Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Farkas, Attila; Gerner‐Smidt, Christian; Lili, Loukia N.; Nusrat, Asma; Capaldo, Christopher

doi:10.3791/53112

Cited by 5 publications

(7 citation statements)

References 16 publications

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“…For example, Hnf4a signal levels within the array positively correlate with claudin 7 (Pearson's correlation coefficient r D 0.88, at p-value D 0.004). In Figure 1C, differentially regulated claudin genes, as well as correlated transcription factors, were confirmed by qPCR using tissue collected by cryosectioning method as illustrated (as described in 21 ). Like LCM, crypt cryosectioning preserves the native cellular environment until RNA collection and is a high yield method that does not require RNA amplification.…”

Section: Gene Ontology Analysis Of Differentially Regulated Genes Alomentioning

confidence: 64%

“…Cell/Tissue collection and RNA extraction for real-time PCR validation Confirmation of microarray candidates was performed on RNA extracted from serial cryosections of whole mount distal colonic tissue as previously described. 21 Briefly, distal colon samples were obtained from a 2 cm segment spanning 2-4 cm from the anus. RNA was extracted using Trizol reagent according to manufacturer's instruction (Life Technologies), subjected to secondary purification (Epoch Life Sciences, Sugarland TX), DNAse treatment (Qiagen), and converted to cDNA (Thermo Fisher, Waltham, MA).…”

Section: Tissue Collection By Lcm Rna Extraction and Amplificationmentioning

confidence: 99%

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Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades

et al. 2016

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Colonic enterocytes form a rapidly renewing epithelium and barrier to luminal antigens. During renewal, coordinated expression of the claudin family of genes is vital to maintain the epithelial barrier. Disruption of this process contributes to barrier compromise and mucosal inflammatory diseases. However, little is known about the regulation of this critical aspect of epithelial cell differentiation. In order to identify claudin regulatory factors we utilized high-throughput gene microarrays and correlation analyses. We identified complex expression gradients for the transcription factors Hopx, Hnf4a, Klf4 and Tcf7l2, as well as 12 claudins, during differentiation. In vitro confirmatory methods identified 2 pathways that stimulate claudin expression; Hopx/Klf4 activation of Cldn4, 7 and 15, and Tcf7l2/Hnf4a up-regulation of Cldn23. Chromatin immunoprecipitation confirmed a Tcf7l2/Hnf4a/Claudin23 cascade. Furthermore, Hnf4a conditional knockout mice fail to induce Cldn23 during colonocyte differentiation. In conclusion, we report a comprehensive screen of colonic claudin gene expression and discover spatiotemporal Hopx/Klf4 and Tcf7l2/Hnf4a signaling as stimulators of colonic epithelial barrier differentiation.

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Section: Gene Ontology Analysis Of Differentially Regulated Genes Alomentioning

confidence: 64%

Section: Tissue Collection By Lcm Rna Extraction and Amplificationmentioning

confidence: 99%

Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades

et al. 2016

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“…The colonic wall can be histologically divided into the colonic mucosa and the external muscle layers (Farkas et al, 2015). The colonic mucosa consists of the epithelial layer, lamina propria and muscularis mucosae (M. mucosae), a very thin layer of smooth muscle that separates mucosa from underlying submucosa and smooth muscle layers (Fig.…”

Section: Resultsmentioning

confidence: 99%

Seq-Scope: Submicrometer-resolution spatial transcriptomics for single cell and subcellular studies

Cho

Park

et al. 2021

Preprint

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Spatial barcoding technologies have the potential to reveal histological details of transcriptomic profiles; however, they are currently limited by their low resolution. Here we report Seq-Scope, a spatial barcoding technology with a resolution almost comparable to an optical microscope. Seq-Scope is based on a solid-phase amplification of randomly barcoded single-molecule oligonucleotides using an Illumina sequencing-by-synthesis platform. The resulting clusters annotated with spatial coordinates are processed to expose RNA-capture moiety. These RNA-capturing barcoded clusters define the pixels of Seq-Scope that are approximately 0.5-1 μm apart from each other. From tissue sections, Seq-Scope visualizes spatial transcriptome heterogeneity at multiple histological scales, including tissue zonation according to the portal-central (liver), crypt-surface (colon) and inflammation-fibrosis (injured liver) axes, cellular components including single cell types and subtypes, and subcellular architectures of nucleus, cytoplasm and mitochondria. Seq-scope is quick, straightforward and easy-to-implement, and makes spatial single cell analysis accessible to a wide group of biomedical researchers.

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“…We thus sought to test the possibility that expression of several major PMN chemokines, including CXCL1, CXCL2 and CXCL5, varies in the different strata. To quantitate this, we used a previously established protocol 42 to serially section control and LPS-inflamed intestinal tissue. This allowed us to harvest and pool representative sections from each of the intestinal layers in question (schematic shown in Fig 6C).…”

Section: Resultsmentioning

confidence: 99%

“…Intestinal layers were separated as previously described 42 . Briefly, a segment of a small intestine (~0.5 × 1 cm) was longitudinally opened, laid flat between two metal plates and fixed in OCT. Intestinal tissue was then sectioned en face along the long axis with section width set to 10 μm.…”

Section: Materials/methodsmentioning

confidence: 99%

In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines

et al. 2018

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Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of gastrointestinal inflammation, with significant implications for host defense, injury and repair. However, phenotypic and mechanistic aspects of PMN recruitment in inflamed intestines have not been explored in vivo. Using novel epithelial/PMN fluorescence reporter mice, advanced intravital imaging and 3D reconstruction analysis, we mapped the microvasculature architecture across the intestinal layers and determined that in response to Salmonella/endotoxin-induced inflammation, PMN transendothelial migration (TEM) was restricted to submucosal vessels. PMN TEM was not observed in villus or crypt vessels, proximal to the epithelium that underlies the intestinal lumen, and was partially dependent on (C-X-C motif) ligands 1 (CXCL1) and 2 (CXCL2) expression, which was found to be elevated in the submucosa layer. Restricted PMN extravasation at the submucosa and subsequent PMN interstitial migration may serve as a novel regulatory step of PMN effector function and recruitment to the luminal space in inflamed intestines.

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Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Cited by 5 publications

References 16 publications

Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades

Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades

Seq-Scope: Submicrometer-resolution spatial transcriptomics for single cell and subcellular studies

In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines

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