Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Erythematous capillary-lymphatic malformations mimicking blood vascular anomalies.

Superficial erythematous cutaneous vascular malformations are assumed to be blood vascular in origin, but cutaneous lymphatic malformations can contain blood and appear red. Management may be different and so an accurate diagnosis is important. Cutaneous malformations were investigated through 2D histology and 3D whole-mount histology. Two lesions were clinically considered as port-wine birthmarks and another 3 lesions as erythematous telangiectasias. The aims were (i) to demonstrate that cutaneous erythematous malformations including telangiectasia can represent a lymphatic phenotype, (ii) to determine if lesions represent expanded but otherwise normal or malformed lymphatics, and (iii) to determine if the presence of erythrocytes explained the red color. Microscopy revealed all lesions as lymphatic structures. Port-wine birthmarks proved to be cystic lesions, with nonuniform lymphatic marker expression and a disconnected lymphatic network suggesting a lymphatic malformation. Erythematous telangiectasias represented expanded but nonmalformed lymphatics. Blood within lymphatics appeared to explain the color. Blood-lymphatic shunts could be detected in the erythematous telangiectasia. In conclusion, erythematous cutaneous capillary lesions may be lymphatic in origin but clinically indistinguishable from blood vascular malformations. Biopsy is advised for correct phenotyping and management. Erythrocytes are the likely explanation for color accessing lymphatics through lympho-venous shunts.

[Genetics, diagnostics and clinical presentation of primary lymphoedema]. / Genetik, Diagnostik und Klinik des primären Lymphödems.

Primary lymphoedema is a hereditary genetic disorder of the lymphatic system. These genetic disorders can result in malformation or dysfunction of the lymphatic system, which leads to an accumulation of fluid in the tissue and, thus to the formation of oedema. The most common form is peripheral lymphoedema of the lower limbs, but systemic manifestations such as intestinal lymphangiectasia, ascites, chylothorax or hydrops fetalis may also occur. The clinical presentation and the degree of lymphoedema varies depending on the causative gene and the specific gene alteration. Primary lymphoedema is divided into five categories: (1) disorders with somatic mosaicism and segmental growth abnormality, (2a) syndromal disorders, (2b) disorders with systemic involvement, (2c) congenital lymphoedema and (2d) disorders that occur after the first year of life (late onset lymphoedema). Targeted genetic diagnosis is based on the patient's clinical presentation and classification into one of the five categories. In general, the diagnosis usually starts with basic diagnostics, which include cytogenetic and molecular genetic testing. Subsequently, a molecular genetic diagnosis is made by performing single-gene analyses, gene panel examinations, exome sequencing or whole genome sequencing. This allows the identification of genetic variants or mutations that are considered to be causative for the presenting symptoms. Combined with human genetic counselling, the genetic diagnosis allows for conclusions about inheritance, the risk of recurrence and potential concomitant symptoms. In many cases, only this approach allows the definite form of primary lymphoedema to be described.

Three-Dimensional Histological Characterization of the Placental Vasculature Using Light Sheet Microscopy.

The placenta is the first embryonic organ, representing the connection between the embryo and the mother, and is therefore necessary for the embryo's growth and survival. To meet the ever-growing need for nutrient and gas exchange, the maternal spiral arteries undergo extensive remodeling, thus increasing the uteroplacental blood flow by 16-fold. However, the insufficient remodeling of the spiral arteries can lead to severe pregnancy-associated disorders, including but not limited to pre-eclampsia. Insufficient endovascular trophoblast invasion plays a key role in the manifestation of pre-eclampsia; however, the underlying processes are complex and still unknown. Classical histopathology is based on two-dimensional section microscopy, which lacks a volumetric representation of the vascular remodeling process. To further characterize the uteroplacental vascularization, a detailed, non-destructive, and subcellular visualization is beneficial. In this study, we use light sheet microscopy for optical sectioning, thus establishing a method to obtain a three-dimensional visualization of the vascular system in the placenta. By introducing a volumetric visualization method of the placenta, we could establish a powerful tool to deeply investigate the heterogeneity of the spiral arteries during the remodeling process, evaluate the state-of-the-art treatment options, effects on vascularization, and, ultimately, reveal new insights into the underlying pathology of pre-eclampsia.

