Whole-mount Immunofluorescence Research Articles

Functional alteration of ribbon synapses in inner hair cells by noise exposure causing hidden hearing loss

For decades, studies on noise-induced hearing loss have been focusing on the loss of sensory hair cells and/or auditory afferent fibers following severe noise exposure. Recently, a condition of hidden hearing loss was characterized, in which moderate noise exposure that causes only temporary threshold elevation could induce persistent reduction in auditory brainstem response (ABR) amplitudes and loss of ribbon synapses in inner hair cells (IHCs). However, it is not clear whether and how moderate noise exposure alters the functionality of surviving and/or recovering ribbon synapses in IHCs. To address this issue, we applied moderate noise exposure to mice and combined auditory systems physiology, whole-mount immunofluorescence staining and patch-clamp electrophysiology to characterize changes of ribbon synapse functions in IHCs. After the noise exposure, the ABR threshold was elevated and then recovered, while the ABR Wave I amplitude was reduced but did not recover. Coincidently, whole-mount cochlea staining revealed the loss and recovery of ribbon synapses in IHCs. We then performed whole-cell patch-clamp recording in IHCs and we found that the Ca2+ current, the sustained exocytosis of synaptic vesicles, and the replenishment of synaptic vesicles were all significantly reduced one day after the noise exposure. Fourteen days after the noise exposure, however, only the sustained exocytosis failed to recover, and further examination revealed that this persistent reduction is due to a decrease in the Ca2+ efficiency of triggering exocytosis. In conclusion, our results suggest temporary and persistent alterations of ribbon synapse functions in IHCs contribute to the hidden hearing loss.

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos.

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

A new member of the runt domain family from Pacific oyster Crassostrea gigas (CgRunx) potentially involved in immune response and larvae hematopoiesis

The Runx family is a kind of heteromeric transcription factors, which is defined by the presence of a runt domain. As transcriptional regulator during development and cell fate specification, Runx is best known for its critical roles in hematopoiesis. In the present study, a Runx transcription factor (designed as CgRunx) was identified and characterized from the oyster Crassostrea gigas. The complete coding sequence of CgRunx was of 1638 bp encoding a predicted polypeptide of 545 amino acids with one conserved runt domain, which shared high similarity with other reported Runx proteins. CgRunx was highly expressed in hemocytes, gill and mantle both at the protein and nucleic acid levels. CgRunx protein was localized specifically in the cell nuclei of hemocytes, and distributed at the tubule lumen of gill filament. During the larval developmental stages, the mRNA transcripts of CgRunx gradually increased after fertilization, reached to a relative high level at the 8 cell embryos and the blastula stage of 2–4 hpf (hours post fertilization) (about 40-fold), and peaked at early trochophore larvae (10 hpf) (about 60-fold). Whole-mount immunofluorescence assay further revealed that the abundant immunofluorescence signals of CgRunx distributed through the whole embryo at blastula stage (5 hpf), and progressively reduced with the development to a ring structure around the dorsal region in trochophore larvae (10 hpf). Scattered positive immunoreactivity signals finally appeared in the velum region of D-veliger larvae. After LPS and Vibrio splendidus stimulations, the expression levels of CgRunx mRNA in hemocytes were up-regulated significantly compared with that in the control (0 h), which were 2.98- and 2.46-fold (p < 0.05), 2.67- and 1.5-fold (p < 0.05), 2.36- and 1.38-fold (p < 0.05) at 3 h, 6 h and 12 h, respectively. These results collectively suggested that CgRunx involved in immune response and might participate in larvae hematopoiesis in oyster.

Myocardial infarction induces bone marrow megakaryocyte proliferation, maturation and platelet production

Platelet, apart from its classic role of homeostasis, serves also as a crucial immune cell component that contributes to the aggravation of atherosclerosis. It has been reported that myocardial infarction (MI) triggers leukocytosis in the bone marrow and spleen, which accelerates post-MI atherosclerosis. However, it remains unclear whether thrombopoiesis is also enhanced after MI. Here, using flow cytometry and bone marrow whole-mount immunofluorescence staining combined with three-dimensional (3D) reconstruction, we for the first time demonstrated an enhanced thrombopoiesis and megakaryopoiesis in a mouse model of coronary artery ligation as a mimic of MI. We showed that MI leads to increasing number of peripheral platelets, as well as elevating number and larger size of bone marrow MKs. We also observed more proplatelets and fragmented MKs, and a closer spatial distribution of MK populations to the bone marrow vascular niche after MI. This study provides direct evidence that MI induces bone marrow megakaryocyte proliferation, maturation and platelet production. It opens a new scope that targeting platelet production might become a novel therapeutic approach for attenuating post-MI atherosclerosis.

