Whole-mount Immunostaining Research Articles

Intronic regulatory sequences regulate epicardial gene expression and EMT during heart development

Abstract Background During organogenesis, cells from the outer layer of the heart, the epicardium, undergo epithelial-to-mesenchymal transition (EMT), contributing essential paracrine signals and different cell types to the growing heart. Epicardial cells are integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties during heart development. Purpose Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is widely accepted, the upstream regulatory mechanisms underpinning its activation remain elusive. Our previous research has led to the identification of two intronic evolutionary conserved regions (ECRs) shared between mouse and human, located within intron 1 of the Wt1 locus, i.e. +4.0kbECR and +5.8kbECR. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Methods & Results Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR (Wt1Δ+4.0kbECR and Wt1Δ+5.8kbECR mice) or a deletion comprising both ECRs (Wt1Δ+4-5.8kbECR mouse). Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation in these models. Mendelian ratios indicated an overall underrepresentation of Wt1ΔECR/ΔECR mutants recovered in the offspring arising from heterozygous inter-crosses. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular or membranous ventricular septum defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal formation/patterning of coronary artery stems in ΔECR/ΔECR hearts. Expression of Wt1 was markedly reduced in ΔECR/ΔECR hearts, but not in the kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue-section immunostaining combined with confocal microscopy revealed abnormal coronary vessel and innervation extension and patterning along the subepicardial space, as well as reduced epicardial EMT and myocardial non-compaction/hyper-trabeculation in Wt1ΔECR/ΔECR hearts. Conclusions Together, we demonstrated a requirement for novel intronic enhancers regulating Wt1 locus activity and impacting on essential epicardial EMT and associated biological processes during normal heart development. Importantly, observation of septum and aortic valve defects in ΔECR mutants suggests an hitherto unrecognised role for WT1/epicardial EMT and opens new avenues of research to improve our understanding of congenital heart disease that affects at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

Whole-mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

FOXC1 and FOXC2 Ablation Causes Abnormal Valvular Endothelial Cell Junctions and Lymphatic Vessel Formation in Myxomatous Mitral Valve Degeneration.

Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix). Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC-Foxc-DKO and lymphatic EC-Foxc-DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection. EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc-DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC-Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC-Foxc1/c2 mutant MVs. Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration.

Lymphatic endothelial cell-specific NRAS p.Q61R mutant embryos show abnormal lymphatic vessel morphogenesis.

Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.

Abstract Or122: Noncoding regulation of epicardial gene expression and epithelial-to-mesenchymal transition during heart development

During organogenesis, epicardial cells undergo epithelial-to-mesenchymal transition (EMT), contributing essential cell types and paracrine signalling to the growing heart. The epicardium is integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties in development. Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is established, the mechanisms underpinning its activation remain elusive. We identified two e volutionary c onserved r egions (ECRs) shared in mouse and human, located within intron 1 of Wt1 locus. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR or a deletion comprising both ECRs. Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation. Mendelian ratios indicated an overall underrepresentation of Wt1 ΔECR/ΔECR mutants in adulthood. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular and membranous defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal patterning of coronary artery stems in mutant hearts. Expression of Wt1 was markedly reduced in Wt1 ΔECR/ΔECR hearts but not kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue immunostaining revealed abnormal coronary vessel and innervation patterning in the subepicardium, as well as reduced EMT and myocardial non-compaction/hyper-trabeculation in Wt1 ΔECR/ΔECR . Collectively, we demonstrated a requirement for novel Wt1 intronic enhancers regulating locus activity and essential epicardial EMT-associated biological processes in normal heart development. Importantly, observation of septum and semilunar valve defects in Δ ECR hearts suggests an hitherto unrecognised role for WT1-driven EMT, opening new avenues of research to improve our understanding of congenital heart disease affecting at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

The 5HT4R agonist velusetrag efficacy on neuropathic chronic intestinal pseudo-obstruction in PrP-SCA7-92Q transgenic mice.

