Jeremiah Bernier-Latmani

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.

Stability and function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium

The signalling pathways operational in quiescent, post-development vasculature remain enigmatic. Here we show that unlike neovascularization, endothelial Akt signalling in established vasculature is crucial not for endothelial cell (EC) survival, but for sustained interactions with pericytes and vascular smooth muscle cells (VSMCs) regulating vascular stability and function. Inducible endothelial-specific Akt1 deletion in adult global Akt2KO mice triggers progressive VSMC apoptosis. In hearts, this causes a loss of arteries and arterioles and, despite a high capillary density, diminished vascular patency and severe cardiac dysfunction. Similarly, endothelial Akt deletion induces retinal VSMC loss and basement membrane deterioration resulting in vascular regression and retinal atrophy. Mechanistically, the Akt/mTOR axis controls endothelial Jagged1 expression and, thereby, Notch signalling regulating VSMC maintenance. Jagged1 peptide treatment of Akt1ΔEC;Akt2KO mice and Jagged1 re-expression in Akt-deficient endothelium restores VSMC coverage. Thus, sustained endothelial Akt1/2 signalling is critical in maintaining vascular stability and homeostasis, thereby preserving tissue and organ function.

Intestinal lymphatic vasculature: structure, mechanisms and functions

The mammalian intestine is richly supplied with lymphatic vasculature, which has functions ranging from maintenance of interstitial fluid balance to transport of antigens, antigen-presenting cells, dietary lipids and fat-soluble vitamins. In this Review, we provide in-depth information concerning the organization and structure of intestinal lymphatics, the current view of their developmental origins, as well as molecular mechanisms of intestinal lymphatic patterning and maintenance. We will also discuss physiological aspects of intestinal lymph flow regulation and the known and emerging roles of intestinal lymphatic vessels in human diseases, such as IBD, infection and cancer.

High-resolution 3D analysis of mouse small-intestinal stroma

Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation. In contrast to alternative current techniques, our protocol enables the entire villus to be visualized with increased spatial resolution of cell location, morphology and cell–cell interactions, thus allowing for easy quantification of phenotypes. The technique, which takes 7 d from mouse dissection to microscopic examination, will be useful for researchers who are interested in most aspects of intestinal biology, including mucosal immunology, infection, nutrition, cancer biology and intestinal microbiota.

Cx47 fine-tunes the handling of serum lipids but is dispensable for lymphatic vascular function

Mutations in the gap junction protein connexin47 (Cx47) are associated with lymphedema. However, the role of Cx47 in lymphatic pathophysiology is unknown. We demonstrate that Cx47 is expressed in lymphatic endothelial cells by whole-mount immunostaining and qPCR. To determine if Cx47 plays a role in lymphatic vessel function we analysed Cx47-/- mice. Cx47-deficiency did not affect lymphatic contractility (contractile amplitude or frequency) or lymphatic morphology (vessel diameter or number of valves). Interstitial fluid drainage or dendritic cell migration through lymphatic vessels was also not affected by Cx47-deficiency. Cx47 is dispensable for long-chain fatty acid absorption from the gut but rather promotes serum lipid handling as prolonged elevated triglyceride levels were observed in Cx47-deficient mice after oral lipid tolerance tests. When crossed with Apolipoprotein E-deficient (Apoe-/-) mice, LDL-cholesterol was decreased in young Cx47-/-Apoe-/- adults as compared to Apoe-/- mice, which was inverted later in life. Finally, advanced atherosclerotic plaques in thoracic-abdominal aortas of 15 months-old mice tended to be larger in Cx47-/-Apoe-/- mice. These plaques contained fewer macrophages but similar amounts of T lymphocytes, collagen and lipids than plaques of Apoe-/- mice. In conclusion, Cx47 is expressed in lymphatic endothelium and seems modestly implicated in multiple aspects of lymphatic pathophysiology.

No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.

Background Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease. Methodology/Principal Findings DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing. Conclusions/Significance As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.

Meet Me in the Middle Dual Origins of Dermal Lymphatic Vasculature in Mammals

A network of thin-walled lymphatic vessels is present in virtually every tissue of the body, where it carries out several important functions, such as transport of antigen-presenting cells to lymph nodes, uptake of dietary fat, and maintenance of interstitial fluid balance. The importance of the lymphatic vasculature to human pathology is only now beginning to be appreciated, as it becomes increasingly clear that lymphatic vessels play important roles in a host of common diseases, ranging from cancer metastasis to atherosclerosis and hypertension.1 Research in the past 2 decades has tremendously improved our understanding of molecular mechanisms involved in the regulation of lymphatic vasculature and its function. In particular, the atypical homeobox transcription factor Prox1 plays a key role in establishing and maintaining mammalian lymphatic endothelial cell (LEC) identity,2–4 whereas the Ccbe1/Adamts3/Vegf-c/Vegfr-3 signaling cascade is essential for LEC proliferation, migration, and survival.5–7 Novel therapies, which are a direct result of this knowledge, are now being developed for treatments that both promote lymphatic vessel regeneration and block excessive lymphangiogenesis. However, full comprehension into the intricacies of LEC biology is still being developed. The work of Martinez-Corral et al,8 based on lineage-tracing analyses of genetic mouse models, now provides first insights into an unexpected complexity of LEC origins in mammals. Article, see p 1649 Historically, there have been 2 main hypotheses about the origin of the lymphatic vasculature. One idea proposed by Sabin9 was that lymphatic vessels arise from veins during embryogenesis. On the basis of India ink injections in pig embryos, Sabin9 demonstrated expansion of ink-filled lymphatic vessels from the areas of primitive lymph sacs, located near cardinal veins, toward the periphery. These data suggested that lymphatic vessels grow in a centrifugal manner by sprouting from the pre-existing venous endothelium. A second …

Testing advances in molecular discrimination among Chinook salmon life histories: evidence from a blind test

Summary The application of DNA‐based markers toward the task of discriminating among alternate salmon runs has evolved in accordance with ongoing genomic developments and increasingly has enabled resolution of which genetic markers associate with important life‐history differences. Accurate and efficient identification of the most likely origin for salmon encountered during ocean fisheries, or at salvage from fresh water diversion and monitoring facilities, has far‐reaching consequences for improving measures for management, restoration and conservation. Near‐real‐time provision of high‐resolution identity information enables prompt response to changes in encounter rates. We thus continue to develop new tools to provide the greatest statistical power for run identification. As a proof of concept for genetic identification improvements, we conducted simulation and blind tests for 623 known‐origin Chinook salmon (Oncorhynchus tshawytscha) to compare and contrast the accuracy of different population sampling baselines and microsatellite loci panels. This test included 35 microsatellite loci (1266 alleles), some known to be associated with specific coding regions of functional significance, such as the circadian rhythm cryptochrome genes, and others not known to be associated with any functional importance. The identification of fall run with unprecedented accuracy was demonstrated. Overall, the top performing panel and baseline (HMSC21) were predicted to have a success rate of 98%, but the blind‐test success rate was 84%. Findings for bias or non‐bias are discussed to target primary areas for further research and resolution.

All TIEd up: mechanisms of Schlemm’s canal maintenance

Glaucoma is a leading cause of blindness, with an estimated world-wide prevalence of 3.5% in members of the population older than 40 years of age. Elevated intraocular pressure as the result of abnormal resistance to aqueous humor drainage is a major contributing, and the only preventable, factor in glaucoma development. Schlemms canal (SC), a lymphatic-like vessel encircling the anterior portion of the eye, plays a key role in promoting aqueous humor outflow and maintenance of normal intraocular pressure. The risk of developing glaucoma increases with age; therefore, understanding mechanisms of SC maintenance and how aging affects SC function are of special importance, both for prevention and novel treatment approaches to glaucoma. Using a compelling array of genetic models, Kim et al. report in this issue of the JCI that continuous angiopoietin/TIE2 signaling is required for maintaining SC identity and integrity during adulthood and show that its age-related changes can be rescued by a TIE2 agonistic antibody.

Development and Differentiation of the Lymphatic Vascular System

The lymphatic vasculature is critical for the maintenance of homeostasis, and performs essential roles in the trafficking of fluids, immune cells, and dietary fats. This importance is highlighted by the contribution of the lymphatic vasculature to several human diseases, including lymphedema, tumor metastasis, and inflammation. In this chapter, we seek to review basic principles of lymphatic vessel biology and discuss the current understanding of molecular mechanisms involved in lymphatic endothelial cell identity, embryonic and postnatal lymphatic development and maturation. Knowledge of the underlying molecular mechanisms controlling lymphatic vessel biology may lead to targeted treatments to improve patient care in the future.