Muscle-Derived Stem Cell-Enriched Scaffolds Are Capable of Enhanced Healing of a Murine Volumetric Muscle Loss Defect.

Volumetric muscle loss secondary to traumatic or surgical causes can lead to functional and aesthetic impairments. The authors hypothesize that an implantable muscle-derived stem cell-enriched collagen scaffold could significantly augment muscle regeneration in a murine model of volumetric muscle loss. Murine muscle-derived stem cells were isolated using a modified preplating technique and seeded onto type 1 collagen scaffolds to create the muscle-derived stem cell-enriched collagen scaffolds. Murine rectus femoris defects of 5 mm were created and randomized to one of three conditions (n = 6 per group): untreated controls, collagen scaffold only, and muscle-derived stem cell-enriched collagen scaffolds. In vivo muscle healing was quantified using micro-computed tomography. Muscle explants were analyzed using standard histology and whole-mount immunofluorescence at 8 weeks. In vivo experiments demonstrated significantly greater quadriceps cross-sectional area in the muscle-derived stem cell-enriched collagen scaffold group compared with controls on micro-computed tomography (0.74 ± 0.21 versus 0.55 ± 0.06 versus 0.49 ± 0.04 ratio of experimental to naive quadriceps cross-sectional area; p < 0.05). Muscle explants of the muscle-derived stem cell-enriched collagen scaffold group demonstrated significantly higher cellular density compared with controls (1185 ± 360 versus 359 ± 62 versus 197 ± 68 nuclei/high-power field; p < 0.01). Immunofluorescence for laminin and myosin heavy chain confirmed formation of organized muscle fibers within the defect of the muscle-derived stem cell-enriched collagen scaffold group only. However, appreciable confocal colocalization of myosin heavy chain with green fluorescent protein expression was low. The results of this study indicate that muscle-derived stem cell-enriched scaffolds significantly improved skeletal muscle regeneration in a murine muscle defect model. Based on the low fluorescent colocalization, host progenitor cells appear to contribute significantly to intradefect myogenesis, suggesting that deployment of a viable muscle-derived stem cell-enriched scaffold stimulates a regenerative mitogen response in native tissues.

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

RNAi analysis of Doublesex1 expression in Daphnia pulex Leydig, 1860 (Cladocera)

Abstract To explore the role of the Doublesex gene (Dsx1) in the switching between reproductive modes in Daphnia pulex Leydig, 1860, we performed gene silencing using RNA interference (RNAi). We also investigated the expression of gfp (Green Fluorescent Protein) by quantitative-PCR, and the expression of Dsx1 mRNA after RNAi by real-time PCR. Dsx1 expression was significantly greater in males than in females, and was down-regulated in experimental groups compared with the control group. The decrease was more significant in females (35% of controls) than males (47% of controls). The gene fragment of Dsx1 amplified by PCR was ligated to the pEASY-Blunt vector to obtain the recombinant plasmid expressed recombinant protein induced by isopropyl-β-D-thiogalactoside (IPTG). After having been purified by Ni-column affinity chromatography, the recombinant protein as antigen was used to immune rabbits. The antiserum was successfully purified by the protein A method to obtain the Dsx1 polyclonal antibody, an IgG concentration of 2.65 mg/ml, titer 240 000. Western blotting showed that RNAi treatment significantly reduced DSX1 protein levels. Whole-mount immunofluorescence analysis revealed that the fluorescence intensity of appendages was significantly lower following RNAi treatment than the negative control group, and comparable with the blank control group, further confirming the decrease in Dsx1 expression after RNAi treatment. The number of offspring in all experimental groups was measured, and was significantly greater in the RNAi-treated group compared with the control group. These results provide a foundation for further work on the molecular basis of switching between the two modes of reproduction in cladocerans. The findings also further increase our understanding of the regulatory effects of Dsx1 in the differing sexes in these crustaceans.

