Lymphatic vessels in cancer.

The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules, and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.

Single-cell mapping reveals new markers and functions of lymphatic endothelial cells in lymph nodes.

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.

KIT is dispensable for physiological organ vascularisation in the embryo.

Blood vessels form vast networks in all vertebrate organs to sustain tissue growth, repair and homeostatic metabolism, but they also contribute to a range of diseases with neovascularisation. It is, therefore, important to define the molecular mechanisms that underpin blood vessel growth. The receptor tyrosine kinase KIT is required for the normal expansion of hematopoietic progenitors that arise during embryogenesis from hemogenic endothelium in the yolk sac and dorsal aorta. Additionally, KIT has been reported to be expressed in endothelial cells during embryonic brain vascularisation and has been implicated in pathological angiogenesis. However, it is neither known whether KIT expression is widespread in normal organ endothelium nor whether it promotes blood vessel growth in developing organs. Here, we have used single-cell analyses to show that KIT is expressed in endothelial cell subsets of several organs, both in the adult and in the developing embryo. Knockout mouse analyses revealed that KIT is dispensable for vascularisation of growing organs in the midgestation embryo, including the lung, liver and brain. By contrast, vascular changes emerged during late-stage embryogenesis in these organs from KIT-deficient embryos, concurrent with severe erythrocyte deficiency and growth retardation. These findings suggest that KIT is not required for developmental tissue vascularisation in physiological conditions, but that KIT deficiency causes foetal anaemia at late gestation and thereby pathological vascular remodelling.

Sialoglycans on lymphatic endothelial cells augment interactions with Siglec-1 (CD169) of lymph node macrophages.

Cellular interactions between endothelial cells and macrophages regulate macrophage localization and phenotype, but the mechanisms underlying these interactions are poorly understood. Here we explored the role of sialoglycans on lymphatic endothelial cells (LEC) in interactions with macrophage-expressed Siglec-1 (CD169). Lectin-binding assays and mass spectrometric analyses revealed that LEC from human skin express more sialylated glycans than the corresponding blood endothelial cells. Higher amounts of sialylated and/or sulfated glycans on LEC than BEC were consistently observed in murine skin, lung and lymph nodes. The floor LEC of the subcapsular sinus (SCS) in murine lymph nodes (LN) displayed sialylated glycans at particularly high densities. The sialoglycans of LN LEC were strongly bound by Siglec-1. Such binding plays an important role in the localization of Siglec-1+ LN-SCS macrophages, as their numbers are strongly reduced in mice expressing a Siglec-1 mutant that is defective in sialoglycan binding. The residual Siglec-1+ macrophages are less proliferative and have a more anti-inflammatory phenotype. We propose that the densely clustered, sialylated glycans on the SCS floor LEC are a key component of the macrophage niche, providing anchorage for the Siglec-1+ LN-SCS macrophages.

Enlarged Field of View in Spatially Modulated Selective Volume Illumination Microscopy.

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.

Epigenetic regulation of the lineage specificity of primary human dermal lymphatic and blood vascular endothelial cells.

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more 'repressed' histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.

Lymphatic MAFB regulates vascular patterning during developmental and pathological lymphangiogenesis.

MAFB is a transcription factor involved in the terminal differentiation of several cell types, including macrophages and keratinocytes. MAFB is also expressed in lymphatic endothelial cells (LECs) and is upregulated by VEGF-C/VEGFR-3 signaling. Recent studies have revealed that MAFB regulates several genes involved in lymphatic differentiation and that global Mafb knockout mice show defects in patterning of lymphatic vessels during embryogenesis. However, it has remained unknown whether this effect is LEC-intrinsic and whether MAFB might also be involved in postnatal lymphangiogenesis. We established conditional, lymphatic-specific Mafb knockout mice and found comparable lymphatic patterning defects during embryogenesis as in the global MAFB knockout. Lymphatic MAFB deficiency resulted in increased lymphatic branching in the diaphragm at P7, but had no major effect on lymphatic patterning or function in healthy adult mice. By contrast, tumor-induced lymphangiogenesis was enhanced in mice lacking lymphatic MAFB. Together, these data reveal that LEC-expressed MAFB is involved in lymphatic vascular morphogenesis during embryonic and postnatal development as well as in pathological conditions. Therefore, MAFB could represent a target for therapeutic modulation of lymphangiogenesis.

Lymphatic endothelium contributes to colorectal cancer growth via the soluble matrisome component GDF11.

