Whole-mount Imaging Research Articles

E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning

Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.

Whole-organism cellular gene-expression atlas reveals conserved cell types in the ventral nerve cord of Platynereis dumerilii

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed “Profiling by Signal Probability Mapping” algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants.

Notch signalling restricts inflammation and serpine1 expression in the dynamic endocardium of the regenerating zebrafish heart.

The zebrafish heart regenerates after ventricular damage through a process involving inflammation, fibrotic tissue deposition/removal and myocardial regeneration. Using 3D whole-mount imaging, we reveal a highly dynamic endocardium during cardiac regeneration, including changes in cell morphology, behaviour and gene expression. These events lay the foundation for an initial expansion of the endocardium that matures to form a coherent endocardial structure within the injury site. We studied two important endocardial molecules, Serpine1 and Notch, which are implicated in different aspects of endocardial regeneration. Notch signalling regulates developmental gene expression and features of endocardial maturation. Also, Notch manipulation interferes with attenuation of the inflammatory response and cardiomyocyte proliferation and dedifferentiation. serpine1 is strongly expressed very early in the wound endocardium, with decreasing expression at later time points. serpine1 expression persists in Notch-abrogated hearts, via what appears to be a conserved mechanism. Functional inhibition studies show that Serpine1 controls endocardial maturation and proliferation and cardiomyocyte proliferation. Thus, we describe a highly dynamic endocardium in the regenerating zebrafish heart, with two key endocardial players, Serpine1 and Notch signalling, regulating crucial regenerative processes.

Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes.

Local anatomy of lymphoid tissues during infection has emerged as a critical regulator of immunity; thus, studying the cellular choreography in the context of an intact tissue environment in situ is crucial. Following an infection, the local pathogen-specific T cell migration and the subsequent egress of effector T cells from the draining lymph nodes are important and complex biological processes. The mechanisms that regulate this complex process can now be investigated by directly visualizing T cell dynamics in vivo using intravital two-photon (2P) microscopy. In addition, static whole-mount imaging technique can provide us with a comprehensive assessment of global changes in the distribution of cellular populations within an intact tissue. Thus, in this chapter, we detail methods to visualize the migration and egress of endogenous antigen-specific CD8 T cells following viral infection using two methods-intravital 2P microscopy and multicolor whole-mount in situ tetramer staining.

Angiodysplasia in embryo lacking protein arginine methyltransferase 1 in vascular endothelial cells.

Protein arginine methyltransferase 1 (PRMT1) is involved in multiple cellular functions including proliferation and differentiation. Although PRMT1 is expressed in vascular endothelial cells (ECs), which are responsible for angiogenesis during embryonic development, its role has remained elusive. In this study, we generated endothelial-specific prmt1-knockout (Prmt1-ECKO) mice, and found that they died before embryonic day 15. The superficial temporal arteries in these embryos were poorly perfused with blood, and whole-mount 3D imaging revealed dilated and segmentalized luminal structures in Prmt1-ECKO fetuses in comparison with those of controls. Our findings provide evidence that PRMT1 is important for embryonic vascular formation.