Donation of mitochondria by iPSC-derived mesenchymal stem cells protects retinal ganglion cells against mitochondrial complex I defect-induced degeneration.

Rationale: Retinal ganglion cell (RGC) degeneration is extremely hard to repair or regenerate and is often coupled with mitochondrial dysfunction. Mesenchymal stem cells (MSCs)-based treatment has been demonstrated beneficial for RGC against degeneration. However, underlying mechanisms of MSC-provided RGC protection are largely unknown other than neuroprotective paracrine actions. In this study, we sought to investigate whether mitochondrial donation from induced pluripotent stem cell-derived MSC (iPSC-MSCs) could preserve RGC survival and restore retinal function.Methods: iPSC-MSCs were injected into the vitreous cavity of one eye in NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) knockout (KO) and wild type mice. Phosphate buffer saline (PBS) or rotenone treated iPSC-MSCs were injected as control groups. Retinal function was detected by flash electroretinogram (ERG). Whole-mount immunofluorescence (IF), morphometric analysis, confocal microscopy imaging, polymerase chain reaction (PCR) of the retinas were conducted to investigate mitochondrial transfer from human iPSC-MSCs to mouse retina. Quantitative mouse cytokine arrays were carried out to measure retinal inflammatory response under difference treatments.Results: RGC survival in the iPSC-MSC injected retina of Ndufs4 KO mice was significantly increased with improved retinal function. GFP labelled human mitochondria from iPSC-MSC were detected in the RGCs in the retina of Ndufs4 KO mice starting from 96 hours post injection. PCR result showed only human mitochondrial DNA without human nuclear DNA could be detected in the mouse retinas after iPSC-MSC treatment in Ndufs4 KO mice eye. Quantitative cytokine array analysis showed pro-inflammatory cytokines was also downregulated by this iPSC-MSC treatment.Conclusion: Intravitreal transplanted iPSC-MSCs can effectively donate functional mitochondria to RGCs and protect against mitochondrial damage-induced RGC loss.

C-Type allatostatin and its putative receptor from the mud crab serve an inhibitory role in ovarian development.

C-Type allatostatins are a family of peptides that characterized by a conserved unblocked PISCF motif at the C-terminus. In insects, it is well known that C-type allatostatin has a potent inhibitory effect on juvenile hormone biosynthesis by the corpora allata. C-Type allatostatin has been widely identified from crustacean species but little is known about its roles. Therefore, this study investigated the tissue distribution patterns of C-type allatostatin and its putative receptor in the mud crab Scylla paramamosain, and further explored its potential effect on vitellogenesis. Firstly, cDNAs encoding C-type allatostatin (Sp-AST-C) precursor and its putative receptor (Sp-AST-CR) were isolated. Subsequently, RT-PCR revealed that Sp-AST-C was mainly expressed in the nervous tissue, middle gut and heart, whereas Sp-AST-CR had extensive expression in all tissues tested except the eyestalk ganglion and hepatopancreas. In addition, in situ hybridization in the cerebral ganglion showed that Sp-AST-C was localized in clusters 6 and 8 of the protocerebrum, clusters 9, 10 and 11 of the deutocerebrum, and clusters 14 and 15 of the tritocerebrum. Whole-mount immunofluorescence revealed a similar distribution pattern. Synthetic Sp-AST-C had no effect on the abundance of S. paramamosain vitellogenin (Sp-Vg) in the hepatopancreas and ovary in vitro but significantly reduced the expression of its receptor (Sp-VgR) in the ovary in a dose-dependent manner. Furthermore, Sp-VgR expression, vitellin content and oocyte diameter in the ovary were reduced 16 days after the first injection of Sp-AST-C. Finally, in situ hybridization showed that Sp-AST-CR transcript was specifically localized in the oocytes, which further indicated that the oocytes are the target cells for Sp-AST-C. In conclusion, our results suggested that the Sp-AST-C signaling system is involved in the regulation of ovarian development, possibly by directly inhibiting the uptake of yolk by oocytes and obstructing oocyte growth.

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence.

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The Effect of Aging on Nerve Morphology and Substance P Expression in Mouse and Human Corneas.