Colorectal cancer (CRC) is one of the most malignant tumors worldwide. Stromal cells residing in the tumor microenvironment strongly contribute to cancer progression through their crosstalk with cancer cells and extracellular matrix. Here we provide the first evidence that CRC-associated lymphatic endothelium displays a distinct matrisome-associated transcriptomic signature, which distinguishes them from healthy intestinal lymphatics. We also demonstrate that CRC-associated human intestinal lymphatic endothelial cells regulate tumor cell growth via growth differentiation factor 11, a soluble matrisome component which in CRC patients was found to be associated with tumor progression. Our data provide new insights into lymphatic contribution to CRC growth, aside from their conventional role as conduits of metastasis.

MFSD2A Promotes Endothelial Generation of Inflammation-Resolving Lipid Mediators and Reduces Colitis in Mice.

BACKGROUND & AIMS: Alterations in signaling pathways that regulate resolution of inflammation (resolving pathways) contribute to pathogenesis of ulcerative colitis (UC). The resolution process is regulated by lipid mediators, such as those derived from the ω-3 docosahexaenoic acid (DHA), whose esterified form is transported by the major facilitator superfamily domain containing 2A (MFSD2A) through the endothelium of brain, retina, and placenta. We investigated if and how MFSD2A regulates lipid metabolism of gut endothelial cells to promote resolution of intestinal inflammation. METHODS: We performed lipidomic and functional analyses of MFSD2A in mucosal biopsies and primary human intestinal microvascular endothelial cells (HIMECs) isolated from surgical specimens from patients with active, resolving UC and healthy individuals without UC (controls). MFSD2A was knocked down in HIMECs with small hairpin RNAs or overexpressed from a lentiviral vector. Human circulating endothelial progenitor cells that overexpress MFSD2A were transferred to CD1 nude mice with dextran sodium sulfate-induced colitis, with or without oral administration of DHA. RESULTS: Colonic biopsies from patients with UC had reduced levels of inflammation-resolving DHA-derived epoxy metabolites compared to healthy colon tissues or tissues with resolution of inflammation. Production of these metabolites by HIMECs required MFSD2A, which is required for DHA retention and metabolism in the gut vasculature. In mice with colitis, transplanted endothelial progenitor cells that overexpressed MFSD2A not only localized to the inflamed mucosa but also restored the ability of the endothelium to resolve intestinal inflammation, compared with mice with colitis that did not receive MFSD2A-overexpressing endothelial progenitors. CONCLUSIONS: Levels of DHA-derived epoxides are lower in colon tissues from patients with UC than healthy and resolving mucosa. Production of these metabolites by gut endothelium requires MFSD2A; endothelial progenitor cells that overexpress MFSD2A reduce colitis in mice. This pathway might be induced to resolve intestinal inflammation in patients with colitis.

Vascular endothelial growth factor C disrupts the endothelial lymphatic barrier to promote colorectal cancer invasion.

BACKGROUND & AIMS: Colorectal cancer (CRC) is highly metastatic. Metastases spread directly into local tissue or invade distant organs via blood and lymphatic vessels, but the role of lymphangiogenesis in CRC progression has not been determined. Lymphangiogenesis is induced via vascular endothelial growth factor C (VEGFC) activation of its receptor, VEGFR3; high levels of VEGFC have been measured in colorectal tumors undergoing lymphangiogenesis and correlated with metastasis. We investigated VEGFC signaling and lymphatic barriers in human tumor tissues and mice with orthotopic colorectal tumors. METHODS: We performed immunohistochemical, immunoblot, and real-time polymerase chain reaction analyses of colorectal tumor specimens collected from patients; healthy intestinal tissues collected during operations of patients without CRC were used as controls. CT26 CRC cells were injected into the distal posterior rectum of BALB/c-nude mice. Mice were given injections of an antibody against VEGFR3 or an adenovirus encoding human VEGFC before orthotopic tumors and metastases formed. Lymph node, lung, and liver tissues were collected and evaluated by flow cytometry. We measured expression of vascular endothelial cadherin (CDH5) on lymphatic vessels in mice and in human intestinal lymphatic endothelial cells. RESULTS: Levels of podoplanin (a marker of lymphatic vessels), VEGFC, and VEGFR3 were increased in colorectal tumor tissues, compared with controls. Mice that expressed VEGFC from the adenoviral vector had increased lymphatic vessel density and more metastases in lymph nodes, lungs, and livers, compared with control mice. Anti-VEGFR3 antibody reduced numbers of lymphatic vessels in colons and prevented metastasis. Expression of VEGFC compromised the lymphatic endothelial barrier in mice and endothelial cells, reducing expression of CDH5, increasing permeability, and increasing trans-endothelial migration by CRC cells. Opposite effects were observed in mice and cells when VEGFR3 was blocked. CONCLUSIONS: VEGFC signaling via VEGFR3 promotes lymphangiogenesis and metastasis by orthotopic colorectal tumors in mice and reduces lymphatic endothelial barrier integrity. Levels of VEGFC and markers of lymphatic vessels are increased in CRC tissues from patients, compared with healthy intestine. Strategies to block VEGFR3 might be developed to prevent CRC metastasis in patients.

Advances in therapeutic interventions targeting the vascular and lymphatic endothelium in inflammatory bowel disease.

PURPOSE OF REVIEW: The review summarizes the current knowledge of the roles played by the vascular and lymphatic endothelium throughout the gut in the pathogenesis of inflammatory bowel disease (IBD) and gives an update on emerging strategies targeting both vasculatures. RECENT FINDINGS: Enormous efforts have been made to understand the mechanisms underlining the origin, development and maintenance of intestinal chronic inflammation. In particular, new studies focused their attention on the role played by the microvascular and lymphatic endothelium in the pathogenesis of IBD. During inflammation, whereas the microvasculature is responsible for the entry and distribution of immune cells in the mucosa, the lymphatic system controls leukocyte exit, bacterial clearance and edema absorption. The study of these events, which are aberrant during chronic inflammation, has resulted in the identification and validation of several targets for the treatment of experimental colitis, some of which have translated into effective treatments for patients with IBD. SUMMARY: Although much attention has been paid to the microvascular endothelium and to antiangiogenic therapies, specific studies on the lymphatic vasculature and its functions in IBD are still at the initial stage, and other molecular mechanisms, genes, molecules and new pathways must definitely be explored.

The p300/CBP Inhibitor A485 Normalizes Psoriatic Fibroblast Gene Expression In Vitro and Reduces Psoriasis-Like Skin Inflammation In Vivo.

Psoriasis is a chronic inflammatory skin disease that often recurs at the same locations, indicating potential epigenetic changes in lesional skin cells. In this study, we discovered that fibroblasts isolated from psoriatic skin lesions retain an abnormal phenotype even after several passages in culture. Transcriptomic profiling revealed the upregulation of several genes, including the extra domain A splice variant of fibronectin and ITGA4 in psoriatic fibroblasts. A phenotypic library screening of small-molecule epigenetic modifier drugs revealed that selective CBP/p300 inhibitors were able to rescue the psoriatic fibroblast phenotype, reducing the expression levels of extra domain A splice variant of fibronectin and ITGA4. In the imiquimod-induced mouse model of psoriasis-like skin inflammation, systemic treatment with A485, a potent CBP/p300 blocker, significantly reduced skin inflammation, immune cell recruitment, and inflammatory cytokine production. Our findings indicate that epigenetic reprogramming might represent a new approach for the treatment and/or prevention of relapses of psoriasis.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

SEMA6A drives GnRH neuron-dependent puberty onset by tuning median eminence vascular permeability.

Innervation of the hypothalamic median eminence by Gonadotropin-Releasing Hormone (GnRH) neurons is vital to ensure puberty onset and successful reproduction. However, the molecular and cellular mechanisms underlying median eminence development and pubertal timing are incompletely understood. Here we show that Semaphorin-6A is strongly expressed by median eminence-resident oligodendrocytes positioned adjacent to GnRH neuron projections and fenestrated capillaries, and that Semaphorin-6A is required for GnRH neuron innervation and puberty onset. In vitro and in vivo experiments reveal an unexpected function for Semaphorin-6A, via its receptor Plexin-A2, in the control of median eminence vascular permeability to maintain neuroendocrine homeostasis. To support the significance of these findings in humans, we identify patients with delayed puberty carrying a novel pathogenic variant of SEMA6A. In all, our data reveal a role for Semaphorin-6A in regulating GnRH neuron patterning by tuning the median eminence vascular barrier and thereby controlling puberty onset.

Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling.

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.

Antibody-Mediated Delivery of VEGF-C Promotes Long-Lasting Lymphatic Expansion That Reduces Recurrent Inflammation.

The lymphatic vascular system plays a fundamental role in inflammation by draining interstitial fluid, immune cells, antigens, and inflammatory mediators from peripheral tissues. Site-specific delivery of the lymphangiogenic growth factor VEGF-C alleviates acute inflammation in mouse models of psoriasis and chronic colitis by enhancing local drainage. However, it is unclear whether therapeutically induced lymphangiogenesis is transient or long-lasting and whether it might prevent relapses of inflammation. Here, we investigated the long-term effects of targeted VEGF-C delivery in a chronic dermatitis model in mice. Congruent with our previous results, intravenous injection with a VEGF-C fusion protein targeted to the EDA domain of fibronectin initially resulted in reduced inflammation. Importantly, we found that targeted VEGF-C-mediated expansion of lymphatic vessels in the skin persisted for more than 170 days, long after primary inflammation had resolved. Furthermore, the treatment markedly decreased tissue swelling upon inflammatory re-challenge at the same site. Simultaneously, infiltration of leukocytes, including CD4+ T cells, macrophages, and dendritic cells, was significantly reduced in the previously treated group. In conclusion, our data show that targeted delivery of VEGF-C leads to long-lasting lymphatic expansion and long-term protection against repeated inflammatory challenge, suggesting that it is a promising new approach for the treatment of chronic, recurrent inflammatory diseases.

Mediators of Capillary-to-Venule Conversion in the Chronic Inflammatory Skin Disease Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and hyperkeratosis, immune cell infiltration and vascular remodeling. Despite the emerging recognition of vascular normalization as a potential strategy for managing psoriasis, an in-depth delineation of the remodeled dermal vasculature has been missing. In this study, we exploited 5' single-cell RNA sequencing to investigate the transcriptomic alterations in different subpopulations of blood vascular and lymphatic endothelial cells directly isolated from psoriatic and healthy human skin. Individual subtypes of endothelial cells underwent specific molecular repatterning associated with cell adhesion and extracellular matrix organization. Blood capillaries, in particular, showed upregulation of the melanoma cell adhesion molecule as well as its binding partners and adopted postcapillary venule‒like characteristics during chronic inflammation that are more permissive to leukocyte transmigration. We also identified psoriasis-specific interactions between cis-regulatory enhancers and promoters for each endothelial cell subtype, revealing the dysregulated gene regulatory networks in psoriasis. Together, our results provide more insights into the specific transcriptional responses and epigenetic signatures of endothelial cells lining different vessel compartments in chronic skin inflammation.

Lymphatic PD-L1 Expression Restricts Tumor-Specific CD8+ T-cell Responses.

Lymph node (LN)-resident lymphatic endothelial cells (LEC) mediate peripheral tolerance by self-antigen presentation on MHC-I and constitutive expression of T-cell inhibitory molecules, including PD-L1 (CD274). Tumor-associated LECs also upregulate PD-L1, but the specific role of lymphatic PD-L1 in tumor immunity is not well understood. In this study, we generated a mouse model lacking lymphatic PD-L1 expression and challenged these mice with two orthotopic tumor models, B16F10 melanoma and MC38 colorectal carcinoma. Lymphatic PD-L1 deficiency resulted in consistent expansion of tumor-specific CD8+ T cells in tumor-draining LNs in both tumor models, reduced primary tumor growth in the MC38 model, and increased efficacy of adoptive T-cell therapy in the B16F10 model. Strikingly, lymphatic PD-L1 acted primarily by inducing apoptosis in tumor-specific CD8+ central memory T cells. Overall, these findings demonstrate that LECs restrain tumor-specific immunity via PD-L1, which may explain why some patients with cancer without PD-L1 expression in the tumor microenvironment still respond to PD-L1/PD-1-targeted immunotherapy. SIGNIFICANCE: A new lymphatic-specific PD-L1 knockout mouse model reveals that lymphatic endothelial PD-L1 expression reduces tumor immunity, inducing apoptosis in tumor-specific CD8+ central memory cells in tumor-draining lymph nodes.

KIT Is Required for Fetal Liver Hematopoiesis.

In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial-to-hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed in hematopoietic progenitors that arise via endoHT from yolk sac hemogenic endothelium to generate early, transient hematopoietic waves. However, it remains unclear whether KIT has essential roles in early hematopoiesis. Here, we have combined single-cell expression studies with the analysis of knockout mice to show that KIT is dispensable for yolk sac endoHT but required for transient definitive hematopoiesis in the fetal liver.