High-throughput single-molecule RNA imaging analysis reveals heterogeneous responses of cardiomyocytes to hemodynamic overload.

J Mol Cell Cardiol. 2019 Jan 3.

2019 Jan 03

Satoh M, Nomura S, Harada M, Yamaguchi T, Ko T, Sumida T, Toko H, Naito AT, Takeda N, Tobita T, Fujita T, Ito M, Fujita K, Ishizuka M, Kariya T, Akazawa H, Kobayashi Y, Morita H, Takimoto E, Aburatani H, Komuro I.
PMID: 30611794 | DOI: 10.1016/j.yjmcc.2018.12.018

Abstract BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2 weeks, TAC2W) and at a late heart failure stage (8 weeks, TAC8W). Expression of myosin heavy chain β (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.

Simulating transplant small-for-size graft using human liver monosegments: impact of portal perfusion pressures.

Transplantation Proceedings (2019)

2019 Jan 09

Mohamed M, Kang L, Zhang C, Edenfield B, Sykes J, Brown T, Johnson JL, Rehman F, Nguyen JH.
| DOI: doi. 10.1016/j.transproceed.2018.12.028

Small-for-size liver grafts (SFSG) in adult transplant recipients have elevated risk of graft dysfunction and graft failure, limiting its application in clinical liver transplantation. Relevant preclinical model of SFSG for deceased-donor split liver transplant is lacking. In this study, we present our initial characterization of SFSG model using monosegments of a discarded deceased-donor human liver.

Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure

Nat Commun. 2018 Oct 30;9(1):4435.

2018 Oct 30

Nomura S, Satoh M, Fujita T, Higo T, Sumida T, Ko T, Yamaguchi T, Tobita T, Naito AT, Ito M, Fujita K, Harada M, Toko H, Kobayashi Y, Ito K, Takimoto E, Akazawa H, Morita H, Aburatani H, Komuro I.
PMID: 30375404 | DOI: 10.1038/s41467-018-06639-7

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.

Lineage dynamics of murine pancreatic development at single-cell resolution.

Nat Commun.

2018 Sep 25

Byrnes LE, Wong DM, Subramaniam M, Meyer NP, Gilchrist CL, Knox SM, Tward AD, Ye CJ, Sneddon JB.
PMID: 30254276 | DOI: 10.1038/s41467-018-06176-3

Organogenesis requires the complex interactions of multiple cell lineages that coordinate their expansion, differentiation, and maturation over time. Here, we profile the cell types within the epithelial and mesenchymal compartments of the murine pancreas across developmental time using a combination of single-cell RNA sequencing, immunofluorescence, in situ hybridization, and genetic lineage tracing. We identify previously underappreciated cellular heterogeneity of the developing mesenchyme and reconstruct potential lineage relationships among the pancreatic mesothelium and mesenchymal cell types. Within the epithelium, we find a previously undescribed endocrine progenitor population, as well as an analogous population in both human fetal tissue and human embryonic stem cells differentiating toward a pancreatic beta cell fate. Further, we identify candidate transcriptional regulators along the differentiation trajectory of this population toward the alpha or beta cell lineages. This work establishes a roadmap of pancreatic development and demonstrates the broad utility of this approach for understanding lineage dynamics in developing organs.

Single-Cell Analysis Reveals a Hair Follicle Dermal Niche Molecular Differentiation Trajectory that Begins Prior to Morphogenesis.

Dev Cell. 2018 Dec 19.

2018 Dec 19

Gupta K, Levinsohn J, Linderman G, Chen D, Sun TY, Dong D, Taketo MM, Bosenberg M, Kluger Y, Choate K, Myung P.
PMID: 30595533 | DOI: 10.1016/j.devcel.2018.11.032

Delineating molecular and cellular events that precede appendage morphogenesis has been challenging due to the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/β-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent progeny of selectively proliferating cells present prior to morphogenesis and that are later identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation.

Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish.

Nat Commun.

2016 Dec 08

Xiao C, Gao L, Hou Y, Xu C, Chang N, Wang F, Hu K, He A, Luo Y, Wang J, Peng J, Tang F, Zhu X, Xiong JW.
PMID: 27929112 | DOI: 10.1038/ncomms13787

The zebrafish possesses a remarkable capacity of adult heart regeneration, but the underlying mechanisms are not well understood. Here we report that chromatin remodelling factor Brg1 is essential for adult heart regeneration. Brg1 mRNA and protein are induced during heart regeneration. Transgenic over-expression of dominant-negative Xenopus Brg1 inhibits the formation of BrdU+/Mef2C+ and Tg(gata4:EGFP) cardiomyocytes, leading to severe cardiac fibrosis and compromised myocardial regeneration. RNA-seq and RNAscope analyses reveal that inhibition of Brg1 increases the expression of cyclin-dependent kinase inhibitors such as cdkn1a and cdkn1c in the myocardium after ventricular resection; and accordingly, myocardial-specific expression of dn-xBrg1 blunts myocardial proliferation and regeneration. Mechanistically, injury-induced Brg1, via its interaction with Dnmt3ab, suppresses the expression of cdkn1c by increasing the methylation level of CpG sites at the cdkn1c promoter. Taken together, our results suggest that Brg1 promotes heart regeneration by repressing cyclin-dependent kinase inhibitors partly through Dnmt3ab-dependent DNA methylation.

	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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