2016 AHA Late-Breaking Basic Science Abstracts.

### 23633 ### The Transcription Factor Early B-cell Factor 1 is Critical for Proper Formation of the Cardiac Ventricular Conduction System Eugene Kim, Akshay Shekhar, Jie Zhang, Fang-Yu Liu, Wilson Young, Glenn I Fishman; New York Univ Sch of Medicine, New York, NY Transcriptional regulation of cardiac ventricular conduction system (VCS) development is incompletely understood. Using gene expression profiling of the VCS using the Cntn2-EGFP transgenic VCS fluorescent reporter mouse, we identified Early B-cell Factor 1, Ebf1 , as being significantly enriched in the VCS. Surface electrocardiography of the Ebf1 knockout mouse revealed ventricular conduction slowing as evidences by QRS widening; echocardiography using strain imaging revealed marked left ventricular dyssynchrony; and wholemount imaging of the Ebf1 KO heart demonstrated dramatic hypoplasia of the VCS. Gene expression profiling was performed on E13.5 embryonic hearts using deep sequencing and a number of transcription factors known to be critical for cardiogenesis were markedly reduced, including Nkx 2–5, Gata4, Hand1, Irx3 , and Irx5 . Histological analysis of embryonic hearts revealed marked cellular hyperplasia and staining for markers of cell cycle (Ki-69; phospho-histone H3) were consistent with exuberant cellular proliferation. Quantitative immunofluorescence for several markers of terminally differentiated VCS cells was performed and demonstrated reduced expression of Nkx 2–5, Scn5a , and Cx40 , particularly within the terminal Purkinje fibers of the VCS. Finally, analysis of the Nkx 2–5 promoter revealed a highly conserved Ebf1 binding site in a region previously described as critical for proper expression of Nkx 2–5 in the VCS. Using CRISPR-Cas9 mediated gene editing, a 200 bp region flanking this site was deleted and the mutant mice generated also demonstrated marked QRS widening, phenocopying the Ebf1 KO. Author Disclosures: E. Kim: None. A. Shekhar: None. J. Zhang: None. F. Liu: None. W. Young: None. G.I. Fishman: None. Key Words: Cardiac development; Biology, developmental; Cardiovascular development; Electrophysiology; Heart conduction system ### 23347 ### Intramyocardial Delivery of miR-199a Reduces Scar Size and Preserves Contractile Function In Infarcted Pig Hearts Khatia Gabisonia; Sant’Anna Sch of Advanced Studies, Pisa, Italy We have previously …

Loss of Adrenergic Nerves in the Bone Marrow Microenvironment Drives an Aging HSC Niche Phenotype

Hematopoietic stem cell (HSC) aging is accompanied by an expansion of myeloid-biased HSCs with declined long-term self-renewal functions, contributing to the development of hematologic malignancies. Recent studies have suggested a role for the bone marrow microenvironment in HSC aging but the extrinsic molecular mechanisms driving their aging remain largely unknown. Mesenchymal stem cells (MSCs) form a critical component of the HSC niche, promoting HSC quiescence and balanced differentiation. Some MSC activity is found associated with arterioles, which are densely innervated by the sympathetic nervous system (SNS). We used tridimensional whole-mount bone marrow confocal immunofluorescence imaging of HSCs and vascular structures to investigate the effects of aging on the HSC microenvironment. Comparative bone marrow analyses of aged (20-24 months) C57BL/6 and Nestin-GFP transgenic mice (where GFP marks MSCs) with young (2 month-old) C57BL/6 mice, revealed significant reductions in sympathetic innervation (3-fold, p<0.01) as determined by staining for tyrosine hydroxylase expressing nerve fibers. The SNS neuropathy of aged mice was accompanied with vascular remodeling characterized by an increase in total vascular density marked by CD31+/CD144+ (2-fold, p<0.01) and shortening of arterioles marked by CD31+/CD144+ and Nestin-GFP+ (5-fold reduction, p<0.001), reduction in α-smooth muscle actin (αSMA) pericytes (12-fold, p<0.01) and expansion of Nestin+ MSCs (4 fold, p<0.05). Whereas a subset of HSCs (~35%) are associated with arterioles in young mice, analyses of HSC localization in aged bone marrow revealed that HSCs were redistributed and expanded away from arterioles (p=0.0047). Functional analysis revealed a diminished in vitro clonal capacity of aged MSCs (determined by CFU-F and mesensphere assays; 3-fold, p<0.0001), reduced HSC maintenance activity determined by the ability to produce HSC maintenance factors (Cxcl12, Scf and Angpt1, 2-fold, p<0.01), and increased proliferative state (2-fold, p<0.0001), all indicative of reduced activity. Since SNS nerves regulate circadian egress of HSCs through regulation of MSC activity, we evaluated the circadian oscillations of HSCs in blood and niche factors in Nestin-GFP+ stromal cells. Aged mice exhibited ablated oscillations of progenitor release determined by peripheral blood colony-forming units in culture (CFU-C/ml blood) and phenotypic progenitors (LSK/ml blood). Furthermore, unlike the robust circadian oscillations of Cxcl12 levels of young MSCs, no circadian expression pattern was detected in aged mice, suggesting that the SNS neuropathy associated with aging has functional consequences by leading to disrupted circadian rhythms and expanded MSC populations with reduced HSC maintenance activity. To determine whether the loss of SNS nerves drives bone marrow vascular remodeling and HSC aging, we surgically sympathectomized young Nestin-GFP mice by unilateral microsection of both the sciatic and femoral nerves, while the sham-operated contralateral side served as control. Strikingly, four months following surgery, phenotypic HSCs selectively expanded in sympathectomized femurs (2-fold, p<0.05) and exhibited myeloid-biased differentiation upon competitive bone marrow transplantation compared to control (3-fold, p<0.01). Furthermore, similar to our observations in aged mice, chronic surgical sympathectomy (16 weeks) induced dramatic "aging-like" alterations of bone marrow arteriolar structures characterized with arteriolar shortening and loss of αSMA+ and Nestin-GFP+ arterioles (4-fold, p<0.01; 3 fold, p<0.001). These results thus suggest that aging-associated sympathetic neuropathy and the loss of β-adrenergic signals in the bone marrow microenvironment drive aging of HSCs by remodeling vascular niches and thus represent a potent therapeutic target for stem cell rejuvenation. DisclosuresFrenette:Pfizer: Consultancy; GSK: Research Funding; PHD Biosciences: Research Funding.

Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods.

BackgroundHigh-resolution 3D imaging of intact tissue facilitates cellular and subcellular analyses of complex structures within their native environment. However, difficulties associated with immunolabelling and imaging fluorescent proteins deep within whole organs have restricted their applications to thin sections or processed tissue preparations, precluding comprehensive and rapid 3D visualisation. Several tissue clearing methods have been established to circumvent issues associated with depth of imaging in opaque specimens. The application of these techniques to study the elaborate architecture of the mouse mammary gland has yet to be investigated.MethodsMultiple tissue clearing methods were applied to intact virgin and lactating mammary glands, namely 3D imaging of solvent-cleared organs, see deep brain (seeDB), clear unobstructed brain imaging cocktails (CUBIC) and passive clarity technique. Using confocal, two-photon and light sheet microscopy, their compatibility with whole-mount immunofluorescent labelling and 3D imaging of mammary tissue was examined. In addition, their suitability for the analysis of mouse mammary tumours was also assessed.ResultsVarying degrees of optical transparency, tissue preservation and fluorescent signal conservation were observed between the different clearing methods. SeeDB and CUBIC protocols were considered superior for volumetric fluorescence imaging and whole-mount histochemical staining, respectively. Techniques were compatible with 3D imaging on a variety of platforms, enabling visualisation of mammary ductal and lobulo-alveolar structures at vastly improved depths in cleared tissue.ConclusionsThe utility of whole-organ tissue clearing protocols was assessed in the mouse mammary gland. Most methods utilised affordable and widely available reagents, and were compatible with standard confocal microscopy. These techniques enable high-resolution, 3D imaging and phenotyping of mammary cells and tumours in situ, and will significantly enhance our understanding of both normal and pathological mammary gland development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0754-9) contains supplementary material, which is available to authorized users.

Optimized SIFTFlow for registration of whole-mount histology to reference optical images.

The registration of two-dimensional histology images to reference images from other modalities is an important preprocessing step in the reconstruction of three-dimensional histology volumes. This is a challenging problem because of the differences in the appearances of histology images and other modalities, and the presence of large nonrigid deformations which occur during slide preparation. This paper shows the feasibility of using densely sampled scale-invariant feature transform (SIFT) features and a SIFTFlow deformable registration algorithm for coregistering whole-mount histology images with blockface optical images. We present a method for jointly optimizing the regularization parameters used by the SIFTFlow objective function and use it to determine the most appropriate values for the registration of breast lumpectomy specimens. We demonstrate that tuning the regularization parameters results in significant improvements in accuracy and we also show that SIFTFlow outperforms a previously described edge-based registration method. The accuracy of the histology images to blockface images registration using the optimized SIFTFlow method was assessed using an independent test set of images from five different lumpectomy specimens and the mean registration error was [Formula: see text].

