Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain.

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.

The adaptor protein Grb2b is an essential modulator for lympho-venous sprout formation in the zebrafish trunk.

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

Sox7 controls arterial specification in conjunction with hey2 and efnb2 function.

SoxF family members have been linked to arterio-venous specification events and human pathological conditions, but in contrast to Sox17 and Sox18, a detailed in vivo analysis of a Sox7 mutant model is still lacking. In this study we generated zebrafish sox7 mutants to understand the role of Sox7 during vascular development. By in vivo imaging of transgenic zebrafish lines we show that sox7 mutants display a short circulatory loop around the heart as a result of aberrant connections between the lateral dorsal aorta (LDA) and either the venous primary head sinus (PHS) or the common cardinal vein (CCV). In situ hybridization and live observations in flt4:mCitrine transgenic embryos revealed increased expression levels of flt4 in arterial endothelial cells at the exact location of the aberrant vascular connections in sox7 mutants. An identical circulatory short loop could also be observed in newly generated mutants for hey2 and efnb2. By genetically modulating levels of sox7, hey2 and efnb2 we demonstrate a genetic interaction of sox7 with hey2 and efnb2. The specific spatially confined effect of loss of Sox7 function can be rescued by overexpressing the Notch intracellular domain (NICD) in arterial cells of sox7 mutants, placing Sox7 upstream of Notch in this aspect of arterial development. Hence, sox7 levels are crucial in arterial specification in conjunction with hey2 and efnb2 function, with mutants in all three genes displaying shunt formation and an arterial block.

Divergence of zebrafish and mouse lymphatic cell fate specification pathways.

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

Mutations in KIF11 cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy.

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.

Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema.

RATIONALE: Mutations in vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3 or FLT4) cause Milroy disease, an autosomal dominant condition that presents with congenital lymphedema. Mutations in VEGFR3 are identified in only 70% of patients with classic Milroy disease, suggesting genetic heterogeneity. OBJECTIVE: To investigate the underlying cause in patients with clinical signs resembling Milroy disease in whom sequencing of the coding region of VEGFR3 did not reveal any pathogenic variation. METHODS AND RESULTS: Exome sequencing of 5 such patients was performed, and a novel frameshift variant, c.571_572insTT in VEGFC, a ligand for VEGFR3, was identified in 1 proband. The variant cosegregated with the affected status in the family. An assay to assess the biological function of VEGFC activity in vivo, by expressing human VEGFC in the zebrafish floorplate was established. Forced expression of wild-type human VEGFC in the floorplate of zebrafish embryos leads to excessive sprouting in neighboring vessels. However, when overexpressing the human c.571_572insTT variant in the floorplate, no sprouting of vessels was observed, indicating that the base changes have a marked effect on the activity of VEGFC. CONCLUSIONS: We propose that the mutation in VEGFC is causative for the Milroy disease-like phenotype seen in this family. This is the first time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema.

A fisheye view on lymphangiogenesis.

Zebrafish have been widely used to study vasculogenesis and angiogenesis, and the vascular system is one of the most intensively studied organ systems in teleosts. It is a little surprising, therefore, that the development of the zebrafish lymphatic network has only been investigated in any detail for less than a decade now. In those last few years, however, significant progress has been made. Due to favorable imaging possibilities within the early zebrafish embryo, we have a very good understanding of what cellular behavior accompanies the formation of the lymphatic system and which cells within the vasculature are destined to contribute to lymphatic vessels. The migration routes of future lymphatic endothelial cells have been monitored in great detail, and a number of transgenic lines have been developed that help to distinguish between arterial, venous, and lymphatic fates in vivo. Furthermore, both forward and reverse genetic tools have been systematically employed to unravel which genes are involved in the process. Not surprisingly, a number of known players were identified (such as vegfc and flt4), but work on zebrafish has also distinguished genes and proteins that had not previously been connected to lymphangiogenesis. Here, we will review these topics and also compare the equivalent stages of lymphatic development in zebrafish and mice. We will, in addition, highlight some of those studies in zebrafish that have helped to identify and to further characterize human disease conditions.

CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo.

RATIONALE: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. OBJECTIVE: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. METHODS AND RESULTS: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1(+) lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C-mediated lymphangiogenesis in a corneal micropocket assay. CONCLUSIONS: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1(+) lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.

Patient-derived stem cell line UKMi005-A (hiPSC) harboring a non-synonymous heterozygous KCNJ5 gene variant.

A published heterozygous gain-of-function variant in the KCNJ5 gene (p.Trp101Cys) encoding the G-protein-activated inward-rectifier potassium channel 4 subunit of the IK,ACh channel is associated with human sinus node dysfunction (SND). Differentiated hiPSC-cardiomyocytes may serve as an in-vitro model to study SND and to develop pharmacological rescue strategies. Therefore, a mutant hiPSCs line from patient-derived peripheral blood mononuclear cells (PBMCs) were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit. The hiPSC line (KCNJ5 K8) showed a regular karyotype, a typical hiPSC morphology, expressed pluripotency-associated markers in immunofluorescence stainings and RT-qPCR analysis. The ability for differentiation into all three germ layers was shown.

