Whole-mount Staining Research Articles

A novel whole-embryo culture model for pharmaceutical and developmental studies.

This report introduces a new vertebrate whole-embryo culture model for the direct application of pharmaceuticals and/or toxins into developing embryos. This method uses a terrestrial amphibian system and therefore has eliminated the problem of mammalian placental and uterine concerns. To test the utility and effectiveness of this method, we investigated the effect of fluoxetine on craniofacial development. Fluoxetine is one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) on the market and treatment of depression during pregnancy is commonly deemed necessary. Previous studies have shown that SSRIs may promote developmental defects and congenital malformations of the heart. This model utilized the egg/embryos of the directly developing Puerto Rican coquí frog, Eleutherodactylus coqui. The E. coqui embryo clutches were placed on filter paper in a Petri dish and were directly exposed (chronically) to fluoxetine concentrations ranging from 0.10mM to 1.0mM. Traditional whole-mount bone (Alizarin red) and cartilage (Alcian blue) staining was utilized to show the effect of fluoxetine on craniofacial development. Whole-mount staining revealed profound defects in cartilage development, particularly in the nasal capsule, mandible, and the brain case. Further, fluoxetine-treated embryos developed significantly slower compared to control animals. We found that the E. coqui culture model was an effective and sensitive technique for pharmaceutical studies, particularly since it allows the direct application of drugs and toxins into the developing embryo without the hindrance of the uterus and placenta. Chromatographic analysis revealed that fluoxetine infiltrated and penetrated embryonic tissue. It was found that altering serotonergic activity during development, via fluoxetine, stunted craniofacial development and organization.

A rapid and convenient method for fluorescence analysis of in vitro cultivated metacestode vesicles from Echinococcus multilocularis.

We here describe a convenient method for preparation, fixation and fluorescence analysis of in vitro cultivated metacestode vesicles from E. multilocularis. Parasite materials could be prepared in one hour, did not need to be sectioned, and were subsequently utilized for further whole-mount staining assays directly. Using these preparations, in combination with conventional fluorescence staining techniques, we could detect the expression and subcellular localization of a specific protein and identify in situ proliferative or apoptotic cells in the germinal layer of metacestode vesicles. Based on this approach, future molecular and cellular analysis of Echinococcus metacestode vesicles in the in vitro system will be greatly facilitated.

Absence of Wolbachia endobacteria in Sri Lankan isolates of the nematode parasite of animals Setaria digitata

Setaria digitata is an animal filarial parasite with natural hosts of cattle and buffaloes that causes mild disease conditions. Infection of non-permissive hosts such as goats, sheep and horses, by this nematode can cause cerebrospinal nematodiasis that leads to lumbar paralysis and the eventual death of the animals and inflicts considerable economic losses on livestock farmers. Wolbachia are obligate mutualistic endosymbionts for some filarial nematodes and are currently being targeted for the control of diseases caused by these parasites. However, little is known about the occurrence of this endosymbiont in the Setariidae family. In this work, worms collected from infected cattle in Sri Lanka were morphologically identified as S. digitata and tested for the presence of Wolbachia by PCR screening using the WSP- and Wolbachia-specific 16S rRNA and multilocus sequence typing primers that were designed to amplify the gatB, coxA, hcpA, ftsZ and fbpA sequences of Wolbachia. The presence of endobacteria in S. digitata was also examined by whole-mount immunofluorescence staining of the parasites and transmission electron microscopic studies. These analyses did not produce evidence of presence of Wolbachia or any other endosymbiotic bacteria in S. digitata, whereas such evidence was found in Brugia malayi, which was used as a positive control in this study.

A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-κB and the PKCα/PI3-K/Akt pathways