Redefining WILD syndrome: a primary lymphatic dysplasia with congenital multisegmental lymphoedema, cutaneous lymphovascular malformation, CD4 lymphopaenia and warts.

BACKGROUND: Primary lymphoedema (PL) syndromes are increasingly recognised as presentations of complex genetic disease, with at least 20 identified causative genes. Recognition of clinical patterns is key to diagnosis, research and therapeutics. The defining criteria for one such clinical syndrome, 'WILD syndrome' (Warts, Immunodeficiency, Lymphoedema and anogenital Dysplasia), have previously depended on a single case report. METHODS AND RESULTS: We present 21 patients (including the first described case) with similar clinical and immunological phenotypes. All had PL affecting multiple segments, with systemic involvement (intestinal lymphangiectasia/pleural or pericardial effusions) in 70% (n=14/20). Most (n=20, 95%) had a distinctive cutaneous lymphovascular malformation on the upper anterior chest wall. Some (n=10, 48%) also had hyperpigmented lesions resembling epidermal naevi (but probably lymphatic in origin). Warts were common (n=17, 81%) and often refractory. In contrast to the previous case report, anogenital dysplasia was uncommon-only found in two further cases (total n=3, 14%). Low CD4 counts and CD4:CD8 ratios typified the syndrome (17 of 19, 89%), but monocyte counts were universally normal, unlike GATA2 deficiency. CONCLUSION: WILD syndrome is a previously unrecognised, underdiagnosed generalised PL syndrome. Based on this case series, we redefine WILD as 'Warts, Immunodeficiency, andLymphatic Dysplasia' and suggest specific diagnostic criteria. The essential criterion is congenital multisegmental PL in a 'mosaic' distribution. The major diagnostic features are recurrent warts, cutaneous lymphovascular malformations, systemic involvement (lymphatic dysplasia), genital swelling and CD4 lymphopaenia with normal monocyte counts. The absence of family history suggests a sporadic condition, and the random distribution of swelling implicates mosaic postzygotic mutation as the cause.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Lymphatic/blood vessel plasticity: motivation for a future research area based on present and past observations.

The lymphatic system plays a significant role in homeostasis and drainage of excess fluid back into venous circulation. Lymphatics are also associated with a number of diseases including lymphedema, tumor metastasis, and various lymphatic malformations. Emerging evidence suggests that lymphatics might have a bigger connection to the blood vascular system than originally presumed. As these two systems are often studied in isolation, several knowledge gaps exist surrounding what constitutes lymphatic vascular plasticity, under what conditions it arises, and where structures characteristic of plasticity can form. The objective of this review is to overview current structural, cell lineage-based, and cell identity-based evidence for lymphatic plasticity. These examples of plasticity will then be considered in the context of potential clinical and surgical implications of this evolving research area. This review details our current understanding of lymphatic plasticity, highlights key unanswered questions in the field, and motivates future research aimed at clarifying the role and therapeutic potential of lymphatic plasticity in disease.

Biallelic variants in ADAMTS15 cause a novel form of distal arthrogryposis.

PURPOSE: We aimed to identify the underlying genetic cause for a novel form of distal arthrogryposis. METHODS: Rare variant family-based genomics, exome sequencing, and disease-specific panel sequencing were used to detect ADAMTS15 variants in affected individuals. Adamts15 expression was analyzed at the single-cell level during murine embryogenesis. Expression patterns were characterized using in situ hybridization and RNAscope. RESULTS: We identified homozygous rare variant alleles of ADAMTS15 in 5 affected individuals from 4 unrelated consanguineous families presenting with congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. Radiographic investigations showed physiological interphalangeal joint morphology. Additional features included knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. Analysis of mouse whole-embryo single-cell sequencing data revealed a tightly regulated Adamts15 expression in the limb mesenchyme between embryonic stages E11.5 and E15.0. A perimuscular and peritendinous expression was evident in in situ hybridization in the developing mouse limb. In accordance, RNAscope analysis detected a significant coexpression with Osr1, but not with markers for skeletal muscle or joint formation. CONCLUSION: In aggregate, our findings provide evidence that rare biallelic recessive trait variants in ADAMTS15 cause a novel autosomal recessive connective tissue disorder, resulting in a distal arthrogryposis syndrome.