Chronic intestinal pseudo-obstruction (CIPO) is a type of intestinal dysfunction with symptoms of intestinal blockage but without the actual mechanical obstruction. Currently, there are no drugs available to treat this disease. Herein, we report the characterization of the PrP-SCA7-92Q transgenic (Tg) line as a valuable CIPO mouse model and investigated the tolerability and efficacy of the 5-hydroxytryptamine type-4 receptor (5HT4R) agonist velusetrag as a promising pharmacological treatment for CIPO. To test the pharmacodynamics of velusetrag, 8-week-old SCA7 Tg mice, which express human mutated Ataxin-7 gene containing 92 CAG repeats under the mouse prion protein promoter, were treated for 5weeks by oral route with velusetrag at 1 and 3mg/kg doses or vehicle. Body weight was monitored throughout the treatment. After sacrifice, the small intestine and proximal colon were collected for whole-mount immunostaining. Untreated, age-matched, C57BL/6J mice were also used as controls in comparison with the other experimental groups. Analysis of SCA7 Tg mice showed tissue damage and alterations, mucosal abnormalities, and ulcers in the distal small intestine and proximal colon. Morphological changes were associated with significant neuronal loss, as shown by decreased staining of pan-neuronal markers, and with accumulation of ataxin-7-positive inclusions in cholinergic neurons. Administration of velusetrag reversed intestinal abnormalities, by normalizing tissue damage and re-establishing the normal level of glia/neuron's count in both the small and large intestines. We demonstrated that the PrP-SCA7-92Q Tg line, a model originally developed to mimic spinocerebellar ataxia, is suitable to study CIPO pathology and can be useful in establishing new therapeutic strategies, such as in the case of velusetrag. Our results suggest that velusetrag is a promising compound to treat patients affected by CIPO or intestinal dysmotility disease.

Tunneling nanotube-driven complete regeneration of murine fetal skin

This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 μm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

MODL-01. IN VITRO MODELING OF AND AUTOMATED DRUG SCREENING IN EMBRYONAL BRAIN TUMORS USING TUMOR-BRAIN ORGANOIDS

Abstract BACKGROUND Embryonal brain tumors (EBTs) such as medulloblastoma, atypical teratoid rhabdoid tumor (AT/RT), and embryonal tumor with multilayered rosettes (ETMR) uniquely arise in the developing brain. Traditional 2D models often fall short in capturing the tumoral microenvironment (TME) and intratumoral heterogeneity, which may limit their translational potential. METHODS To address this, we co-aggregated EBT cells from AT/RT (Chla02 and SHH310) and ETMR (BT183 and murine GEMM cells) with forebrain organoids derived from human induced pluripotent stem cells and developed an automated, screening-ready in vitro model (EBT-FBO). We used immunohistochemistry, whole-mount immunostaining, and single-cell RNA sequencing to characterize our model and compared it with a developmental forebrain atlas. We further used our model for cell type-specific drug screening and validated the results in vivo. RESULTS Our EBT-FBO model mimicked the differentiation states of a mid-gestational fetal forebrain, providing a natural immature neuronal environment for supratentorial EBT cells. This environment allowed the EBT cells from both AT/RT and ETMR to mimic in vivo tumor cell states and avoided the cycling cell selection common in other models. We screened over 160 drugs in ETMR-FBO and identified antracyclines and Triptolide as having anti-tumor, yet non-neurotoxic effects. Our in-depth transcriptomic analysis identified new therapeutic targets, including PDGFR-signaling for ETMR. Building on this insight, we disrupted tumor-TME interactions and observed significant anti-tumor effects both in vitro and in vivo. CONCLUSIONS EBT-FBO provide embryonal brain tumors like ETMR or AT/RT with their intrinsic, immature neuronal microenvironment essential for recapitulating natural tumor heterogeneity. This marks a significant advancement towards targeted therapy and patient-specific models in precision medicine.

Immunolabeling-compatible PEGASOS tissue clearing for high-resolution whole mouse brain imaging.

Novel brain clearing methods revolutionize imaging by increasing visualization throughout the brain at high resolution. However, combining the standard tool of immunostaining targets of interest with clearing methods has lagged behind. We integrate whole-mount immunostaining with PEGASOS tissue clearing, referred to as iPEGASOS (immunostaining-compatible PEGASOS), to address the challenge of signal quenching during clearing processes. iPEGASOS effectively enhances molecular-genetically targeted fluorescent signals that are otherwise compromised during conventional clearing procedures. Additionally, we demonstrate the utility of iPEGASOS for visualizing neurochemical markers or viral labels to augment visualization that transgenic mouse lines cannot provide. Our study encompasses three distinct applications, each showcasing the versatility and efficacy of this approach. We employ whole-mount immunostaining to enhance molecular signals in transgenic reporter mouse lines to visualize the whole-brain spatial distribution of specific cellular populations. We also significantly improve the visualization of neural circuit connections by enhancing signals from viral tracers injected into the brain. Last, we show immunostaining without genetic markers to selectively label beta-amyloid deposits in a mouse model of Alzheimer's disease, facilitating the comprehensive whole-brain study of pathological features.