IntroductionThe natural product tetramethylpyrazine (TMP) has a variety of biologic activities, including neuroprotection. Nitrones are powerful free radical scavengers. We have designed and synthesized a TMP derivative, TN-2, which is armed with two nitrone moieties. AimsIn this study, we investigated the neuroprotective effect of TN-2 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro and in zebrafish. MethodsPC12 cells, zebrafish and rats were exposed to 6-OHDA challenge. MTT assay, LDH release, Hoechst staining, DAF-FM staining, luciferase reporter construct transfection, and western blotting were applied to detect cell viability, apoptosis, intracellular nitric oxide (NO), NF-κB transcriptional activity and proteins expression. In zebrafish, whole-mount staining and real-time PCR were performed to quantify dopaminergic neurons and mRNA expression. Hematoxylin and eosin staining and immunohistochemistry for glial fibrillary acidic protein were used to detect the astrocyte activation in the unilateral 6-OHDA rat model. ResultsTN-2 but not TMP exhibited potent neuroprotective effect against 6-OHDA-induced apoptosis in PC12 cells. Moreover, TN-2 prevented dopaminergic neuron loss and suppressed mRNA expression of pro-inflammatory genes, including IL-1β, TNF-α and COX-2, in 6-OHDA-treated zebrafish. TN-2 remarkably attenuated microglial/astrocyte activation in the unilateral 6-OHDA rat model. The mechanistic study demonstrated that TN-2 inhibited over-production of intracellular NO and protein expression of inducible nitric oxide synthase through down-regulating NF-κB activity. Additionally, the PKCα/PI3-K/Akt pathway was also involved in the neuroprotection of TN-2. ConclusionThese results suggest that TN-2 protected against 6-OHDA-induced neurotoxicity via modulating the NF-κB-medicated neuroinflammation and PKCα/PI3-K/Akt pathways.

Anatomy and ontogeny of a novel hemodynamic organ in zebrafish.

The zebrafish maxillary barbel can protract and retract in response to stimuli, and appears connected to a prominent blood sinus on the lateral aspect of the maxillary bone. However, the mechanism of barbel movement is not described. Using whole-mount phalloidin staining of the sinus region, we observed long filamentous actin cables, suggesting highly organized vascular smooth muscle cells, surrounding an endothelial chamber. Although the chamber is variably filled by erythrocytes in vivo, cardiac injection of fluorescent dextrans shows that it consistently contains plasma. Full-thickness confocal imaging of dextran-injected adults containing EGFP(+) endothelial cells revealed a vascular complex with three compartments, here named the distal bulb, central chamber, and accessory chamber. The early ontogeny of all three compartments was confirmed in a whole-mount series of Tg(fli1a:EGFP) juveniles. In wild type adults, the fine structure of each chamber was studied using paraffin- and plastic-section histochemistry and transmission electron microscopy. The distal bulb and central chamber have smooth muscle coats with luminally-elongated septa, forming semi-detached blood-filled lacunae. The central chamber walls and septa are extensively innervated by small, unmyelinated axons, as confirmed by immunohistochemical detection of acetylated tubulin, a component of axonal cytoplasm. The accessory chamber appears neither innervated nor muscularized, but is an endothelial cul-de-sac with a thickened elastic adventitia, suggesting an extensible fluid reservoir. We propose that we have identified a new organ in zebrafish, the maxillary barbel blood sinus, whose neurovascular specializations may contribute to zebrafish sensory biology and appendage control.

Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival.

To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival. Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy. Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells. Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.

Diabetes mellitus leads to accumulation of dendritic cells and nerve fiber damage of the subbasal nerve plexus in the cornea.

To evaluate whether nerve fibers of the subbasal nerve plexus (SNP) and dendritic cells (DCs) are in association with each other leading to neuropathy in the diabetic cornea. BALB/c mice were injected with streptozotocin (STZ) for 5 days for induction of diabetes mellitus (DM) or with vehicle solution (control). B6.VLep(ob/ob) (ob/ob) mice served as an obese and glucose-intolerant DM type 2 (DM II) model and lean B6.VLep(ob/+) (ob/+) mice as respective controls. Using in vivo corneal confocal microscopy (CCM), nerve fibers and DCs were quantified over a period of 9 weeks and additionally analyzed by in vitro immunofluorescence whole-mount staining. In STZ-diabetic mice, CCM revealed an increase of DC density (DCD) in contrast to controls, whereas nerve fiber density (NFD) was decreased with duration of DM. In ob/ob mice, DCD was 3-fold higher than in both ob/+ mice and STZ-diabetic mice. Whole-mount staining displayed CD11c(+) and major histocompatibility complex (MHC) class II(+) mature DCs in colocalization with class III β-tubulin(+) nerve fibers in the cornea. Hyperglycemia leads to corneal DC infiltration, and obesity aggravates this immune response. The direct contact between DCs and the SNP can be assumed to be a trigger of nerve fiber damage and thus a contributing factor to polyneuropathy in diabetic corneas.