Distinct vascular niches determine hematopoietic stem cell fate

Identification of cellular constituents of the hematopoietic stem cell (HSC) niche has recently been the subject of intensive investigation. To investigate the spatial localization of the HSC niches in bone marrow, we have established a whole-mount immunofluorescence imaging technique in which the 3D spatial relationships between stromal structures and HSCs in the BM can be precisely determined. The imaging assessment combined with computational simulations has uncovered a significant association between HSCs and arterioles, ensheathed exclusively by rare Nestin(bright) NG2⁺ pericytes (Nes(peri) cells), distinct from sinusoid-associated Nestin(dim) Leptin receptor (LepR)⁺ (Nes(retic)) cells which reportedly represent peri-vascular niche cells. Depletion of NG2⁺ cells using NG2-creERTM / inducible diphtheria toxin receptor (iDTR) mice changed HSC localization away from arterioles, induced HSC cycling and reduced long-term repopulation of HSCs in BM, suggesting that periarteriolar NG2⁺ cells form quiescent niches for HSCs. These results form the basis of studies that will allow us to genetically dissect the functions of distinct vascular niches. This vascular niche model, in which arterioles and sinusoids differentially regulate HSC quiescence and proliferation, respectively, have implications for the behavior of healthy HSC and may be useful in the future for evaluating the niches for cancer (leukemic) stem cells.

Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice

Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1+/− on the C57BL/6Jx129 and ICR backgrounds, Bmp4+/− ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1+/− and Bmp4+/− heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma.

Neuroanatomy and Neurochemistry of Mouse Cornea.

PurposeTo investigate the entire nerve architecture and content of the two main sensory neuropeptides in mouse cornea to determine if it is a good model with similarities to human corneal innervation.MethodsMice aged 1 to 24 weeks were used. The corneas were stained with neuronal-class βIII-tubulin, calcitonin gene–related peptide (CGRP), and substance P (SP) antibodies; whole-mount images were acquired to build an entire view of corneal innervation. To test the origin of CGRP and SP, trigeminal ganglia (TG) were processed for immunofluorescence. Relative corneal nerve fiber densities or neuron numbers were assessed by computer-assisted analysis.ResultsBetween 1 and 3 weeks after birth, mouse cornea was mainly composed of a stromal nerve network. At 4 weeks, a whorl-like structure (or vortex) appeared that gradually became more defined. By 8 weeks, anatomy of corneal nerves had reached maturity. Epithelial bundles converged into the central area to form the vortex. The number and pattern of whorl-like structures were different. Subbasal nerve density and nerve terminals were greater in the center than the periphery. Nerve fibers and terminals that were CGRP-positive were more abundant than SP-positive nerves and terminals. In trigeminal ganglia, the number of CGRP-positive neurons significantly outnumbered those positive for SP.ConclusionsThis is the first study to show a complete map of the entire corneal nerves and CGRP and SP sensory neuropeptide distribution in the mouse cornea. This finding shows mouse corneal innervation has many similarities to human cornea and makes the mouse an appropriate model to study pathologies involving corneal nerves.

Reawakening the Duct Cell Progenitor?

The generation of new -cells from progenitors is an area of immense research interest in diabetes, given the potential not only for therapeutic -cell replacement by transplantation but also regeneration of endogenous -cells. Defining the progenitors that serve as sources of new -cells in vivo has been an area of considerable debate and research effort (1, 2). Replication of existing -cells has been shown to be an important source of new -cells in rodents throughout life, in health and in response to injury (3). Evidence for transdifferentiation of non-cells, including -cells (4) and pancreatic acinar cells (5, 6) has also emerged in rodents. Most studies have employed models of -cell regeneration after severe -cell loss or pancreatic injury, potentially inducing “facultative” stem cell differentiation, leaving questions as to the importance of these putative progenitors in a physiological context. Pancreatic ductal cells have perhaps received the most interest and study as a potential -cell progenitor. Lineage tracing studies in mice in which carbonic anhydrase-expressing (and therefore likely ductal) cells were marked, provided persuasive evidence that ductal cells can differentiate into -cells in mice after ductal ligation (7), but limitations to such studies, such as the fidelity of the carbonic anhydrase promoter, raised questions that have lingered for a number of years. Although numerous histological studies have shown islets in close association with ducts, and are suggestive that new islets originate from ducts (8) and in response to -cell loss, there have been near as many reports that do not support the idea that ductal cells can give rise to -cells in adult mice (9, 10). An innovative whole-mount imaging approach with 3-dimensional reconstruction employed by El-Gohary et al in this issue of Endocrinology (11) sheds new light on this debate and provides compelling evidence that, at least under certain conditions, pancreatic duct cells can give rise to -cells postnatally. Using whole-mount immunostaining, small ducts were found to reside within the islets, and moreover these intraislet ducts appear to arise from larger pancreatic ducts outside of the islet. Importantly, wholemount imaging of a limited number of young human pancreas samples demonstrated the presence of intraislet ducts in humans as well. The presence of these intraislet ducts was highly age dependent in both mice and humans, peaking in mice at about 5 weeks of age, and being most prevalent in 1-2 month-old human sample. Notably these are times when -cell genesis is high. Somewhat surprisingly, -cell regeneration induced after partial pancreatectomy did not increase the number of intraislet ducts, at least in older (10-wk-old) mice. Building on their earlier finding that TGF may inhibit -cell neogenesis (12), the authors performed partial pancreatectomy in transgenic mice in which TGF signaling is suppressed by constitutive expression of a dominant negative TGF -type II receptor. This transgenic mouse model phenocopied the young mouse with extensive intraislet ducts and allowed El-Gohary et al to carry out lineage tracing to provide further support for their assertion that -cells can arise from ductal cells. For these studies, the pancreatic ductal tree of a Cre-reporter mouse line was infused with an adeno-associated virus serotype 6 virus expressing Cre recombinase under control of the “ductal” Sox9 promoter. After partial pancreatectomy, insulinpositive cells were observed to be lineage labeled, supporting their conclusion that -cells can be derived by neogenesis from pancreatic ductal cells.