Wake and non-rapid eye movement sleep dysfunction is associated with colonic neuropathology in Parkinson's disease.

The body-first Parkinson's disease (PD) hypothesis suggests initial gut Lewy body pathology initially propagates to the pons before reaching the substantia nigra, and subsequently progresses to the diencephalic and cortical levels, a disease course presumed to likely occur in PD with rapid eye movement sleep behavior disorder (RBD). We aimed to explore the potential association between colonic phosphorylated alpha-synuclein histopathology (PASH) and diencephalic or cortical dysfunction evidenced by non-rapid eye movement (NREM) sleep and wakefulness polysomnographic markers. In a study involving 43 patients with PD who underwent clinical examination, rectosigmoidoscopy, and polysomnography, we detected PASH on colonic biopsies using whole-mount immunostaining. We performed a visual semi-quantitative analysis of NREM sleep and wake electroencephalography (EEG), confirmed it with automated quantification of spindle and slow wave features of NREM sleep, and the wake dominant frequency, and then determined probable Arizona PD stage classifications based on sleep and wake EEG features. The visual analysis aligned with the automated quantified spindle characteristics and the wake dominant frequency. Altered NREM sleep and wake parameters correlated with markers of PD severity, colonic PASH, and RBD diagnosis. Colonic PASH frequency also increased in parallel to probable Arizona PD stage classifications. Colonic PASH is strongly associated with widespread brain sleep and wake dysfunction, suggesting an extensive diffusion of the pathologic process in PD. Visual and automated analyses of polysomnography signals provide useful markers to gauge covert brain dysfunction in PD. Name: SYNAPark, URL: https://clinicaltrials.gov/study/NCT01748409, registration: NCT01748409.

A tridimensional atlas of the developing human head

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Targeting Endothelial PERK-DLL4 Axis to Enhance Hematopoietic Stem Cell and Lymphoid Progenitor Regeneration