A Three-Dimensional Atlas of Human Dermal Leukocytes, Lymphatics, and Blood Vessels

Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31+ blood capillaries, LYVE-1+ lymphatics, discrete populations of CD11c+ myeloid DCs, FXIIIa+ Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0–20 μm beneath the dermoepidermal junction), Mφ were located deeper (40–60 μm), and CD3+ lymphocytes were observed throughout (0–60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0–30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

Effect of neurturin deficiency on cholinergic and catecholaminergic innervation of the murine eye

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123–4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4–6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 μm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

The distribution and degeneration pattern of the cone opsins in rd11 mice

Background The retinal degeneration 11 (rd11) mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 (Lpcatl) mutation.Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rd1 1 mice,while cone degeneration pattern in rd11 mice is unclcar.Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rd1 1 mice.Methods A total of thirty rd1 1 and C57BL/6J mice at postnatal (P) day 14,28,42 (five in each age group) were sacrificed and retinal wholemounts were prepared.Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts,which were photographed with a fluorescent microscope.Cone opsins were compared between rd1 1 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas.Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina.M-opsin positive fluorescent dots in dorsal/temporal,ventral/temporal,dorsal/nasal and ventral/nasal quadrants of rdl 1 retina at P28 were (414±32),(300± 8),(324 ± 22) and (250± 20)/0.037 mm2,which were lower than the age-matched normal C57BL/6J mice (t =4.114,15.225,7.505,17.990,all at P＜0.05).At the same time the S-opsin positive fluorescent dots in P28 rd11 were (8 ±4),(175 ± 16),(74 ± 13) and (315 ±20)/0.037 mm2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice (t =8.555,17.076,21.637,13.498,all at P＜0.05).With the development of retinal degeneration in rd11 mice,the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina.Conclusions Most of the M-opsin and S-opsins,especially the S-opsins in rd11 mice,degenerate in 6 weeks.Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study. Key words: rd11 Mouse/spontaneous retinal degeneration; Cone; Opsin; Retinal wholemount; Immunofluoreseence staining

Effects of High Salt-Exposure on the Development of Retina and Lens in 5.5-Day Chick Embryo

Background/Aims: Excess maternal salt intake during pregnancy may alter fetal development. However, our knowledge on how an increased salt intake during pregnancy influences fetal eye development is limited. In this study, we investigated the effects of high-salt treatment on the developing eyes in chick embryos, especially focusing on the development of the retina and the lens. Methods: 5.5-day chick embryos were exposed to 280mosm/l (n=17), or 300mosm/l (n=16) NaCl. The treated embryos were then incubated for 96 hours before they were fixed with 4% paraformaldehyde for H&E staining, whole-mount embryo immunostaining and TUNEL staining. BrdU and PH3 incorporation experiments were performed on the chick embryos after high-salt treatment. RT-PCR analyses were conducted from chick retina tissues. Results: We demonstrated that high-salt treatment altered the size of eyes in chick embryos, induced malformation of the eyes and impaired the development of the lens and the retina. We found an impaired expression of Paired box 6 (PAX6) and neuronal cells in the developing retina as revealed by neurofilament immunofluorescent staining. There was a reduction in the number of BrdU-positive cells and PH3-positive cells in the retina, indicating an impaired cell proliferation with high-salt treatment. High-salt treatment also resulted in an increased number of TUNEL-positive cells in the retina, indicating a higher amount of cell death. RT-PCR data displayed that the expression of the pro-apoptotic molecule nerve growth factor (NGF) in chick retina was increased and CyclinD1 was reduced with high-salt treatment. The size of the lens was reduced and Pax6 expression in the lens was significantly inhibited. High salt-treatment was detrimental to the migration of neural crest cells. Conclusion: Taken together, our study demonstrated that high-salt exposure of 5.5-day chick embryos led to an impairment of retina and lens development, possibly through interfering with Pax6 expression.

Effect of NSAIDs and Diuretics on Nephrogenesis in an Ex Vivo Embryogenic Kidney Model

The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole-mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short-term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.

Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development

MMP-11 is a bad prognosis paracrine factor in invasive breast cancers. However, its mammary physiological function remains largely unknown. In the present study we have investigated MMP-11 function during postnatal mammary gland development and function using MMP-11-deficient (MMP-11-/-) mice. Histological and immunohistochemical analyses as well as whole-mount mammary gland staining show alteration of the mammary gland in the absence of MMP-11, where ductal tree, alveolar structures and milk production are reduced. Moreover, a series of transplantation experiments allowed us to demonstrate that MMP-11 exerts an essential local paracrine function that favors mammary gland branching and epithelial cell outgrowth and invasion through adjacent connective tissues. Indeed, MMP-11-/- cleared fat pads are not permissive for wild-type epithelium development, whereas MMP-11-/- epithelium transplants grow normally when implanted in wild-type cleared fat pads. In addition, using primary mammary epithelial organoids, we show in vitro that this MMP-11 pro-branching effect is not direct, suggesting that MMP-11 acts via production/release of stroma-associated soluble factor(s). Finally, the lack of MMP-11 leads to decreased periductal collagen content, suggesting that MMP-11 has a role in collagen homeostasis. Thus, local stromal MMP-11 might also regulate mammary epithelial cell behavior mechanically by promoting extracellular matrix stiffness. Collectively, the present data indicate that MMP-11 is a paracrine factor involved during postnatal mammary gland morphogenesis, and support the concept that the stroma strongly impact epithelial cell behavior. Interestingly, stromal MMP-11 has previously been reported to favor malignant epithelial cell survival and promote cancer aggressiveness. Thus, MMP-11 has a paracrine function during mammary gland development that might be harnessed to promote tumor progression, exposing a new link between development and malignancy.