Distinct Contributions By Perivascular Niche Cells in Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood types in response to various demands through life. HSC functions are tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow (BM). Using Nestin-GFP transgenic mice, we have identified Nestin-GFP+ perivascular stromal cells exhibiting a mesenchymal stem/progenitor cell activity as niche cells. Furthermore, we found two types of Nestin-GFP+ cells expressing different surface markers, Nerve/glial antigen 2 (NG2) and Leptin receptor (Lepr) that are associated with arterioles and sinusoid, respectively, in the BM (Kunisaki et al. Nature, 2013). Both arteriolar and sinusoidal niche cells have been reported to show high gene expression of cytokines essential for HSC maintenance such as CXCL12 and stem cell factor (SCF), however, it remains unknown how the distinct niche cells differentially regulate HSC functions. To investigate the mechanisms, we utilized genetic mouse models, in which CXCL12 or SCF can be deleted in specific cell types. CXCL12 deletion in sinusoidal niche cells by using Lepr-cre/Cxcl12fl/− mice mobilized HSCs and lineage− Sca-1+ c-kit+ (LSK) progenitors into spleen (HSC, CT: Control/DL: Deleted: 760±165 / 2193±557 / spleen, n=6, p<0.05) and blood (LSK, CT/DL: 177±36 / 668±156 / mL blood, n=5, p<0.05), but had no effect on HSC numbers in the BM (CT/DL: 1435±101 / 1194±75 / femur, n=6, p=0.085), which is consistent with a previous report (Ding and Morrison, Nature, 2013). Furthermore, assessments of endogenous HSC localization using whole-mount 3D imaging technology revealed that the deletion of CXCL12 in Lepr+ niche cells had no impact on HSC location (KS-test: Two-sample Kolmogorov-Smirnov test, p=0.9981). By contrast, deletion of CXCL12 in NG2-cre derived cells, which recombines efficiently in the entire Nestin-GFP+ non-endothelial stromal fraction including both peri-arteriolar and peri-sinusoidal cells (96.9±1.3%), and overlapping with Lepr+ cells (88.5±1.6%) and CXCL12-abundant reticular cells (90.7±1.4%), led to a robust reduction of HSC numbers in the BM (CT/DL: 1487±87 / 179±40 / femur, n=10, p<0.0001) with HSC and progenitor mobilization into spleen (HSC, CT/DL: 705±262 / 3550±540 / spleen, n=6-8, p<0.01) and blood (LSK, CT/DL: 494±178 / 5357±896 / mL blood, n=5-7, p<0.01). In addition, deletion of CXCL12 in NG2-cre targeted cells led to HSC displacement away from arterioles (KS-test: Two-sample Kolmogorov-Smirnov test; p=0.001). To examine further a role of CXCL12 produced by NG2+ arteriolar niches on HSC maintenance, we generated tamoxifen-inducible NG2-creERTM/Cxcl12fl/− mice. Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduced the number of HSCs in the BM (CT/DL: 1617±160 / 960±95 / femur, n=10-13, p=0.0013), which was confirmed functionally by a competitive repopulation assay. Moreover, 3D imaging revealed that HSCs were located further away from arterioles in NG2-creERTM/Cxcl12fl/− marrow (KS-test: p<0.0001), suggesting a role for arteriolar niches in CXCL12-mediated HSC maintenance. As niche cells synthesize several factors, we evaluated the contribution of arteriolar niches in SCF synthesis, a cytokine shown to be critical for HSC maintenance. As expected, deletion of SCF in NG2-cre targeted cells led to a significant reduction of HSC numbers in the BM (CT/DL: 606±85 / 96±23 / femur, n=5-7, p<0.0001). To further evaluate functions of SCF produced by distinct vascular niches, we also compared these mice with deletions using Lepr-cre or tamoxifen-inducible NG2-creERTM mice. We found that deletion of SCF in Lepr-cre targeted cells showed a significant reduction of HSC numbers in the BM (CT/DL: 690±84 / 220±83 / femur, n=3-4, p<0.0118), consistent with previous studies (Ding et al., Nature, 2012), whereas there was no significant change observed in NG2-creERTM/SCFfl/− mice, suggesting that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important source of SCF in the BM. These results highlight distinct contributions of perivascular cells primarily located in separate vascular niches, arteriolar and sinusoidal, in HSC maintenance and mobilization. DisclosuresNo relevant conflicts of interest to declare.