Despite our current information on hundreds of genes implicated in hematopoietic stem cells (HSC) biology, major gaps still exist in our understanding of HSC physiology and their regeneration mechanism under disease and stress conditions, hindering the development of strategies to enhance HSC regeneration. HSCs reside in a tightly controlled bone marrow HSC niche that regulates and maintains hematopoietic homeostasis. HSC niche is composed of a heterogeneous population of specialized stroma cells. Among these, the endothelial cells and the perivascular stroma cells provide critical pro-hematopoietic factors to support HSC pool and committed lineage progenitors particularly lymphoid progenitors. In addition, evidence suggests that endothelium-expressing Notch ligand may promote Notch-dependent long-term HSC proliferation. Recent single cell transcriptome profiling revealed that Delta-like 4 (DLL4) is expressed by the vascular endothelial cells at a higher level than other Notch ligands while its expression down-regulates in response to myeloablation. However, the molecular mechanism regulating the dynamic DLL4 expression during hematopoietic regeneration in response to myeloablative stimuli is unknown. Unfolded protein response (UPR) (also known as endoplasmic reticulum stress, ER stress) has been increasingly recognized as crucial for HSC maintenance. During hematopoietic regeneration, UPR is critical for meeting the increased demand for protein synthesis required for rapid cellular proliferation and for adapting to pathological conditions such as increased levels of reactive oxygen species following irradiation or chemotherapy. Unlike HSCs, how HSC niche endothelial cells deal with redox imbalance and higher requirements of vessel regeneration to regulate stress hematopoietic regeneration is not clear. In this study, using genetically engineered mouse models, we examined the dynamic expression and function of PERK, one arm of UPR, and its potential downstream target, Dll4, during the stress hematopoiesis triggered by transplantation. We found that PERK is activated in the mouse bone marrow endothelial cells following irradiation. Ablation of endothelial Perk in mice (referred as Perk iΔEC mice) promoted post-transplantation immediate recovery of blood cells derived from wild type donor mice and enhanced HSC and lymphoid progenitor regeneration with the expansion of B lineage progenitors including pre-proB, pro-B, and pre-B cells. In addition, by whole-mount immunostaining and intravital microscopy, we found that endothelial Perk deletion restrained disorganized angiogenesis and vascular leakage provoked by irradiation. Although irradiation itself had no effect on endothelial Dll4, Perk ablation markedly increased endothelial Dll4 expression. We found that Dll4 is essential for the post-irradiation HSC and lymphoid progenitor rejuvenation as ablation of endothelial Dll4 in mice (referred as Dll4 iΔEC mice) led to impaired short-term and long-term hematopoietic regeneration and markedly decreased wild type donor derived peripheral B cell and lymphoid progenitor recovery. We found that endothelial Dll4 is required for HSC quiescence maintenance, HSC niche retention and HSC self-renewal. Further, ablation of endothelial Dll4 led to disorganized bone marrow angiogenesis and increased marrow vascular leakage. Finally, we showed that deletion of Dll4 in Perk iΔEC mice completely abrogated the increased numbers of HSC and lymphoid progenitors mediated by Perk deletion, suggesting that up-regulation of endothelial Dll4 is likely responsible for Perk ablation-mediated enhanced HSC and lymphoid progenitor regeneration. Taken together, our results revealed a novel regulatory mechanism by which endothelial Dll4 expression is suppressed by PERK of UPR during transplantation while ablating Perk increases Dll4 expression. Endothelial DLL4 in the bone marrow endothelial cells is critical for supporting vascular integrity and myeloablation-induced HSC and lymphoid progenitor regeneration. Our findings suggest that targeting endothelial PERK-DLL4 axis could be a viable option to improve post-irradiation regeneration of HSC and lymphoid progenitors.

Site-specific pathophysiology in a neonatal mouse model of gastroparesis.

Early-life events impact maturation of the gut microbiome, enteric nervous system, and gastrointestinal motility. We examined three regions of gastric tissue to determine how maternal separation and gut microbes influence the structure and motor function of specific regions of the neonatal mouse stomach. Germ-free and conventionally housed C57BL/6J mouse pups underwent timed maternal separation (TmSep) or nursed uninterrupted (controls) until 14 days of life. We assessed gastric emptying by quantifying the progression of gavaged fluorescein isothiocyanate (FITC)-dextran. With isolated rings of forestomach, corpus, and antrum, we measured tone and contractility by force transduction, gastric wall thickness by light microscopy, and myenteric plexus neurochemistry by whole-mount immunostaining. Regional gastric sampling revealed site-specific differences in contractile patterns and myenteric plexus structure. In neonatal mice, TmSep prolonged gastric emptying. In the forestomach, TmSep increased contractile responses to carbachol, decreased muscularis externa and mucosa thickness, and increased the relative proportion of myenteric plexus nNOS+ neurons. Germ-free conditions did not appreciably alter the structure or function of the neonatal mouse stomach and did not impact the changes caused by TmSep. A regional sampling approach facilitates site-specific investigations of murine gastric motor physiology and histology to identify site-specific alterations that may impact gastrointestinal function. Delayed gastric emptying in TmSep is associated with a thinner muscle wall, exaggerated cholinergic contractile responses, and increased proportions of inhibitory myenteric plexus nNOS+ neurons in the forestomach. Gut microbes do not profoundly affect the development of the neonatal mouse stomach or the gastric pathophysiology that results from TmSep.

Cerebral Organoid Arrays for Batch Phenotypic Analysis in Sections and Three Dimensions.

Organoids can recapitulate human-specific phenotypes and functions in vivo and have great potential for research in development, disease modeling, and drug screening. Due to the inherent variability among organoids, experiments often require a large sample size. Embedding, staining, and imaging each organoid individually require a lot of reagents and time. Hence, there is an urgent need for fast and efficient methods for analyzing the phenotypic changes in organoids in batches. Here, we provide a comprehensive strategy for array embedding, staining, and imaging of cerebral organoids in both agarose sections and in 3D to analyze the spatial distribution of biomarkers in organoids in situ. We constructed several disease models, particularly an aging model, as examples to demonstrate our strategy for the investigation of the phenotypic analysis of organoids. We fabricated an array mold to produce agarose support with microwells, which hold organoids in place for live/dead imaging. We performed staining and imaging of sectioned organoids embedded in agarose and 3D imaging to examine phenotypic changes in organoids using fluorescence micro-optical sectioning tomography (fMOST) and whole-mount immunostaining. Parallel studies of organoids in arrays using the same staining and imaging parameters enabled easy and reliable comparison among different groups. We were able to track all the data points obtained from every organoid in an embedded array. This strategy could help us study the phenotypic changes in organoids in disease models and drug screening.