P.9.11 Transgenic zebrafish expressing mutant skeletal muscle actin, acta1a, model human nemaline myopathy

The nemaline myopathies (NMs) are a heterogeneous group of congenital myopathies defined by moderate to severe muscle weakness and accumulation of rod-like structures (nemaline bodies) in myofibers. Heterozygous dominant mutations of the skeletal muscle α-actin gene (ACTA1) account for ∼25% of all NM cases. Despite our current understanding of normal actin function, the mechanisms underlying muscle pathology remain unclear and there are no curative therapies available for patients. In order to elucidate the molecular defects behind ACTA1-related NM, we are generating a panel of transgenic zebrafish lines that express a series of disease-linked dominant mutations on the zebrafish acta1a transcript, the predominant actin expressed in fast myofibers. We initially tested whether three of these variants, acta1a H42Y, M134V and V165M would lead to a muscle phenotype by injecting the mutant mRNA into zebrafish embryos. The fish overexpressing mutant actins displayed myopathic phenotypes characterized by delayed hatching from the chorion and curved bodies to variable degrees that corresponded with the severity of disease in patients with these mutations. We next generated an acta1a V165M transgenic line that stably expresses mutant actin in all fast myofibers and characterized its neuromuscular phenotypes by morphological and histological analysis at different time points. The acta1a V165M fish display muscle weakness as evidenced by their thin bodies and curved tails at 2 days-post-fertilization (dpf) and reduced motility in touch-evoked escape response assay at 5 dpf. Whole-mount phalloidin staining at 2 dpf revealed actin aggregates in the affected fish muscle, while electron microscopy demonstrated Z-line thickening and severe myofibrillar disorganization. These results provide proof of concept that zebrafish models of actin mutations recapitulate the human disease and have robust myopathic phenotypes that may be amenable for high-throughput chemical screening.

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

PurposeMacrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages.MethodsAutologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl2MDP-LIP). The presence of CD11b+ F4/80+ macrophages, α-smooth muscle actin+ (α-SMA+) myofibroblasts, CD31+ vascular endothelial cells and NG2 + pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation.ResultsSub-conjunctival Cl2MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl2MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion.ConclusionsMacrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.

Stem Cells from Trabecular Meshwork Home to TM Tissue In Vivo

To investigate the potential of human trabecular meshwork stem cells (TMSCs) for homing to mouse TM tissue and survival in vivo. Human TMSCs and fibroblasts were labeled with fluorescent membrane dye DiO and injected into normal mouse anterior chamber. Stem cell and TM cell markers were identified by immunofluorescent staining of cryosections or tissue whole mounts. Apoptosis was determined by TUNEL assay. Replicating and inflammatory cells were detected by bromodeoxyuridine (BrdU) incorporation and anti-CD45 staining, respectively. Quantitative RT-PCR detected gene expression of injected cells after isolation by fluorescence activated cell sorting. Intraocular pressure was measured using a TonoLab rebound tonometer. Expanded cultures of DiO-labeled TMSCs expressed stem cell markers preferentially in DiO positive cells, demonstrating a slow-cycling, label-retaining stem cell phenotype. DiO-labeled TMSCs injected into the anterior chamber of normal mice localized primarily in TM, remaining in the tissue at least 4 months. Within 1 week, TM-associated TMSCs began expressing TM marker protein CHI3L1. Fibroblasts injected in mouse anterior chamber showed distributed localization in corneal endothelium, lens epithelium, and TM and did not express CHI3L1. Little apoptosis was detected in injected TM tissue and intraocular pressure was not elevated during the experiment. Dividing cells or CD45-staining cells were not detected after TMSC-injection. Stem cells isolated from human TM and expanded in vitro exhibit the ability to home to the TM and differentiate into TM cells in vivo. Such cells present a potential for development of a novel cell-based therapy for glaucoma.

