Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Yokomizo, Tomomasa; Yamada-Inagawa, Tomoko; Yzaguirre, Amanda D.; Chen, Michael; Speck, Nancy A.; Dzierzak, Elaine

doi:10.1038/nprot.2011.441

Cited by 150 publications

(134 citation statements)

References 18 publications

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“…Intestinal perfusion was assessed in newborn mice in an adaptation of the technique of Besner and colleagues (12). In brief, newborn mice were injected with 1 mg/mL fluorescein-labeled tomato lectin (Vector Laboratories; 10 μL/g) for 5 min, and the terminal ileum was freshly harvested, then stained with the endothelial cell marker PECAM-1 (BD Biosciences), and evaluated by whole-mount confocal microscopy as described (23) to evaluate PECAM-1 and tomato lectin fluorescent emission. Images were compiled by using Imaris (Version 7.4; Bitplane AG).…”

Section: Methodsmentioning

confidence: 99%

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

Yazji

Sodhi²,

Lee³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

203

171

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Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate-a precursor for enteral generation of nitrite and nitric oxide-and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.neonatal inflammation | prematurity | infant formula | neonatal nutrition | sepsis N ecrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in the premature infant and is gradually increasing in frequency (1). The defining pathological feature of NEC is the presence of patchy areas of ischemia and necrosis of the small and large intestine (2). Although prematurity is the leading risk factor for NEC development, breast milk administration has been identified as the most important protective strategy (3). Importantly, the mechanisms that lead to the acute development of intestinal necrosis in the premature intestine and factors within breast milk that may prevent NEC remain largely unexplored.In seeking to understand the underlying biological mechanisms that lead to NEC, we and others have identified a critical role for the innate immune receptor toll-like receptor 4 (TLR4) in NEC pathogenesis, because mice deficient in TLR4 showed reduced mucosal inflammation and reduced intestinal necrosis in experimental NEC (4, 5). Microcirculatory perfusion of the premature intestine is primarily regulated by the vasodilator nitric oxide (NO), which is generated through the activity of endothelial NO synthase (eNOS) (6). ...

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Section: Methodsmentioning

confidence: 99%

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

Yazji

Sodhi²,

Lee³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

203

171

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“…Dissociated cells were pelleted at 300 × g for 10 min and resuspended in wash buffer at 1 Whole-Embryo Confocal Microscopy. Embryos were fixed, stained, and analyzed as described (25,67). Briefly, E10.5 embryos were stained for c-Kit using rat anti-mouse c-Kit (BD Biosciences) and Alexa Fluor 647 goat anti-rat IgG (Invitrogen) and then for PECAM1 using biotinylated rat anti-mouse CD31 (BD Biosciences) and Cy3-conjugated streptavidin (Jackson ImmunoResearch).…”

Section: Methodsmentioning

confidence: 99%

Mechanism governing a stem cell-generating cis -regulatory element

Sanalkumar

Johnson

Gào

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The unremitting demand to replenish differentiated cells in tissues requires efficient mechanisms to generate and regulate stem and progenitor cells. Although master regulatory transcription factors, including GATA binding protein-2 (GATA-2), have crucial roles in these mechanisms, how such factors are controlled in developmentally dynamic systems is poorly understood. Previously, we described five dispersed Gata2 locus sequences, termed the −77, −3.9, −2.8, −1.8, and +9.5 GATA switch sites, which contain evolutionarily conserved GATA motifs occupied by GATA-2 and GATA-1 in hematopoietic precursors and erythroid cells, respectively. Despite common attributes of transcriptional enhancers, targeted deletions of the −2.8, −1.8, and +9.5 sites revealed distinct and unpredictable contributions to Gata2 expression and hematopoiesis. Herein, we describe the targeted deletion of the −3.9 site and mechanistically compare the −3.9 site with other GATA switch sites. The −3.9 −/− mice were viable and exhibited normal Gata2 expression and steady-state hematopoiesis in the embryo and adult. We established a Gata2 repression/reactivation assay, which revealed unique +9.5 site activity to mediate GATA factor-dependent chromatin structural transitions. Loss-of-function analyses provided evidence for a mechanism in which a mediator of long-range transcriptional control [LIM domain binding 1 (LDB1)] and a chromatin remodeler [Brahma related gene 1 (BRG1)] synergize through the +9.5 site, conferring expression of GATA-2, which is known to promote the genesis and survival of hematopoietic stem cells.cis element | HSCs W hereas proximal promoter sequences assemble the basal transcriptional machinery and RNA polymerase, distant cis-regulatory elements often confer tissue-specific or contextdependent transcriptional regulation. Enhancer elements reside many kilobases upstream or downstream of a promoter or within introns, and extensive efforts have focused on elucidating "action-at-a-distance" mechanisms (1). Long-range transcriptional control involves physical interactions between proteins bound at distal regions and promoter sequences and higher order structural transitions, including subnuclear relocalization of target loci (2-4). Given the high frequency of long-range mechanisms at mammalian loci and the mutations that disrupt the function of such elements in pathological conditions, elucidating the underlying mechanisms in development,

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“…The protocol for whole mount immunostaining and imaging described below is an adaptation from previously reported methods used for other tissues [16][17][18][19] . Buffers and materials are described in Table 1 and Materials List.…”

Section: Whole-mount Immunofluorescence Staining and 3d Reconstructionmentioning

confidence: 99%

“…Transfer tissues in polypropylene tubes. Clear tissues in methylsalicylate (handle under chemical hood) 18 or as an alternative use BABB solution 19 . Incubate at RT under constant agitation until the tissues become transparent.…”

Section: Whole-mount Immunofluorescence Staining and 3d Reconstructionmentioning

confidence: 99%

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Karkampouna

Kloen

Obdeijn

et al. 2015

JoVE

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Citation: Karkampouna, S., Kloen, P., Obdeijn, M.C., Riester, S.M., van Wijnen, A.J., Kruithof-de Julio, M. Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions. J. Vis. Exp. (98), e52534, doi:10.3791/52534 (2015). AbstractOrgan fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells.As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation. Video LinkThe video component of this article can be found at

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Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos

Cited by 150 publications

References 18 publications

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

Mechanism governing a stem cell-generating cis -regulatory element

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

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