A Sox17 downstream gene Rasip1 is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

BackgroundDuring mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta’s intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity.MethodsRNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells.ResultsThe increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity.ConclusionsRasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.

Methanol-Based Whole-Mount Preparation for the Investigation of Retinal Ganglion Cells

Retinal ganglion cells (RGCs), which are the projection neurons of the retina, propagate external visual information to the brain. Pathological changes in RGCs have a close relationship with numerous retinal degenerative diseases. Whole-mount retinal immunostaining is frequently used in experimental studies on RGCs to evaluate the developmental and pathological conditions of the retina. Under some circumstances, some valuable retina samples, such as those from transgenic mice, may need to be retained for a long period without affecting the morphology or number of RGCs. For credible and reproducible experimental results, using an effective preserving medium is essential. Here, we describe the effect of methanol as an auxiliary fixed medium for retinal whole-mount preparations and long-term storage. In brief, during the isolation process, cold methanol (-20 °C) is pipetted onto the surface of the retina to help fix the tissues and facilitate their permeability, and then the retinas can be stored in cold methanol (-20 °C) before being immunostained. This protocol describes the retina isolation workflow and tissue sample storage protocol, which is useful and practical for the investigation of RGCs.

Abstract 4614: Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors in humans. PDAC has an immunosuppressive tumor microenvironment, constituting for poor immunotherapy outcomes. Lymphatic vessels (LVs) transport leukocytes and antigens from tumor peripheries to draining lymph nodes (dLNs) for activation of anti-tumor immune responses. It is believed that impairment of the lymphatic function may result in delayed and ineffective immune activation against tumors. In this study, we created a three-dimensional (3D) tumor-on-chip model to examine phenotypical and functional changes in 3D engineered LVs, composed of human primary lymphatic endothelial cells (LECs) in co-culture with PDAC cells. Our in vitro studies revealed that PDAC cells induce lymphangiogenesis and tighten cell-cell junctions in LECs, forming “zipper-like” LEC junctions, compared to control LECs with “button-like” junctions. We introduced 70kDa dextran into the LV lumens and showed PDAC-induced zippering of junctions decreased vessel permeability. These results led us to hypothesize that altered LEC junctions may affect fluid drainage into the LVs in vivo. We inoculated PDAC cells into the mouse ear pinnae and performed whole-mount immunostaining and lymphatic drainage experiment by injecting Evans blue into the ears and measured the remained Evans blue in the ears. In the ear experiments, inoculated PDAC cells affected host LVs with originally button-like junctions to become zipper-like and impede lymphatic drainage. To identify a potential signaling pathway that may be responsible for LEC junction zippering, we used phospho-kinase arrays and discovered that PDAC cells induced LECs to increase phosphorylation of GSK3-β, JNK kinases, and enhanced the total protein level of β-catenin, implying that PDAC cells turned on the WNT signaling in LECs. In preliminary screening, we treated LECs co-cultured with PDAC cells with WNT-C59, an WNT inhibitor that blocks secretion of all WNT ligands, which resulted in decreased number of LV sprouting and less contractile actin-cytoskeleton in LECs. It is critical to know which molecules were secreted by PDAC cells and influenced the WNT signaling in LECs. With that in mind, we plan to perform mass spectrometry with PDAC-conditioned media to identify PDAC-specific secretome; and run RNA-seq with LECs isolated from mouse ears with or without PDAC to investigate molecular basis of the WNT activation in LECs and LV remodeling. Finally, we plan to examine the egress of leukocytes from primary PDAC tumor site, T cell activation in tumor-dLNs, and PDAC tumor growth with or without WNT inhibition in Kaeda mice. The identity and function of PDAC-egressed immune cells, and those which stayed within the primary tumor will enhance our understanding of the roles of LVs in orchestrating anti-tumor immunity in PDAC. Citation Format: Anna Maria Kolarzyk, Conor Loy, Renhao Lu, Iwijn De Vlaminck, Deborah Fowell, Esak Lee. Investigating lymphatic vessel remodeling and anti-tumor immunity in pancreatic cancer using tumor-on-chip and mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4614.