Aging impairs corneal nerve density and sensitivity. Substance P (SP), a neuropeptide secreted by sensory nerves, regulates nerve morphology and nociception. Here, we investigate the relationship between aging, nerve morphology, and SP expression in mouse and human corneas. SP levels in mouse corneas (wild type and substance P-knockout) and human corneas and tears were quantified with an ELISA assay. Corneal total nerve length (TNL) was measured with whole-mount β3-tubulin immunofluorescence in mouse and in vivo laser corneal confocal microscopy in humans. SP and β3-tubulin stained cross-sections were used to assess the colocalization of SP and nerves in human and mouse corneas. Ocular surface nociception was assessed with a wiping test in mice. SP colocalizes with sub-basal neurons in mice and humans. In WT mice, SP levels decrease with age (P = 0.0045, 8 vs. 52 weeks; P = 0.004, 26 vs. 52 weeks) as well as TNL (P = 0.018, 8 vs. 26 weeks; P = 0.0001, 8 vs. 52 weeks). Knockout mice show a greater TNL reduction (8 vs. 26 weeks, P = 0.0016) than WT mice. In the oldest WT and age-matched KO mice, nociception is impaired (P = 0.007 and P < 0.0001, respectively), and KO mice sensitivity is restored by topical SP treatment. In humans, SP levels are reduced in old subject corneas and correlate, in tears, with age (P = 0.0368); TNL also decreases in older patients (P = 0.0002). Age-associated corneal nerve loss is paralleled by reduction of SP expression in mice and humans. SP promotes the maintenance of normal nerve morphology in the long term and modulates nociception in the cornea.

Involvement of clustered oyster Wnt genes in gut formation

Genes encoding Wnt ligands play important roles in organ development. The Wnt10 - Wnt6 - Wnt1 - Wnt9 cluster widely presents in many metazoan genomes, indicating the importance of gene arrangement. Hypothesis has been proposed that they may be coordinately regulated. However, few expression correlations were identified in model animals. We analyzed the tissue expression pattern of clustered oyster Wnt10, Wnt6, Wnt1, and Wnt9a genes in this study. The results indicated the highest expression level in adult gut system of these clustered Wnt genes, except for Wnt6, which had highest expression in mantle. Further whole-mount immunofluorescence assay indicated that Wnt6 protein was restricted to gut region in oyster larvae. These results suggest the possible important role of the Wnt10 - Wnt6 - Wnt1 - Wnt9 cluster in oyster gut formation.

The SDF1-CXCR4 Signals Regulate Adipose Tissue Expansion by Modulating Angiogenesis in Diet-Induced Obesity in Mice

Adipose tissue (AT) expands with angiogenesis in obesity. Mature vessels in the epididymal white adipose tissue (eWAT) are tightly wrapped with pericytes (PCs), which attenuates endothelial (ECs) proliferation. We found that obesity-associated increase of PDGF-B promoted detachment of PCs from nature vessels; thereby vessels were prone to neoangiogenesis. In this study, we aimed to clarify the role of stromal cell derived factor 1 (SDF1), another proangiogenic factor in the AT development in obesity. In the whole-mount immunofluorescence study of organ cultured eWAT, treatment with PDGF-B or SDF1 for 24 h significantly promoted detachment of PCs from vessels. The effect of SDF1 on PCs was reproduced by treatment with ATI2341, a potent CXCR4 agonist, but not by VUX11207, a CXCR7 agonist. Interestingly, PCs were well associated with vessels when tissue was treated with both PDGF-B and SDF1. In addition, the association was declined by preincubation with AMD3100, a CXCR4 antagonist or when AT was derived from Pdgfrb knockout mice. To further explore the in vivo effect of SDF1 in AT during obesity, we administrated Anagliptin (Ana), a DPP-4 inhibitor to C57BL/6J mice fed chow or HFD for 12 weeks, since SDF1 is a known substrate for DPP-4. The protein amount of SDF1 was significantly elevated in the eWAT of Ana-treated mice. Importantly, HFD-induced adipose tissue expansion was significantly attenuated with reduced vascular density and PCs detachment in the eWAT by Ana. To clarify the mechanism by which PCs attach even under HFD-fed condition by coexistence of SDF1 and PDGF-B, we isolated ECs, PCs, and macrophages (Mφ) from eWAT of mice. Expression of PDGFB was increased in Mφ and decreased in ECs by HFD, whereas these changes were attenuated and the gradient of PDGFB concentration between PCs and ECs was blunted in Ana-treated eWAT. These findings suggest that both PDGF-B and SDF1 coordinately regulate PCs detachments and adipose tissue expansion with obesity in mice. Disclosure T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. E. Watanabe: None. Y. Kotera: None. Y. Onogi: None. H. Tsuneki: None. T. Sasaoka: None.

