Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.     

Three-Dimensional Imaging of Prox1-EGFP Transgenic Mouse Gonads Reveals Divergent Modes of Lymphangiogenesis in the Testis and Ovary

The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both the temporal and spatial development of the lymphatic vessel network in mouse testes and ovaries. We find that lymphangiogenesis in the testis is initiated during late gestation, but in contrast to other organs, lymphatic vessels remain confined to the testis cap and, unlike blood vessels, do not infiltrate the entire organ. Conversely, lymphatic vessels invade the ovarian tissue, beginning postnatally, and sprouting from preexisting lymphatic vessels at the extraovarian rete. The ovary develops a rich network of lymphatic vessels, extending from the medulla into the surrounding cortex adjacent to developing follicles. This study reveals distinct patterns of lymphangiogenesis in the testes and ovaries and will serve as the basis for the identification of the divergent molecular pathways that control morphogenesis and the function of the lymphatic vasculature in these two organs.     

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2^Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2^Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.     

Schlemm's Canal Is a Unique Vessel with a Combination of Blood Vascular and Lymphatic Phenotypes that Forms by a Novel Developmental Process

Schlemm''s canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name “canalogenesis.” Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision.     

Prox1, master regulator of the lymphatic vasculature phenotype

In contrast to the extensive molecular and functional characterization of blood vascular endothelium, little is known about the mechanisms that control the formation and lineage-specific differentiation and function of lymphatic vessels. The homeobox gene Prox1, the vertebrate homologue of the Drosophila prospero gene, has been recently identified to be required for the induction of lymphatic vascular development from preexisting embryonic veins, and studies in Prox1-deficient mice have confirmed Florence Sabin's original hypothesis about the origin of the lymphatic vascular system from embryonic veins. The recent establishment of cell culture models for the selective propagation of blood vascular and lymphatic endothelial cells, together with the findings that these cells maintain their lineage-specific differentiation in vitro, has led to the discovery that Prox1 expression is sufficient to induce a lymphatic phenotype in blood vascular endothelium. Ectopic expression of Prox1 downregulated blood vascular-associated genes and also upregulated some of the known lymphatic endothelial cell markers. Together, these studies suggest that the blood vascular phenotype represents the default endothelial differentiation and they identify an essential role of Prox1 in the program specifying lymphatic endothelial cell fate.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.     

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

Background

The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.

Methodology/Principal Findings

We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1^Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1^Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.

Conclusions/Significance

These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.     

prox1b Activity is essential in zebrafish lymphangiogenesis

Background

The lymphatic vascular system, draining interstitial fluids from most tissues and organs, exerts crucial functions in several physiological and pathological processes. Lymphatic system development depends on Prox1, the first marker to be expressed in the endothelial cells of the cardinal vein from where lymph vessels originate. Prox1 ortholog in the optically clear, easily manipulated zebrafish model has been previously isolated and its contribution to lymphangiogenesis has been clarified. Because of a round of genome duplication occurred at the base of teleosts radiation, several zebrafish genes have been retained in duplicate through evolution. We investigated for the presence of additional prox1 genes and determined their role in zebrafish lymphangiogenesis.

Methodology/Principal Findings

We isolated a second ortholog, named prox1b, and analyzed its expression during development by whole mount in situ hybridization (WISH). We detected strong prox1b expression in the endothelium of the posterior cardinal vein (PCV) from where lymphatic precursors originate. To analyze prox1b involvement in lymphangiogenesis we utilized the fli1:GFP transgenics and followed the formation of the toracic duct (TD), the primary lymph vessel in fish, after prox1b knockdown. Our findings clearly demonstrated that the absence of prox1b activity severely hampers the formation of the TD.

Conclusions/Significance

This work provides substantial progress toward the understanding of zebrafish lymphangiogenesis. In light of the features shared by the lymphatic systems of zebrafish and higher vertebrates, the establishment of such lymphatic model will provide a powerful tool to study, for instance, disorders of body fluid homeostasis, inflammation and cancer metastasis, and may ultimately contribute to novel therapies.     

Reappraisal of VAChT‐Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers

VAChT‐Cre.Fast and VAChT‐Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT‐Cre lines with a reporter line, CAG‐Syp/tdTomato, in which synaptophysin‐tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co‐localized with osteopontin, a recently discovered motor neuron marker for slow‐twitch fatigue‐resistant (S) and fast‐twitch fatigue‐resistant (FR) types. The fluorescence did not preferentially co‐localize with matrix metalloproteinase‐9, a marker for fast‐twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT‐Cre lines are Cre‐drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT‐Cre.Fast and VAChT‐Cre.Slow to VAChT‐Cre.Early and VAChT‐Cre.Late, respectively. The mouse lines will be useful tools to study slow‐type motor neurons, in relation to physiology and pathology.     

Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.