Neural plate progenitors give rise to both anterior and posterior pituitary cells.

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis harboring hormones producing cells and the posterior pituitary/neurohypophysis, which relays the passage of hormones from the brain to the periphery. It is accepted that the adenohypophysis originates from the oral ectoderm (Rathke's pouch), whereas the neural ectoderm contributes to the neurohypophysis. Single-cell transcriptomics of the zebrafish pituitary showed that cyp26b1-positive astroglial pituicytes of the neurohypophysis and prop1-positive adenohypophyseal progenitors expressed common markers implying lineage relatedness. Genetic tracing identifies that, in contrast to the prevailing dogma, neural plate precursors of zebrafish (her4.3+) and mouse (Sox1+) contribute to both neurohypophyseal and a subset of adenohypophyseal cells. Pituicyte-derived retinoic-acid-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that adenohypophyseal cells are exclusively derived from non-neural ectoderm and demonstrate that crosstalk between neuro- and adeno-hypophyseal cells affects differentiation of pituitary cells.

Specific fibroblast subpopulations and neuronal structures provide local sources of Vegfc-processing components during zebrafish lymphangiogenesis.

Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Neuronal sFlt1 and Vegfaa determine venous sprouting and spinal cord vascularization.

Formation of organ-specific vasculatures requires cross-talk between developing tissue and specialized endothelial cells. Here we show how developing zebrafish spinal cord neurons coordinate vessel growth through balancing of neuron-derived Vegfaa, with neuronal sFlt1 restricting Vegfaa-Kdrl mediated angiogenesis at the neurovascular interface. Neuron-specific loss of flt1 or increased neuronal vegfaa expression promotes angiogenesis and peri-neural tube vascular network formation. Combining loss of neuronal flt1 with gain of vegfaa promotes sprout invasion into the neural tube. On loss of neuronal flt1, ectopic sprouts emanate from veins involving special angiogenic cell behaviours including nuclear positioning and a molecular signature distinct from primary arterial or secondary venous sprouting. Manipulation of arteriovenous identity or Notch signalling established that ectopic sprouting in flt1 mutants requires venous endothelium. Conceptually, our data suggest that spinal cord vascularization proceeds from veins involving two-tiered regulation of neuronal sFlt1 and Vegfaa via a novel sprouting mode.

Atypical E2fs control lymphangiogenesis through transcriptional regulation of Ccbe1 and Flt4.

Lymphatic vessels are derived from venous endothelial cells and their formation is governed by the Vascular endothelial growth factor C (VegfC)/Vegf receptor 3 (Vegfr3; Flt4) signaling pathway. Recent studies show that Collagen and Calcium Binding EGF domains 1 protein (Ccbe1) enhances VegfC-dependent lymphangiogenesis. Both Ccbe1 and Flt4 have been shown to be indispensable for lymphangiogenesis. However, how these essential players are transcriptionally regulated remains poorly understood. In the case of angiogenesis, atypical E2fs (E2f7 and E2f8) however have been recently shown to function as transcriptional activators for VegfA. Using a genome-wide approach we here identified both CCBE1 and FLT4 as direct targets of atypical E2Fs. E2F7/8 directly bind and stimulate the CCBE1 promoter, while recruitment of E2F7/8 inhibits the FLT4 promoter. Importantly, inactivation of e2f7/8 in zebrafish impaired venous sprouting and lymphangiogenesis with reduced ccbe1 expression and increased flt4 expression. Remarkably, over-expression of e2f7/8 rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel in vivo transcriptional regulators of Ccbe1 and Flt4, both essential genes for venous sprouting and lymphangiogenesis.

Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in the Drosophila gastrula.

The Drosophila guanine nucleotide exchange factor Pebble (Pbl) is essential for cytokinesis and cell migration during gastrulation. In dividing cells, Pbl promotes Rho1 activation at the cell cortex, leading to formation of the contractile actin-myosin ring. The role of Pbl in fibroblast growth factor-triggered mesoderm spreading during gastrulation is less well understood and its targets and subcellular localization are unknown. To address these issues we performed a domain-function study in the embryo. We show that Pbl is localized to the nucleus and the cell cortex in migrating mesoderm cells and found that, in addition to the PH domain, the conserved C-terminal tail of the protein is crucial for cortical localization. Moreover, we show that the Rac pathway plays an essential role during mesoderm migration. Genetic and biochemical interactions indicate that during mesoderm migration, Pbl functions by activating a Rac-dependent pathway. Furthermore, gain-of-function and rescue experiments suggest an important regulatory role of the C-terminal tail of Pbl for the selective activation of Rho1-versus Rac-dependent pathways.