A dominant variant in the PDE1C gene is associated with nonsyndromic hearing loss.

Identification of genes with variants causing non-syndromic hearing loss (NSHL) is challenging due to genetic heterogeneity. The difficulty is compounded by technical limitations that in the past prevented comprehensive gene identification. Recent advances in technology, using targeted capture and next-generation sequencing (NGS), is changing the face of gene identification and making it possible to rapidly and cost-effectively sequence the whole human exome. Here, we characterize a five-generation Chinese family with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining population-specific mutation arrays, targeted deafness genes panel, whole exome sequencing (WES), we identified PDE1C (Phosphodiesterase 1C) c.958G>T (p.A320S) as the disease-associated variant. Structural modeling insights into p.A320S strongly suggest that the sequence alteration will likely affect the substrate-binding pocket of PDE1C. By whole-mount immunofluorescence on postnatal day 3 mouse cochlea, we show its expression in outer (OHC) and inner (IHC) hair cells cytosol co-localizing with Lamp-1 in lysosomes. Furthermore, we provide evidence that the variant alters the PDE1C hydrolytic activity for both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Collectively, our findings indicate that the c.958G>T variant in PDE1C may disrupt the cross talk between cGMP-signaling and cAMP pathways in Ca2+ homeostasis.

The Forkhead Transcription Factor FOXC2 Is Required for Maintaining Murine Spermatogonial Stem Cells.

Continuous spermatogenesis from puberty to old age in males relies on spermatogonial stem cells (SSCs) that possess the property of self-renewal and differentiation. The delicate balance between self-renewal and differentiation is of great importance. In mice, SSCs exist as a subpopulation of undifferentiated spermatogonia. SSCs are controlled by intrinsic molecular pathways that can be activated by extrinsic signals. Our results here first show that the expression of forkhead box C2 (FOXC2) is restricted to GFRα1-positive spermatogonia in the testis. Whole-mount immunofluorescence results reveal that FOXC2 is expressed predominately in As and Apr spermatogonia. Reduction of Foxc2 gene expression by shRNA lentivirus treatment significantly impairs the maintenance of SSCs in vitro. Furthermore, knock-down of Foxc2 decreases SSC colonization to only 10.42% compared to the control by transplantation. Reverse transcription and real-time quantitative PCR gene analyses following knock-down of Foxc2 indicate that Foxc2 may act as a suppressor for SSC differentiation. Extrinsic stimuli treatments show that glial cell line-derived neurotrophic factor and retinoic acid act in opposite ways to regulate FOXC2 expression and subsequent SSC property. These results suggest that FOXC2 is a critical intrinsic regulator of SSC self-renewal and differentiation.

The microenvironment of proliferative diabetic retinopathy supports lymphatic neovascularization

Proliferative diabetic retinopathy (PDR) is a major diabetic microvascular complication characterized by pathological angiogenesis. Several retinopathy animal models have been developed to study the disease mechanisms and putative targets. However, knowledge on the human proliferative disease remains incomplete, relying on steady-state results from thin histological neovascular tissue sections and vitreous samples. New translational models are thus required to comprehensively understand the disease pathophysiology and develop improved therapeutic interventions. We describe here a clinically relevant model, whereby the native multicellular PDR landscape and neo(fibro)vascular processes can be analysed ex vivo and related to clinical data. As characterized by three-dimensional whole-mount immunofluorescence and electron microscopy, heterogeneity in patient-derived PDR neovascular tissues included discontinuous capillaries coupled with aberrantly differentiated, lymphatic-like and tortuous endothelia. Spatially confined apoptosis and proliferation coexisted with inflammatory cell infiltration and unique vascular islet formation. Ex vivo-cultured explants retained multicellularity, islet patterning and capillary or fibrotic outgrowth in response to vitreoretinal factors. Strikingly, PDR neovascular tissues, whose matched vitreous samples enhanced lymphatic endothelial cell sprouting, contained lymphatic-like capillaries in vivo and developed Prox1+ capillaries and sprouts with lymphatic endothelial ultrastructures ex vivo. Among multiple vitreal components, vascular endothelial growth factor C was one factor found at lymphatic endothelium-activating concentrations. These results indicate that the ischaemia-induced and inflammation-induced human PDR microenvironment supports pathological neolymphovascularization, providing a new concept regarding PDR mechanisms and targeting options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Recovery of the sub-basal nerve plexus and superficial nerve terminals after corneal epithelial injury in mice