Functional Analyses of TNFR2 in Physiological and Pathological Retina Angiogenesis

To determine the function of tumor necrosis factor receptor-2 (TNFR2) in retinal development and ischemia-induced revascularization in an oxygen-induced retinopathy (OIR) model. Mice with a global deletion of TNFR2 (TNFR2-KO) or with a vascular endothelial cell (EC)-specific TNFR2 transgene (TNFR2-TG) were compared to wild-type C57BL/6 mice (WT). Retinal vasculature development was visualized by whole-mount and cross-sectional isolectin staining. In the OIR model, neonatal mice were subjected to 75% oxygen from postnatal day (P)7 to P12 and then returned to normoxia from P12 to P17. Immunostaining and biochemical analyses were performed to assess the effects of TNFR2 deletion and TNFR2 transgenesis on retinal vascular repair. TNFR2 deletion slightly delayed, while TNFR2 transgenesis weakly promoted, intraretinal vascular development and intraretinal vessel growth. TNFR2 deletion enhanced, while TNFR2 transgene reduced, hyperoxia-induced vaso-obliteration. However, hypoxia-induced revascularization and development of deep intraretinal vessels at P17 were reduced in TNFR2-KO but increased in TNFR2-TG mice without significant increase in preretinal neovascularization (NV). Moreover, TNFR2-TG/KO mice in which only vascular EC express TNFR2 sufficiently rescued the vascular defects of TNFR2-KO in the OIR model. Biochemical analyses of retina tissues showed that the phenotypic changes in retina correlated with TNFR2-dependent activation of Nuclear factor-κB (NF-κB) survival and bone marrow kinase (Bmx)-VEGFR2 angiogenic pathways. TNFR2 plays a marginal role during retinal vascular development. TNFR2 in vascular EC strongly prevents hyperoxia-induced vaso-obliteration by inhibiting cell apoptosis, and promotes retinal repair by enhancing hypoxia-induced revascularization without increasing pathological neovascular tufts. Therefore, activation of TNFR2 signaling may be an ideal strategy for the treatment of OIR.

The effect of hypoxia on facial shape variation and disease phenotypes in chicken embryos

SUMMARYCraniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3–6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4–6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

Localization of the bacterial symbiont Candidatus Midichloria mitochondrii within the hard tick Ixodes ricinus by whole-mount FISH staining

Here, we present an investigation on the spatial distribution of the bacterial symbiont Candidatus Midichloria mitochondrii within Ixodes ricinus, by whole mount fluorescence in situ hybridization (FISH). M. mitochondrii is a peculiar, recently discovered bacterium that resides in the mitochondria of female ticks. We applied a rapid and specific FISH protocol with oligonucleotide probes targeted on the 16S rRNA of M. mitochondrii, 12S rRNA of tick mitochondria, and a probe revealing active mitochondria. In this report that represents the first application of whole mount FISH on ticks, we observed strong, specific fluorescence signals in all the examined life stages, as the optimized protocol allowed us to overcome the autofluorescence interference of the cuticle. Cellular localization and quantification of the symbionts were also assessed with electron microscopy and specific real-time PCR, respectively.

Early lens ablation causes dramatic long-term effects on the shape of bones in the craniofacial skeleton of Astyanax mexicanus.

The Mexican tetra, Astyanax mexicanus, exists as two morphs of a single species, a sighted surface morph and a blind cavefish. In addition to eye regression, cavefish have an increased number of taste buds, maxillary teeth and have an altered craniofacial skeleton compared to the sighted morph. We investigated the effect the lens has on the development of the surrounding skeleton, by ablating the lens at different time points during ontogeny. This unique long-term study sheds light on how early embryonic manipulations on the eye can affect the shape of the adult skull more than a year later, and the developmental window during which time these effects occur. The effects of lens ablation were analyzed by whole-mount bone staining, immunohistochemisty and landmark based morphometric analyzes. Our results indicate that lens ablation has the greatest impact on the skeleton when it is ablated at one day post fertilisation (dpf) compared to at four dpf. Morphometric analyzes indicate that there is a statistically significant difference in the shape of the supraorbital bone and suborbital bones four through six. These bones expand into the eye orbit exhibiting plasticity in their shape. Interestingly, the number of caudal teeth on the lower jaw is also affected by lens ablation. In contrast, the shape of the calvariae, the length of the mandible, and the number of mandibular taste buds are unaltered by lens removal. We demonstrate the plasticity of some craniofacial elements and the stability of others in the skull. Furthermore, this study highlights interactions present between sensory systems during early development and sheds light on the cavefish phenotype.