging to the A haplogroup spread worldwide from the Near East. As opposed to this, paternal lineages experienced a population decline around the 10,000 YBP. The evidence from the Y chromosome suggests that male goats were not affected by the A haplogroup worldwide transmission, which implies sexually unbalanced contribution to the goat trade and population expansion in post-Neolithic period.

Molecular characterization of Barrett's esophagus at single-cell resolution

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

2021 Nov 23

Busslinger, GA;de Barbanson, B;Oka, R;Weusten, BLA;de Maat, M;van Hillegersberg, R;Brosens, LAA;van Boxtel, R;van Oudenaarden, A;Clevers, H;
PMID: 34795059 | DOI: 10.1073/pnas.2113061118

Barrett's esophagus (BE) is categorized, based on morphological appearance, into different stages, which correlate with the risk of developing esophageal adenocarcinoma. More advanced stages are more likely to acquire chromosomal instabilities, but stage-specific markers remain elusive. Here, we performed single-cell DNA-sequencing experiments (scDNAseq) with fresh BE biopsies. Dysplastic BE cells frequently contained chromosomal instability (CIN) regions, and these CIN cells carried mutations corresponding to the COSMIC mutational signature SBS17, which were not present in biopsy-matched chromosomally stable (CS) cells or patient-matched nondiseased control cells. CS cells were predominantly found in nondysplastic BE biopsies. The single-base substitution (SBS) signatures of all CS BE cells analyzed were indistinguishable from those of nondiseased esophageal or gastric cells. Single-cell RNA-sequencing (scRNAseq) experiments with BE biopsies identified two sets of marker genes which facilitate the distinction between columnar BE epithelium and nondysplastic/dysplastic stages. Moreover, histological validation confirmed a correlation between increased CLDN2 expression and the presence of dysplastic BE stages. Our scDNAseq and scRNAseq datasets, which are a useful resource for the community, provide insight into the mutational landscape and gene expression pattern at different stages of BE development.

Single-cell transcriptomic landscape of cardiac neural crest cell derivatives during development

EMBO reports

2021 Sep 27

Chen, W;Liu, X;Li, W;Shen, H;Zeng, Z;Yin, K;Priest, JR;Zhou, Z;
PMID: 34569705 | DOI: 10.15252/embr.202152389

The migratory cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, developmental chronology, and transcriptomic states of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC-associated congenital anomalies. Here, we perform single-cell transcriptomic sequencing of 34,131 CNCC-derived cells in mouse hearts covering eight developmental stages between E10.5 and P7. We report the presence of CNCC-derived mural cells that comprise pericytes and microvascular smooth muscle cells (mVSMCs). Furthermore, we identify the transition from the CNCC-derived pericytes to mVSMCs and the key regulators over the transition. In addition, our data support that many CNCC derivatives had already committed or differentiated to a specific lineage when migrating into the heart. We explore the spatial distribution of some critical CNCC-derived subpopulations with single-molecule fluorescence in situ hybridization. Finally, we computationally reconstruct the differentiation path and regulatory dynamics of CNCC derivatives. Our study provides novel insights into the cell lineages, developmental chronology, and regulatory dynamics of CNCC derivatives during development.

Thymosin β4 protects against aortic aneurysm via endocytic regulation of growth factor signaling

The Journal of clinical investigation

2021 Mar 30

Munshaw, S;Bruche, S;Redpath, AN;Jones, A;Patel, J;Dubé, KN;Lee, R;Hester, SS;Davies, R;Neal, G;Handa, A;Sattler, M;Fischer, R;Channon, KM;Smart, N;
PMID: 33784254 | DOI: 10.1172/JCI127884

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic modulation which increases susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 (Tβ4) may function to maintain healthy vasculature throughout postnatal life. This was supported by the identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been implicated by genome-wide association studies with risk of AAA and other arterial diseases. Tβ4-null mice displayed aortic VSMC and elastin defects, phenocopying LRP1 mutants, and their compromised vascular integrity predisposed to Angiotensin II-induced aneurysm formation. Aneurysmal vessels were characterised by enhanced VSMC phenotypic modulation and augmented platelet-derived growth factor (PDGF) receptor (PDGFR)β signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, associated with dysregulated endocytosis, with increased recycling and reduced lysosomal targeting of LRP1-PDGFRβ. Accordingly, the exacerbated aneurysmal phenotype in Tβ4-null mice was rescued upon treatment with the PDGFRβ antagonist, Imatinib. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health and provides insights into the mechanisms of growth factor-controlled VSMC phenotypic modulation underlying aortic disease progression.

Spatial analysis of organ-wide RNA, protein expression, and lineage tracing in the female mouse reproductive tract

STAR protocols

2021 Dec 17

Gurumurthy, RK;Kumar, N;Chumduri, C;
PMID: 34841280 | DOI: 10.1016/j.xpro.2021.100969

Visualizing precise spatial patterns of an organ-wide gene and protein expression among diverse cell types can provide critical insights into the fundamental processes underlying normal tissue homeostasis and disease development. Here, we describe an optimized protocol for single-molecule RNA in situ hybridization (smRNA-ISH), immunohistochemistry, and cell lineage analysis of the female reproductive tract organs using commercially available smRNA-ISH probes, antibodies, and inducible Cre-mice. The high-resolution multispectral fluorescence imaging is performed using wide-field epifluorescence or confocal microscopy combined with a slide scanner. For complete details on the use and execution of this protocol, please refer to Chumduri et al. (2021).

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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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