Our aim was to compare regeneration of the sub-basal nerve plexus (SBNP) and superficial nerve terminals (SNT) following corneal epithelial injury. We also sought to compare agreement when quantifying nerve parameters using different image analysis techniques. Anesthetized, female C57BL/6 mice received central 1-mm corneal epithelial abrasions. Four-weeks post-injury, eyes were enucleated and processed for PGP9.5 to visualize the corneal nerves using wholemount immunofluorescence staining and confocal microscopy. The percentage area of the SBNP and SNT were quantified using: ImageJ automated thresholds, ImageJ manual thresholds and manual tracings in NeuronJ. Nerve sum length was quantified using NeuronJ and Imaris. Agreement between methods was considered with Bland-Altman analyses. Four-weeks post-injury, the sum length of nerve fibers in the SBNP, but not the SNT, was reduced compared with naïve eyes. In the periphery, but not central cornea, of both naïve and injured eyes, nerve fiber lengths in the SBNP and SNT were strongly correlated. For quantifying SBNP nerve axon area, all image analysis methods were highly correlated. In the SNT, there was poor correlation between manual methods and auto-thresholding, with a trend towards underestimating nerve fiber area using auto-thresholding when higher proportions of nerve fibers were present. In conclusion, four weeks after superficial corneal injury, there is differential recovery of epithelial nerve axons; SBNP sum length is reduced, however the sum length of SNTs is similar to naïve eyes. Care should be taken when selecting image analysis methods to compare nerve parameters in different depths of the corneal epithelium due to differences in background autofluorescence.

Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes.

Asymmetric distribution of mRNA and protein is a hallmark of cell polarity in many systems. The Xenopus laevis oocyte provides many technical advantages to studying such polarity. Thousands of oocytes at different stages of maturity can be harvested from a single ovary and, owing to their relatively large size, even the youngest oocytes can be manually microinjected. Microinjection of fluorescently labeled RNA combined with immunofluorescence of endogenous proteins can provide insight into the cytoplasmic interactions contributing to polarity. Here, we present an updated method to image endogenous protein and microinjected RNA in X. laevis oocytes.

The ancient role for GATA2/3 transcription factor homolog in the hemocyte production of oyster

Hemocytes, the cellular component of invertebrate hemolymph, are essential for invertebrate immunity, but the hematopoiesis and regulation mechanism are still largely unknown. In the present study, a conserved hematopoietic transcription factor Cg-GATA2/3 was identified in Pacific oyster Crassotrea gigas, which was evolutionarily close to the vertebrate GATA1/2/3. Cg-GATA2/3 was mainly distributed in the immune organs, such as gill, hemocytes, and mantle. After Cg-GATA2/3 was interferenced by dsRNA, the mRNA expressions of hemocytes specific gene (EcSOD) and hematopoietic transcription factor (C-Myb) were all significant down-regulated, and the hemocyte renewal rates also decreased both in hemolymph and gill. During the larval developmental stages, the mRNA transcripts of Cg-GATA2/3 increased immediately after fertilization and kept a high level during blastula and early trochophore larvae stage (4-10 hpf, hours post fertilization), then decreased sharply in early D-veliger larvae stage (15 hpf). Whole-mount immunofluorescence assay further revealed that the abundant immunoreactivity of Cg-GATA2/3 was distributed in the whole body of blastula and gastrula embryos, while specialized gradually to a ring structure around the dorsal region in trochophore larvae. In the D-veliger and umbo larvae, scattered positive signals appeared in the specific sinus structure on the dorsal side and velum region. These results demonstrated that Cg-GATA2/3 was a hematopoietic lineage-specific transcription factor to regulate the hemocyte production, and it could also be used as hematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster.