Transgenic Fluorescent Reporter Mice Research Articles

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ Tcells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Serine Racemase Expression by Striatal Neurons.

D-serine is synthesized by serine racemase (SR) and is a co-agonist at forebrain N-methyl-D-aspartate receptors (NMDARs). D-serine and SR are expressed primarily in neurons, but not in quiescent astrocytes. In this study, we examined the localization of D-serine and SR in the mouse striatum and the effects of genetically silencing SR expression in GABAergic interneurons (iSR-/-). iSR-/- mice had substantially reduced SR expression almost exclusively in striatum, but only exhibited marginal D-serine reduction. SR positive cells in the striatum showed strong co-localization with dopamine- and cyclic AMP-regulated neuronal phosphoprotein (DARPP32) in wild type mice. Transgenic fluorescent reporter mice for either the D1 or D2 dopamine receptors exhibited a 65:35 ratio for co-localization with D1and D2 receptor positive cells, respectively. These results indicate that GABAergic medium spiny neurons receiving dopaminergic inputs in striatum robustly and uniformly express SR. In behavioral tests, iSR-/- mice showed a blunted response to the hedonic and stimulant effects of cocaine, without affecting anxiety-related behaviors. Because the cocaine effects have been shown in the constitutive SR-/- mice, the restriction of the blunted response to cocaine to iSR-/- mice reinforces the conclusion that D-serine in striatal GABAergic neurons plays an important role in mediating dopaminergic stimulant effects. Results in this study suggest that SR in striatal GABAergic neurons is synthesizing D-serine, not as a glutamatergic co-transmitter, but rather as an autocrine whereby the GABAergic neurons control the excitability of their NMDARs by determining the availability of the co-agonist, D-serine.

Intravital Imaging of Vaccinia Virus-Infected Mice.

Intravital multiphoton microscopy (MPM) allows the direct visualization of viral infections in real time as they occur in living animals. Here we describe the routes and considerations for murine infection with vaccinia virus (VACV) for imaging, and the preparation of the skin and inner lip (labial mucosa) of infected animals for MPM. Using different recombinant VACVs expressing fluorescent proteins in combination with transgenic fluorescent reporter mice, MPM imaging can be used to examine the movements, interactions, and functions of virus-infected cells or selected immune cell populations after infection.

Microvascular networks in the area of the auditory peripheral nervous system

Using transgenic fluorescent reporter mice in combination with an established tissue clearing method, we detail heretofore optically opaque regions of the spiral lamina and spiral limbus where the auditory peripheral nervous system is located and provide insight into changes in cochlear vascular density with ageing. We found a relatively dense and branched vascular network in young adults, but a less dense and thinned network in aged adults. Significant reduction in vascular density starts early at the age of 180 days in the region of the spiral limbus (SL) and continues into old age at 540 days. Loss of vascular volume in the region of spiral ganglion neurons (SGN) is delayed until the age of 540 days. In addition, we observed that two vascular accessory cells are closely associated with the microvascular system: perivascular resident macrophages and pericytes. Morphologically, perivascular resident macrophages undergo drastic changes from postnatal P7 to young adult (P30). In postnatal animals, most perivascular resident macrophages exhibit a spherical or nodular shape. In young adult mice, the majority of perivascular resident macrophages are elongated and display an orientation parallel to the vessels. In our imaging, some of the perivascular resident macrophages are caught in the act of transmigrating from the blood circulation. Pericytes also display morphological heterogeneity. In the P7 mice, pericytes are prominent on the capillary walls, relatively large and punctate, and less uniform. In contrast, pericytes in the P30 mice are relatively flat and uniform, and less densely distributed on the vascular network. With triple fluorescence labeling, we did not find obvious physical connection between the two systems, unlike neuronal-vascular coupling found in brain. However, using a fluorescent (FITC-conjugated dextran) tracer and the enzymatic tracer horseradish peroxidase (HRP), we observed robust neurovascular exchange, likely through transcytotic transport, evidenced by multiple vesicles present in the endothelial cells. Taken together, our data demonstrate the effectiveness of tissue-clearing methods as an aid in imaging the vascular architecture of the SL and SGNs in whole mounted mouse cochlear preparations. Structure is indicative of function. The finding of differences in vascular structure in postnatal and young adult mice may correspond with variation in hearing refinement after birth and indicate the status of functional activity. The decrease in capillary network density in the older animals may reflect the decreased energy demand from peripheral neural activity. The finding of active transcytotic transport from blood to neurons opens a potential therapeutic avenue for delivery of various growth factors and gene vectors into the inner ear to target SGNs.

Csf1r-mApple Transgene Expression and Ligand Binding In Vivo Reveal Dynamics of CSF1R Expression within the Mononuclear Phagocyte System.

CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r-mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647–labeled CSF1-Fc and CSF1, and a modified ΔCsf1–enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r-driven reporter lines, Csf1r-mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double ΔCsf1r-ECFP-Csf1r-mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r-mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines.

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

Extragonadal oocytes residing in the mouse ovarian hilum contribute to fertility.

The observation of pups born from recipient and donor mice after ovariectomy followed by ovarian transplant poses the interesting possibility of an extraovarian source of oocytes. However, whether mammalian adult oocytes reside in extragonadal tissues remains elusive. Using transgenic fluorescent reporter mice and transplantation surgeries, we demonstrate the presence of both donor- and recipient-derived corpora lutea and recovery of both donor- and recipient-derived offspring from ovariectomized mice after transplantation of donor ovaries. A potential region for extraovarian oocytes is the hilum, a ligament-like structure between the ovary and the reproductive tract. Immunofluorescent confocal microscopy of mouse ovaries and reproductive tracts revealed that a population of primordial follicles resides outside the ovary within the hilum. Ovariectomy-only controls confirmed that oocytes remain in the recipient hilum after surgery. These results provide evidence that the hilum is a reserve source of follicles, which likely return to the ovary for maturation and ovulation. By identifying a new follicle reservoir, our study addresses a long-standing question in reproductive biology and contributes to new conceptual knowledge about ovarian function and fertility.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Evaluation of enzymatically crosslinked injectable glycol chitosan hydrogel.

Enzymatically cross-linkable phenol-conjugated glycol chitosan was prepared by reacting glycol chitosan with 3-(4-hydroxyphenyl)propionic acid (HPP). The chemical modification was confirmed by FTIR, 1H-NMR and UV spectroscopy. Glycol chitosan hydrogels (HPP-GC) with or without rhBMP-2 were prepared by the oxidative coupling of the substituted phenol groups in the presence of hydrogen peroxide and horse radish peroxidase. Rheological characterization demonstrated the feasibility of developing hydrogels with varying storage moduli by changing the polymer concentration. The gel presented a microporous structure with pore sizes ranging from 50-350 μm. The good viability of encapsulated 7F2 osteoblasts indicated non-toxicity of the gelation conditions. In vitro release of rhBMP-2 in phosphate buffer solution showed ∼11% release in 360 h. The ability of the hydrogel to maintain the in vivo bioactivity of rhBMP-2 was evaluated in a bilateral critical size calvarial bone defect model in Col3.6 transgenic fluorescent reporter mice. The presence of fluorescent green osteoblast cells with overlying red alizarin complexone and yellow stain indicating osteoclast TRAP activity confirmed active cell-mediated mineralization and remodelling process at the implantation site. The complete closure of the defect site at 4 and 8 weeks post implantation demonstrated the potent osteoinductivity of the rhBMP-2 containing gel.

Assessment of the developmental toxicity of nanoparticles in an ex vivo 3D model, the murine limb bud culture system

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is an ex vivo 3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype and Col2a1; Col10a1; Col1a1 triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3–100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructures per se or a degradation component released from the complex nanostructure.

Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Gene Silencing in Skin After Deposition of Self-Delivery siRNA With a Motorized Microneedle Array Device

Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.

Developmental niches for embryonic erythroid cells

Primitive erythroid cells (EryP) are the first differentiated cell type to be specified during mammalian embryogenesis. EryP arise from a pool of lineage-restricted progenitors in the yolk sac (YS) and then enter the newly formed embryonic circulation to mature in a stepwise, synchronous fashion. Numbering in the millions in the mid-gestation mouse embryo, EryP are the dominant circulating blood cell prior to the rapid generation of adult-type definitive erythroid (EryD) cells in the fetal liver. The identification of maturational events in this lineage presented a significant challenge, as EryD begin to outnumber EryP in the bloodstream from ∼ E14.5 onwards. We used human epsilon-globin gene regulatory elements to drive lineage-specific expression of a histone-H2B::EGFP fusion protein, allowing us to label the chromatin of EryP during their development and to track and quantify EryP nuclei following their expulsion from the cell. Using this transgenic fluorescent reporter mouse line, we have monitored primitive erythropoiesis in three distinct niches: the YS, where EryP progenitors arise; the circulation, where EryP continue to divide and mature; and the fetal liver, where EryP complete the terminal stages of their differentiation.