Visualization of Organ-Specific Lymphatic Growth: An Efficient Approach to Labeling Molecular Markers in Cleared Tissues

Organ-specific lymphatics are essential for the maintenance of healthy organ function and lymphatic dysfunction can lead to the development of various diseases. However, the precise role of those lymphatic structures remains unknown, mainly due to inefficient visualization techniques. Here, we present an efficient approach to visualizing organ-specific lymphatic growth. We used a modified CUBIC protocol to clear mouse organs and combined it with whole-mount immunostaining to visualize lymphatic structures. We acquired images using upright, stereo and confocal microscopy and quantified them with AngioTool, a tool for the quantification of vascular networks. Using our approach, we then characterized the organ-specific lymphatic vasculature of the Flt4kd/+ mouse model, showing symptoms of lymphatic dysfunction. Our approach enabled us to visualize the lymphatic vasculature of organs and to analyze and quantify structural changes. We detected morphologically altered lymphatic vessels in all investigated organs of Flt4kd/+ mice, including the lungs, small intestine, heart and uterus, but no lymphatic structures in the skin. Quantifications showed that these mice have fewer and dilated lymphatic vessels in the small intestine and the lungs. Our results demonstrate that our approach can be used to investigate the importance of organ-specific lymphatics under both physiological and pathophysiological conditions.

Age-dependent Microglial Disease Phenotype Results in Functional Decline in Gut Macrophages.

Muscularis macrophages (MMs) are tissue-resident macrophages in the gut muscularis externa which play a supportive role to the enteric nervous system. We have previously shown that age-dependent MM alterations drive low-grade enteric nervous system inflammation, resulting in neuronal loss and disruption of gut motility. The current studies were designed to identify the MM genetic signature involved in these changes, with particular emphasis on comparison to genes in microglia, the central nervous system macrophage population involved in age-dependent cognitive decline. Young (3 months) and old (16-24 months) C57BL/6 mice and human tissue were studied. Immune cells from mouse small intestine, colon, and spinal cord and human colon were dissociated, immunophenotyped by flow cytometry, and examined for gene expression by single-cell RNA sequencing and quantitative real-time PCR. Phagocytosis was assessed by in vivo injections of pHrodo beads (Invitrogen). Macrophage counts were performed by immunostaining of muscularis whole mounts. MMs from young and old mice express homeostatic microglial genes, including Gpr34, C1qc, Trem2, and P2ry12. An MM subpopulation that becomes more abundant with age assumes a geriatric state (GS) phenotype characterized by increased expression of disease-associated microglia genes including Cd9, Clec7a, Itgax (CD11c), Bhlhe40, Lgals3, IL-1β, and Trem2 and diminished phagocytic activity. Acquisition of the GS phenotype is associated with clearance of α-synuclein aggregates. Human MMs demonstrate a similar age-dependent acquisition of the GS phenotype associated with intracellular α-synuclein accumulation. MMs demonstrate age-dependent genetic changes that mirror the microglial disease-associated microglia phenotype and result in functional decline.

Antibody development to identify components of IIS and mTOR signaling pathways in lepidopteran species, a set of non-model insects.

Nutritional stress impacts many insect species that have differing reproductive strategies and life histories, yet it is unclear how nutrient-sensing signaling pathways mediate tissue-specific responses to changes in dietary input. In Drosophila melanogaster , insulin/insulin-like growth factor (IIS) and mTOR-mediated signaling within adipocytes regulates oogenesis. To facilitate comparative study of nutrient-sensing pathway activity in the fat body, we developed antibodies to assess IIS (anti-FOXO) and mTOR signaling (anti-TOR) across three nymphalid species (Lepidoptera). By optimizing whole-mount fat body immunostaining, we find FOXO nuclear enrichment in adult adipocytes, like that observed in Drosophila . Additionally, we show a previously uncharacterized TOR localization pattern in the fat body.