3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM).

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities.

Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery, liver and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.

Scalable robust graph and feature extraction for arbitrary vessel networks in large volumetric datasets.

BACKGROUND: Recent advances in 3D imaging technologies provide novel insights to researchers and reveal finer and more detail of examined specimen, especially in the biomedical domain, but also impose huge challenges regarding scalability for automated analysis algorithms due to rapidly increasing dataset sizes. In particular, existing research towards automated vessel network analysis does not always consider memory requirements of proposed algorithms and often generates a large number of spurious branches for structures consisting of many voxels. Additionally, very often these algorithms have further restrictions such as the limitation to tree topologies or relying on the properties of specific image modalities. RESULTS: We propose a scalable iterative pipeline (in terms of computational cost, required main memory and robustness) that extracts an annotated abstract graph representation from the foreground segmentation of vessel networks of arbitrary topology and vessel shape. The novel iterative refinement process is controlled by a single, dimensionless, a-priori determinable parameter. CONCLUSIONS: We are able to, for the first time, analyze the topology of volumes of roughly 1 TB on commodity hardware, using the proposed pipeline. We demonstrate improved robustness in terms of surface noise, vessel shape deviation and anisotropic resolution compared to the state of the art. An implementation of the presented pipeline is publicly available in version 5.1 of the volume rendering and processing engine Voreen.

Adipose Tissue Hypertrophy, An Aberrant Biochemical Profile and Distinct Gene Expression in Lipedema.

BACKGROUND: Lipedema is a common adipose tissue disorder affecting women, characterized by a symmetric subcutaneous adipose tissue deposition, particularly of the lower extremities. Lipedema is usually underdiagnosed, thus remaining an undertreated disease. Importantly, no histopathologic or molecular hallmarks exist to clearly diagnose the disease, which is often misinterpreted as obesity or lymphedema. MATERIALS AND METHODS: The aim of the present study is to characterize in detail morphologic and molecular alterations in the adipose tissue composition of lipedema patients compared with healthy controls. Detailed histopathologic and molecular characterization was performed using lipid and cytokine quantification as well as gene expression arrays. The analysis was conducted on anatomically matched skin and fat tissue biopsies as well as fasting serum probes obtained from 10 lipedema and 11 gender and body mass index-matched control patients. RESULTS: Histologic evaluation of the adipose tissue showed increased intercellular fibrosis and adipocyte hypertrophy. Serum analysis showed an aberrant lipid metabolism without changes in the circulating adipokines. In an adipogenesis gene array, a distinct gene expression profile associated with macrophages was observed. Histologic assessment of the immune cell infiltrate confirmed the increased presence of macrophages, without changes in the T-cell compartment. CONCLUSIONS: Lipedema presents a distinguishable disease with typical tissue architecture and aberrant lipid metabolism, different to obesity or lymphedema. The differentially expressed genes and immune cell infiltration profile in lipedema patients further support these findings.

Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds.

Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations.

BACKGROUND: Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS: VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy-based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS: Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION: VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING: Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

The role of fatty acid ß-oxidation in lymphangiogenesis.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

Podoplanin and CLEC-2 drive cerebrovascular patterning and integrity during development.

Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.

A transgenic Prox1-Cre-tdTomato reporter mouse for lymphatic vessel research.

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.

Fusing VE-cadherin to α-catenin impairs fetal liver hematopoiesis and lymph but not blood vessel formation.

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin-α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin-α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin-α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.