A histamine-independent itch pathway is required for allergic ocular itch

Non-histaminergic TRPA1 neural pathway is required for the development of allergic ocular itch. Pharmacological inhibition of TRPA1 channel can lead to novel therapeutic strategies to treat ocular itch in severe allergic conjunctivitis.

Intravital and Whole-Organ Imaging Reveals Capture of Melanoma-Derived Antigen by Lymph Node Subcapsular Macrophages Leading to Widespread Deposition on Follicular Dendritic Cells

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

Morphology and neurochemistry of rabbit iris innervation

The aim of this study was to map the entire nerve architecture and sensory neuropeptide content of the rabbit iris. Irises from New Zealand rabbits were stained with antibodies against neuronal-class βIII-tubulin, calcitonin gene-related peptide (CGRP) and substance P (SP), and whole-mount images were acquired to build a two-dimensional view of the iridal nerve architecture. After taking images in time-lapse mode, we observed thick nerves running in the iris stroma close to the anterior epithelia, forming four to five stromal nerve rings from the iris periphery to the pupillary margin and sub-branches that connected with each other, constituting the stromal nerve plexus. In the anterior side, fine divisions derivated from the stromal nerves, forming a nerve network-like structure to innervate the superficial anterior border layer, with the pupillary margin having the densest innervation. In the posterior side, the nerve bundles ran along with the pupil dilator muscle in a radial pattern. The morphology of the iris nerves on both sides changed with pupil size. To obtain the relative content of the neuropeptides in the iris, the specimens were double stained with βIII-tubulin and CGRP or SP antibodies. Relative nerve fiber densities for each fiber population were assessed quantitatively by computer-assisted analysis. On the anterior side, CGRP-positive nerve fibers constituted about 61%, while SP-positive nerves constitute about 30.5%, of the total nerve content, which was expressed as βIII tubulin-positive fibers. In addition, in the anterior stroma of the collarette region, there were non-neuronal cells that were positive for SP. On the posterior side, CGRP-positive nerve fibers were about 69% of total nerve content, while SP constituted only up to 20%. Similarly, in the trigeminal ganglia (TG), the number of CGRP-positive neurons significantly outnumbered those that were positive for SP. Also, all the SP-positive neurons were labeled with CGRP. This is the first study to provide a two-dimensional whole mount and a cross-sectional view of the entire iris nerve architecture. Considering the anatomical location, the high expression of CGRP and SP suggests that these neuropeptides may play a role in the pathogenesis of anterior uveitis, glaucoma, cataracts and chronic ocular pain.

Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice

This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Whole-mount imaging of mouse embryo sensory axon projections.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkA(taulacZ) mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkA(taulacZ) line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkA(taulacZ)/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.